Insights to Micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms

Insights to Micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insights to Micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms Tangxin Li, Yue Song, Quan Sun, Qiong Yang, Yunlin Tang, Zigang Shen, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5908051/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in BMC Genomics → Version 1 posted 6 You are reading this latest preprint version Abstract Microsporidia are a group of intracellular and unicellular eukaryotic parasites, which can nearly infect all animals, including human beings. As the first identified microsporidia, Nosema bombycis is a world-wide threat for silkworm eggs production, it can cause the congenital infection via transovarial transmission. It is important for pathogenesis elucidation to unravel the molecular characteristics of N. bombycis proliferation and host immune responses to the congenital infection in embryo and larva stage. Here, we adopted dual RNA-seq approach to investigate and compare the dynamic molecular pattern of pathogen proliferation and host immune responses between diapause and non-diapause silkworm eggs. Our results showed the N. bombycis proliferation in non-diapause silkworm eggs is a continuous process, many parasites enter the sporogony stage at 2 days post-oviposition (dpo). For newly hatched larva (1dph), the abundance of pathogen mRNA sequences is up to 2.32% in non-diapause strain, far higher than 0.34% of diapause strain, the main reason is the hot HCl bath treatment at 24 hours post-oviposition for diapause silkworm eggs with the aim to free the egg diapause. As to immune responses, whatever for diapause strain or non-diapause strain, there is stronger immune responses to congenital infection in larva stage than that of embryo stage, however, the host immune responses to congenital infection are fairly different between non-diapause and diapause strains of silkworms, especially in embryo stage. We found the surprising “First day Chaos” that there are 6,071 differential expressed genes (DEGs) at 1 dpo for non-diapause strain between infection group and uninfected group, but decreases dramatically to 109 DEGs at 2 dpo. In non-diapause strain, the earliest DEGs of antimicrobial peptides were up-regulated at 1 dpo, then is 5 dpo with up-regulated lebocin , 7 dpo with morLP-B1, morLP-B4. For non-diapause strain, the well-established immune responses were observed in newly hatched larvae. On the contrast, for diapause strain, the earliest DEGs of AMPs appear at 5 dph, the mature immune responses are well established at 5 dph too. In non-diapause silkworms, we observed obvious pathogen’s regulation in the main immune pathways including Toll, IMD, JAK-STAT and melanization at the different steps such as immune recognition, signal modulation and transduction, effectors. Taken together, our results for the first time provide a global molecular view of microsporidia proliferation and innate immunity responses in a congenital infection system and provide some new insights into immune development and establishment in the embryo and early larva stage of Bombyx mori . Bombyx mori microsporidia Nosema bombycis congenital infection immune response transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Regarded as “the Master Parasites” [1], Microsporidia are a group of fungi-related obligate intracellular and opportunistic parasites that infect a broad range of hosts, even immunocompromised humans [2–4]. To date, more than 1,700 species belonging to over 200 genera have been identified [5]. There are two modes of transmission of microsporidia, one is horizontal transmission and the other is vertical transmission [6, 7]. Vertical transmission is common in insect microsporidiosis. The insect microsporidia can reduce its virulence and can be transmitted to the next generation without affecting the ovarian development of the female host. Antonospora locustae infects embryonic tissue through vertical transmission, resulting in low hatching rate and high embryo mortality [7]. Vertical transmission of the Nosema fumiferanae can result in congenital infection that delay the hatching of Choristoneura fumiferana larvae [8]. As the first reported Microsporidia, Nosema bombycis is a major pathogen that causes a highly fatal silkworm disease, pébrine [9, 10]. The infection of N. bombycis in silkworm ovaries results in the parasite transovarial transmission, which causes congenital infection in silkworm embryos and larvae [11]. The congenital infection system composed of N. bombycis and silkworms becomes a good model to explore the characteristics of pathogen proliferation and host immune responses. In our previous work, Song et al. found that congenital N. bombycis proliferated mainly around yolk granules and in the intestinal lumen during the development of silkworm embryo, meanwhile, a small amount of infection presented in embryonic tissues [12]. Recently, Shen et al. investigated molecular proliferation in congenital infected embryo and larva of non-diapause silkworms, however, very limited data of only one time point (5 dpo) of embryo was obtained [13], the dynamic molecular proliferation characteristics of N. bombycis during the whole process of embryo development are not known yet. In addition to N. bombycis proliferation pattern in congenital infection, the host immune responses are very vital to understand the pathogenesis of pébrine disease. Despite the lack of adaptive immunity, invertebrates have an efficient innate immunity to recognize and eliminate invading pathogens [14, 15]. Innate immunity, including humoral and cellular responses, is activated during pathogen invasion and infection inside the host [16–19]. Generally, innate immunity in insects mainly includes the Toll pathway, immune deficiency pathway (IMD pathway), Janus kinase-signal transducer and activator of transcription signaling pathway (JAK-STAT signaling pathway), and melanization cascade, etc. [18]. Considering its economic value and the availability of genetic information [20], B. mori has become a valuable and well-characterized model system in Lepidoptera for studying insect innate immunity [21–23]. In 2013, Ma et al. found that N. bombycis oral infection induced a strong and complicated host response in silkworm larvae. Analysis of immune-related genes showed that the Toll pathway, JAK-STAT pathway, cellular immunity, and ROS response were induced, while the melanization of silkworm was inhibited [24]. For N. bombycis congenital infected silkworms, Shen et al. found that the expression of immune related genes, such as βGRP 2 , Spz 3 and pro-phenol oxidase , decreased in 5-day embryos with N. bombycis congenital infection compared with uninfected samples. While most immune genes, such as peptidoglycan recognition protein like ( PGRP-L ), Toll-like receptor 3 and antimicrobial peptide genes, were up-regulated in larvae exposed to the congenital N. bombycis challenge compared with uninfected samples [25]. However, also because the limited data of only one time point (5 dpo) in the embryo immune responses in congenital infection are available, it is necessary to explore the dynamic host immune responses during the whole process of embryo development. What’s more, although studies on B. mori innate immunity have progressed dramatically in post-genomics era, it remains unclear when silkworm establishes an immunocompetent immune system during the embryo development and how it responds to pathogen congenital infection. Thus, here we collected the silkworm eggs and larvae with Nosema bombycis congenital infection, dual RNA-seq was adopted to explore and compare the pathogen proliferation characteristics and host immune responses of the diapause and non-diapause silkworm embryos and larvae, we are also looking forward to investigate the temporal and spatial clues of innate immune system development and establishment in Bombyx mori . Material and methods Preparation of eggs congenitally infected by N. bombycis N. bombycis isolate CQ1 (Chongqing, China) was purified form infected silkworms and conserved in the China Veterinary Culture Collection Center (CVCC No. 102059). Spores were isolated from silkworm pupae that were challenged at the fourth instar stage by oral infection (approximately 10 4 spores per larvae) [26]. The silkworms of strain Chun 5 (diapause silkworm) and 305 (non-diapause silkworm), were reared at 26℃ under natural lighting in a dedicated room. Fifth instar larvae were challenged with mature spores by oral inoculation (approximately 10 6 spores per larva). Surviving pupae closed at approximately 17 days post inoculation. Approximately 2 hours post mating, the majority of moths completed oviposition and the eggs were collected and mixed immediately as pooled samples. Eggs from infected females were transferred to a sterile climate incubator at 28 ℃. Eggs were collected from uninfected moths and were prepared under the same conditions. RNA preparation and Illumina RNA-sequencing For diapause silkworm (strain: Chun 5), deep RNA sequencing from infected and uninfected eggs was performed at 1 day post-oviposition (dpo), 26 hours post-oviposition (hpo), and 2, 4, 6, 8 dpo and 1 days post-hatched (dph) from the same pooled samples. Those above newly hatched larvae (1 dph) were collected directly without feeding mulberry leaves. Silkworm larvae at 5 dph and 10 dph from another batch of congenitally infected pooled samples were collected as larva stages samples. Approximately 24 hpo, eggs were treated with an HCl solution (specific gravity 1.075) at 46℃ for 5 min to prevent eggs from diapausing [27, 28]. In our study, samples at 1 dpo represented eggs without HCl solution treatment, and samples at 26 hpo represented eggs treated with HCl solution for 5 min at 24 hours and collected 2 h later. Infected and uninfected samples were treated identically. For non-diapause silkworm (strain: 305), the samples were collected at 1, 2, 3, 5, 7 dpo and 1, 2, 3 dph from the same pooled samples. Newly hatched larvae (1 dph) were collected immediately with no feeding of mulberry leaves. All samples were treated with liquid nitrogen and stored at -80℃. Three replicates were included at each time point, and each sample included 50 eggs or larvae. Illumina RNA-seq for diapause silkworm was conducted by the Biomarker Technology Company, Beijing, China. And Illumina RNA-seq for non-diapause silkworm was conducted by the GeneDenovo Technology Company, Guangzhou, China. Total RNA was prepared from a mixture of 50 silkworm eggs or 50 larvae from each time point with three biological repeats using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA purity and integrity, the generation of sequencing libraries and the clustering of index-coded samples were performed as previously described [29]. After total RNA was extracted, the host and parasite mRNA were enriched by Oligo(dT) beads. Then the enriched mRNAs were fragmented into short fragments and reverse transcribed into cDNAs with random primers. Subsequently, the cDNA fragments were purified, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were sequenced using Illumina HiSeq2500 platform and paired-end reads were generated. The rRNA removed reads of each sample were then individually mapped to the reference genome by HISAT2 (version 2. 4) [30]. N. bombycis reference genome is download from https://silkpathdb.swu.edu.cn/ [20]. B. mori reference genome is download from https://silkdb.bioinfotoolkits.net [31]. The alignment parameters were: (1) Maximum read mismatch is 2; (2) Maximum distance between mate-pair reads is 50 bp; (3) The error of distance between mate-pair reads is ± 80 bp. Gene abundances were quantified by software StringTie (version 1.3.1) [32, 33] and normalized by using FPKM (Fragments Per Kilobase of transcript per Million mapped reads. \(\:\:\text{F}\text{P}\text{K}\text{M}=\frac{10E6C}{NL/10E3}\) , E: scientific notation, C: number of fragments mapped to target gene, N: total number of fragments that mapped to reference genes, L: number of bases on target gene) method. Therefore, the calculated gene expression was directly used for comparing the difference of gene expression among samples. The sequence data generated in this study have been submitted to the NCBI Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/sra ) with the accession number PRJNA549766 (for diapause silkworms) and PRJNA1215458 (for non-diapause silkworms). cDNA preparation and qRT-PCR detection of differential expression genes The RNA samples of non-diapause silkworm returned from the GeneDenovo Technology Company were reverse-transcribed by Hifair®Ⅱ 1st Strand cDNA Synthesis SuperMix for qRT-PCR (Yeasen Biotech Co., Ltd) to get the same concentration of cDNA. The obtained cDNA samples were used as a template for qRT-PCR. qRT-PCR were done by using Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotech Co., Ltd), and the primers are listed in Table S1 . Data analysis methods RNAs differential expression analysis was performed by DESeq2 [34] software between two different groups. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Principal component analysis (PCA) was performed with R package gmodels ( http://www.r-project.org/ ) in this experience. PCA is a statistical procedure that converts hundreds of thousands of correlated variables (gene expression) into a set of values of linearly uncorrelated variables called principal components. PCA is largely used to reveal the relationship of the samples. All the heatmaps were done with TBtools [35], all phylogenetic tree were done with MEGA11. All the protein sequences of N. bombycis were downloaded from NCBI ( https://www.ncbi.nlm.nih.gov/datasets/taxonomy/27978/ ) and all the protein sequences of B. mori were downloaded from SilkDB 3.0 ( https://silkdb.bioinfotoolkits.net/main/species-info/-1 ). Signal peptides prediction were all done by SignalP 6.0 ( https://services.healthtech.dtu.dk/services/SignalP-6.0/ ). Results Molecular proliferation characteristics of N. bombycis during the development process of non-diapause silkworm eggs with congenital infection To characterize parasite proliferation in non-diapause silkworm embryos and larvae from a molecular viewpoint, we obtained 645.02 Gb Clean Data from the non-diapause silkworm eggs and larvae, with 0.51–5.20% of these reads mapped to the N. bombycis genome (Fig. 1 A), totally, we get 4,413 genes expressed of the 4,486 annotated genes in N. bombycis . For diapause strain Chun 5, 300.42 Gb Clean Data from the infected eggs were obtained, with 0.07–0.89% of these reads mapped to the N. bombycis genome [12] (Fig. 1 B). It is obvious that for the diapause eggs, the 24 hrs’ hot HCl treatment to lift the diapause has a big impact on the proliferation of N. bombycis inside the eggs, there was a sharp down at 2 dpo for the detected mRNA of N. bombycis . Hot HCl bath treatment killed many parasites in congenital infected eggs, finally contribute the big difference in pathogen load in newly hatched larva between the non-diapause strain 305 and the diapause strain Chun 5, similar results also were verified by qRT-PCR in 2019 [36]. Then, we performed principal component analysis (PCA) of N. bombycis in non-diapause silkworm eggs based on the original RNA-seq data (Fig. 2 A). The samples of 2 dpo, 3 dpo, 5 dpo, 7 dpo and 1 dph gather into a big cluster, among them, samples of 5 dpo, 7 dpo and 1 dph form a small and closer cluster. The samples of 1 dpo, 2 dph and 3 dph are isolated from each other. Histone H4 and Spore wall protein 1 were the signature genes for merongony and sporogony stages respectively in the proliferation of N. bombycis , the expression level of Histone H4 was relatively high and stable, which suggest the pathogen proliferation rate was rapid. The expression level of SWP 1 increased sharply at 2 dpo, then decreased a little, and reached a peak at 3 dph (Fig. 2 B), SWP 1 become the top 1 highest transcribed gene except the 2 dph as top 2 gene, which suggest the more moronts start to enter the sporogony state from 2 dpo. In addition, we listed the number of DEG sets at each time point of N. bombycis (Fig. 2 C). We found that there were more up-regulated genes at 2dpo-vs-3dpo and 3dpo-vs-5dpo, and more genes were down-regulated in the larval stage. To explore transcriptional signatures of the life cycles of N. bombycis , we analyzed the top 100 highly expressed genes of N. bombycis from congenitally infected non-diapause silkworm samples (S1 Dataset). According to Venn diagrams from each time point, we identified 41 highly expressed core genes (Fig. 3 A) including eleven ribosomal proteins, ten histone proteins, four hypothetical proteins, three tubulin proteins, three actin proteins and ten genes related to essential elements of cell biology, involved in transcription and translation, cellular component, protein transport. The identification of genes related to ribosome synthesis, DNA replication and translation factors showed N. bombycis rapid proliferation in the embryo. After removing hypothetical proteins, ribosomal proteins and histone related genes from the top 100 gene sets of all time points, we conducted continuous statistics on the transcription of SWPs ( spore wall proteins ) [37], PTPs ( polar tube proteins ) [38] and tubulin proteins in each gene set (Fig. 3 B). At 1 dpo, cellular component ycf1 ( yeast cadmium factor 1 ) [39] was the highest transcribed gene, however SWP 1 become the highest transcribed gene in the following time points except in 2 dph as the second highest transcribed gene. In the data of 1 dpo, we did not find SWP 1 in Top 100 highly expressed genes, but ranked 1083th. What’s more, we did not find PTP 1, PTP 2, PTP 3 in 1 dpo data either. Above results suggest that most of parasites in 1 dpo just start their meront proliferation. However, from 2 dpo, more and more parasites enter into sporogony stage with the high expressed marker gene SWP 1 . For the sampling time between non-diapause silkworm 305 and diapause silkworm Chun 5, they share three common time points, 1 dpo, 2 dpo and 1 dph, which provide us an opportunity to compare N. bombycis proliferation tendency between them. For the 1 dpo, we did not observe the marker genes of sporogony, such as SWP 1, PTP 1, PTP 2, PTP 3 in Top 100 highly expressed genes neither in diapause nor non-diapause samples, which suggest that for 1 dpo, the main parasites were in meront stage inside diapause and non-diapause eggs, which is in consistent with the IFA (indirect immunefluorescence assay) results. The IFA results revealed that only a few scattered early development stages N. bombycis were situated around the yolk granules and germ band at 1 dpo, with no mature spores being found [12]. For 2 dpo, in non-diapause samples, sporogony marker genes, SWP 1, SWP 2, PTP 1, PTP 2 have been listed in Top 100 highly expressed genes, however for diapause samples, these genes were not listed in Top 100 highly expressed genes, for examples, SWP 1 was ranked 709th, two copies of PTP 1, NBO_79g0015, NBO_943g004 are ranked 785th and 1,861th respectively. These above results suggest Hot HCl treatment at 24 hrs (1 dpo) killed many parasites in the diapause silkworm eggs, Wang et al also verified the similar killing effect by qPCR method [36]. At 1dph, for the N. bombycis Top 100 highly expressed genes, the diapause and non-diapause samples share 58 genes including SWP 1, SWP 2, PTP 1, PTP 2 , which indicates that the parasite proliferation tendency is similar in newly hatched larvae of diapause and non-diapause silkworms. The expression characteristics of N. bombycis secreteome during the process of non-diapause silkworm egg development As the intracellular parasite, N. bombycis secreteome plays an important role in interaction between pathogen and host, especially some secreted effectors behaving in host manipulation. Here, a novel secreteome of N. bombycis was collected by SignalP6.0 prediction. Totally, 277 of 4,486 annotated proteins are predicted with signal peptides (Table S2 ). 264 of 277 secreteome genes were detected with transcription, 194 of 264 coding genes were annotated as hypothetical proteins (Fig. 4 A). 66 annotated genes were detected expression at each time-points (Fig. 4 B). We also listed the number of DEG sets of secreteome genes between two adjacent time points (Fig. 4 C). Among these genes with signal peptides, a part of genes are coding some structural proteins, such as PTP 1 ~ 3, SWP 1, SWP 5, ycf1 , most of them are belonging to highly expressed genes. What’ more, there are some enzymes with signal peptides, such as peptidase (NBO_65g0001), threonyl-tRNA synthetase (NBO_76gi004), Isoleucyl-tRNA synthetase (NBO_71g0001), Hexokinase-2 (NBO_1320g0001), Trehalase (NBO_10g0113), polysaccharide deactylase (NBO_53g0005), Acidic endochitinase sp2 (NBO_41g0043), Thioredoxin (NBO_10g0070). Here we carried out comparative genomics of Thioredoxin in microsporidia and found that Thioredoxin (with the signal peptide) is conserved in nearly all microsporidia, all orthologs in other microsporidia are with the predicted signal peptides (Fig. S1 ). Specifically, the thioredoxin in N. bombycis may be secreted and modulate the host's redox balance, and create favorable conditions for the pathogen's survival and proliferation [40, 41]. Thus, microsporidia thioredoxin’s localization and function are worthy of being investigated further. Now, we have confirmed that the N. bombycis secreted effectors, such as serpin 6 involved in host immunity regulation, serpin 14 inhibiting host cell apoptosis [42, 43]. It is believed that there are more secreted effectors in the secreteome that would be the arsenals of N. bombycis . Serpin family is very unique in Nosema genus, 19 NbSerpin members were annotated in the N. bombycis genome [44]. Among them, ten were predicted with the signal peptide. Here, the transcription of NbSerpins were analyzed (Fig. 4 D, Table S6 ). On the whole, the expression of NbSerpins in silkworm eggs were relatively low, and the transcription of serpin 9 and serpin 14 were not detected. Serpin 2 , serpin 3 , serpin 4 , serpin 6 and serpin 10 were expressed relatively higher than other serpins , coincidentally, these five serpins are all with predicted signal peptides. Meanwhile, these results are very similar to the Nbserpins expressions in diapause silkworm samples [12]. At 1 dpo, the transcription of Serpin 2 , serpin 8 , serpin 12, serpin 15, serpin 16 , serpin 18 and serpin 19 was not detected, serpin 4, serpin 5, serpin 6, serpin 10 were expressed relatively higher. For the newly hatched larva at 1 dph (9 dpo), only serpin 7 transcription was not detected. The function of NbSerpins family during the embryo stage still need more verification in future. Differential expression genes screening in host responses to microsporidia congenital infection In this study, we extracted total RNA from three replicates of uninfected and infected silkworm eggs or larvae at different time points. For diapause silkworm strain, nine time-points including 1 dpo, 26 hpo, 2, 4, 6, 8 dpo in embryo stages, 1 dph (9 dpo) and 5 dph, 10 dph of larva stages were covered. For non-diapause silkworm eggs or larvae, eight time-points (1, 2, 3, 5, 7 dpo and 1, 2, 3 dph) were designed. Then we utilized the HiSeq Illumina platform to obtain a global and continuous profile of transcriptome related to embryo and larva response to N. bombycis congenital infection. After the sequence analysis, we obtained 684.03 Gb Clean data from diapause silkworm and 645.02 Gb Clean Data from non-diapause silkworm. All of the clean reads were mapped with B. mori reference genome, the individual mapped rate from all samples was ranged from 54.11–86.05% in diapause silkworms (Table S3 ) and from 79.43–87.72% in non-diapause silkworms (Table S4 ). In addition, we also obtained information for 13,727 transcribed genes (93.8%) in diapause silkworm and 14,372 transcribed genes (98.28%) in non-diapause silkworm, compared with the total 14,623 annotated genes in B. mori . Pairwise correlation coefficients (Fig. 5 A) and principal component analysis (Fig. 5 B) of transcripts across all diapause silkworm egg samples illustrated that the reproducibility between samples was high. For both diapause silkworms and non-diapause silkworms, both uninfected groups and infected groups at the same time-point were clustered together (Fig. 5 C), suggesting that the congenital infection of N. bombycis did not significantly impact embryonic development in silkworm. For non-diapause silkworm data, we chose 32 differentially expressed genes randomly for relative qRT-PCR to verify the accuracy of data. The qRT-PCR results of 25 genes were consistent with the trend of transcriptome differences (Fig. 5 D), which proved that the differential genes screened by RNA-seq data were reliable. For the accuracy of diapause silkworm data, it had been verified by our previous work [12]. Analysis of DEGs (FDR ≤ 0.05, Fold change ≥ 2, S2 Dataset) at different host stages provides a number of potential clues to host stage-specific response against the N. bombycis congenital infection (Table 1 , Fig. S2 ). In general, the congenital infection of N. bombycis in diapause silkworm induces a weak embryo response with a total of 527 DEGs, including 342 up-regulated and 145 down-regulated in embryo stages (6 time-points), compared to a strong larva response with a total of 2,671 DEGs including 1,171 up-regulated and 1,500 down-regulated in larva stages (3 time-points) (Database S3). For non-diapause silkworm, we found 7,311 DEGs from eggs and 7,431 DEGs from larvae (Database S4). The surprising result is that there were so many DEGs (3,447 up-regulated and 2,624 down-regulated) at 1 dpo, we call it “First day Chaos”, the reason is not still known. We repeated the RNA-seq with same samples of 1dpo, including the infected and uninfected sample, we obtained similar results. In the remaining seven time points of the DEGs, 372 genes were up-regulated and 868 genes were down-regulated in all four time points of embryo stages, 1,827 genes up-regulated and 5,604 genes were down-regulated in all three time-points of larva stages. Table 1 A The number of DEGs in diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2) 1 dpo 26 hpo 2 dpo 4 dpo 6 dpo 8 dpo 1 dph 5 dph 10 dph Up 17 253 5 2 61 4 23 979 169 Down 26 61 14 28 3 13 17 907 576 Total 43 314 19 30 64 17 40 1886 745 Table 1 B The number of DEGs in non-diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2) 1 dpo 2 dpo 3 dpo 5 dpo 7 dpo 1 dph 2 dph 3 dph Up 3,447 117 15 119 121 417 234 1,176 Down 2,624 72 126 274 396 406 1,652 3,546 Total 6,071 189 141 393 517 823 1,886 4,722 Due to limited length of this paper, it is difficult to cover all host dynamic responses such as cell division, tissue and organ development, host metabolism and other responses according to the DEGs collected. Here we mainly analyze the host immune responses to the congenital infection in diapause and non-diapause silkworms. Besides the analysis of DEGs, we have screened most innate immunity-related genes involved in pathogen recognition (Fig. 6 ), classic immune pathway (Fig. 7 – 10 ) and AMPs (Fig. 11 ) in embryo and larva stages. The numbers of DEGs related to immune response in diapause silkworms and non-diapause silkworms were listed in Table 2 , IDs of genes involved in immune response were listed in Table S5 . Table 2 A The number of DEGs involved in immune response in diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2) 1 dpo 26 hpo 2 dpo 4 dpo 6 dpo 8 dpo 1 dph 5 dph 10 dph Up 0 5 0 0 0 0 1 38 2 Down 2 3 0 0 0 1 0 17 17 Total 2 8 0 0 0 1 1 55 19 Table 2 B The number of DEGs involved in immune response in non-diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2) 1 dpo 2 dpo 3 dpo 5 dpo 7 dpo 1 dph 2 dph 3 dph Up 41 0 0 7 9 22 15 20 Down 26 4 3 2 6 3 18 51 Total 67 4 3 9 15 25 33 71 Comparison of immune responses in embryo stage between diapause and non-diapause silkworms Based on the heat map, most of the immune-related genes in the diapause silkworms were expressed at a low level in embryo stages, among them, only a few genes that belonged to DEGs were significantly changed. For immune recognition genes, βGRP 1 ( β - Glucan recognition protein 1 ) [21, 45] was down-regulated at 1 dpo, CTL 16 ( C-type lectin 16 ) [46] and SCRC ( Scavenger receptor-C ) [21] were down-regulated at 26 hpo. CTL 15 was up-regulated at 8 dpo in diapause silkworm eggs (Fig. 6 ). For non-diapause silkworms (Fig. 6 ), a number of recognition genes were up-regulated at 1 dpo, such as PGRP-L5 ( peptidoglycan recognition protein-L5 ) [47], PGRP-S2, CTL 2/7/9/10/13/15/16/19, SCRB 1/5/6/8/9/13 and SCRC . Meanwhile, the βGRP 1/3, PGRP-L6, PGRP-S1,CTL 4/5, SCRAC 1, SCRB 3 and SCRB 12 were down-regulated at 1 dpo. Different from diapause silkworms, more recognition genes were significantly differentially expressed in the embryonic stage of non-diapause silkworms. However, at 2 dpo, only three genes were down-regulated, respectively were βGRP 2 , CTL 5 , SCRB 7 . At 3 dpo, CTL 5 , SCRB 8 were down-regulated. There were no up-regulated DEGs observed at 2 dpo and 3 dpo. At 5 dpo, PGRP-L1 , SCRB 13 were up-regulated while PGRP-S2 and CTL 20 were down-regulated. In silkworm, PGRP-S2 had been reported to be involved in the activation of IMD pathway [48], and PGRP-L1 has no interaction with IMD [49]. At 7 dpo, PGRP-S1 was up-regulated, it had been reported that PGRP-S1 can bind to PGN resulting the activation of the PPO cascade [47]. More analyses of PGRPs, CTLs and SCRs with signal peptides and domain information were showed in Fig. S3 /4/5. After the pathogens were recognized by pattern recognition receptors (PRRs), it would be presented to the signaling molecules of the immune pathways. For diapause silkworms (Fig. 7 ), there were no significantly expressed genes in Toll pathway in the embryonic stage. Notably, a universal adapter protein used by almost all tolls , Myd88 , was not expressed at embryonic stages. For non-diapause silkworms (Fig. 7 ), at 1 dpo, Spz 1 ( Spätzle 1 ) [50], Spz 2, Toll receptor 8/9 − 2/10 − 2/10 − 3 [23, 50]and Pelle were up-regulated, only Toll 3–3 and TRAF 2 were down-regulated. For the rest time points of the embryonic stage, only Spz 3 was significantly down-regulated at 7 dpo. It is interesting that in non-diapause silkworms, Myd88 expression was detected in all time points but at a relatively low level. In diapause silkworms, genes of IMD pathway were not induced significantly yet in the embryonic stage (Fig. 8 ). In non-diapause silkworms (Fig. 8 ), there were five DEGs ( Fadd was up-regulated, IAP 2, Tak 1, ikk β and Relish were down-regulated) in the embryonic stage, they were all belong to 1 dpo DEGs sets. For JAK-STAT pathway (Fig. 9 ), there were no DEGs in diapause silkworm in the embryos, and two down-regulated DEGs ( Socs and Fos ) in non-diapause silkworms at 1 dpo, however, no DEG in the following time points in embryonic stage. As to the PPO pathway, because now most CLIPs and SPNs function are not identified with detail, here we temporarily classify all CLIPs and SPNs into PPO pathway though some CLIPs are also behave in Toll and other pathways [51]. For diapause silkworms, only SPN 24 was down-regulated at 1 dpo and SPN 20 was down-regulated at 2 dpo, CLIP 2/4/8/9 were down-regulated at 2 dpo (Fig. 10 ). In non-diapause silkworm (Fig. 10 ), at 1dpo, CLIP 1/3/5/15 were up-regulated, CLIP 2/4/9 were down regulated. SPN 1/18/20/21/24 were up-regulated, SPN 3/5/6/7/22/26 were down regulated. We also found PPAE was up-regulated in 1 dpo. A putative defense protein precursor named Reeler 1 (BGIBMGA014360) was listed as up-regulated DEG at 1 dpo, which was verified as the reeler protein involved in regulating the PPO activity in PPO pathway [52].After 1 dpo, DEGs decreased a lot, there was only one DEG, SPN 9 (down-regulated) at 2 dpo, CLIP 4 was down regulated at 3 dpo, CLIP 7 , CLIP 15 , Lysozyme and Reeler 1 were up-regulated at 5 dpo. At 7 dpo, there are three up-regulated gene ( SPN 22, PPO 1, PPO 2 ) and five down-regulated genes ( CLIP 5/6/14, SPN 18/21 ). More analyses of CLIPs and SPNs with signal peptides and domain information were showed in Fig. S6 and S7. As to the antimicrobial peptides (Fig. 11 ), there was no AMP gene listed as DEG in diapause silkworms. On the contrary, in non-diapause silkworms, the earliest AMPs listed in DEGs were up-regulated gloverin 2, gloverin 3, gloverin 4 and defensin at 1 dpo. Lebocin was up-regulated at 5 dpo. At 7 dpo, morLP-B1 , morLP-B4 were up-regulated. Although some genes exhibited with big changes, but not listed as DEGs because FDR value was not ≤ 0.01. Comparison of immune responses in larva stage between diapause and non-diapause silkworms Immune responses to congenital infection in larva stage of diapause silkworms For diapause silkworms, compared with embryonic stage, it was evident that immune responses of larvae were induced strongly by N. bombycis congenital infection, mainly reflected by the significant expression changes of host immune-related genes. At 1 dph, there was only one DEG, Reeler 1 involved in regulating the PPO activity in PPO pathway. Although the expression of cecropin A1 / 3 , cecropin B3 , and gloverin 2 were higher in the infected group than the expression in the uninfected group but not significantly different (Fig. 11 ), suggesting the congenital infection of N. bombycis induced the innate immunity. By contrast, as shown in the heat map at 5 dph, a dozen of recognition related genes such as βGRP 2 , PGRP-S1 / S3 / S5 / S6 , CTL 11 , CTL 19, SCRAL1, SCRB 6 / 7 / 10 / 13 were up-regulated, but CTL 1 / 2 / 4 / 12 / 16 / 18 and SCRASP 4 were down-regulated (Fig. 6 ). In the Toll pathway (Fig. 7 ), Spz 2/3/4/6 were down-regulated but only Spz 4/6 were listed as DEGs, Toll 9 − 1 and TRAF 3 were up-regulated significantly. Interestingly, the Cactus , a negative regulatory factor in the Toll pathway, was significantly up-regulated. In addition, there was no DEG in IMD pathway (Fig. 8 ) and two up-regulated DEGs ( Dome and Fos ) in JAK-STAT pathway (Fig. 9 ). However, the PPO pathway was another sight (Fig. 10 ), CLIP 4/11 / 12 , SPN 5 / 7 / /19 / 22 and Reeler 1 were up-regulated; however, CLIP 5 / 14 , SPN 10 / 12 / 15 / 17 / 20 / 21 were down-regulated. Lysozyme and Lysozyme like protein 2 were significantly up-regulated. Effectors related genes including Cec B1 ~ B6 , gloverin 2 , defensin , moricin , morLP-B2 , and morLP-B5 were all up-regulated (Fig. 11 ). All results above showed that the innate immune system has been fully initiated by the congenital infection, and the pathogens also behave a lot to manipulate the host immunity through regulating the immune recognition and classic immune pathways. At 10 dph, the congenital infection became more serious, the infected silkworms were very weak and near to die. At this time point, recognition related genes such as CTL 1 / 2 / 5 / 9 and SCRB 8 were significantly down-regulated, βGRP 1 / 3 / 4 and PGRP-L2 / S3 / S5 / S6 , CTL 3 were up-regulated but not significant. In modulation related genes, CLIP 5 / 9 / 10 / 14 and SPN 6 / 20 were significantly down-regulated. At this time, most of Toll pathway genes did not show significant differences in expression, only Spz 2 , Spz 4 and Spz 6 were significantly down-regulated, Spz 3 was also down-regulated but not significantly (Fig. 7 ). In the PPO pathway (Fig. 10 ), only Reeler 1 was significantly up-regulated, the other genes listed as DEGs were all down-regulated, they were CLIP 5/9/10 , SPN 6/20 and PPO 1/2 , suggesting that the PPO pathway was inhibited by pathogens at this time. Moreover, effectors related genes such as enb 1, enb 2, gloverin 1, gloverin 2, leb and morLP-B1 were up-regulated but only gloverin 3 was significant. Notably, although the expression of Cec B1 ~ B6 were down-regulated, but not significantly, which formed an obvious contrast with the data of 5 dph. DEGs aforementioned implied that the host (larva stage) significantly enhanced the immunity against N. bombycis infection. Simultaneously, the pathogen may evade or manipulate the host’s innate immunity supported by these regulated genes. Immune responses to congenital infection in larva stage of non-diapause silkworms Similar to dispause silkworms, the immune response in the larva stage of non-diapause silkworm was stronger than embryo stage. At 1 dph, immune recognition genes (Fig. 6 ), PGRP-L1/S1/S3 and SCRB 13 were up-regulated, PGRP-L4 , SCRB 10 and SCRB 11 were down-regulated. The signal transduction genes in Toll pathway (Fig. 7 ), IMD pathway (Fig. 8 ) and JAK-STAT pathway (Fig. 9 ) had no DEG at 1dph. In PPO pathway (Fig. 10 ), there seven genes up-regulated but only three genes ( SPN 17 , Lysozyme and Reeler 1 ) were significant. Most of antimicrobial peptides genes were up-regulated, cec A1 , cec B1 ~ B6 , gloverin 2/4 and morLP-B1 ~ B5 significantly up-regulated with big changes (Fig. 11 ). At 2 dph, there were two up-regulation DEGs ( CTL 7 and SCRC ) and six down-regulation DEGs ( PGRP-S4 , SCRAC 2 , SCRASP 2 , SCRB 7 , SCRB 8 and SCRB 13 ) listed in the recognition related genes (Fig. 6 ). There were six Tolls ( Toll 6/7 − 2/7 − 3/8/10 − 2/12 ) were down-regulated while Toll 3–3 was up-regulated, implied that the Toll pathway was inhibited at this time. In JAK-STAT pathway (Fig. 9 ), it was the similar case, two genes ( Dome and Hem ) were significantly down-regulated. Inhibition of JAK-STAT pathway resulted in decreased survival rate and antibacterial activity of silkworm, we also observed that the larvae began to die at 2 dph. According to the transcriptome data, the surviving larvae at this time were also weak and the function of the immune system was deteriorated. Although the positive regulator of PPO pathway PGRP-S4 was down-regulated at this time point, the PPO pathway seemed as activated (Fig. 10 ). CLIP 3/13 , SPN 16 PPO 1/2 and Reeler 1 were up-regulated, CLIP 8 and SPN 10/20/21 were down-regulated. In addition, SPN 15 was up-regulated which was the negative regulator of PPO pathway. At this time point, it was interesting that the morLP-B1 ~ B5 were up-regulated, which same as 1 dph, while the other AMPs were down-regulated (Fig. 11 ). At 3 dph, most of immune-related DEGs were down-regulation. The recognition related genes (Fig. 6 ), only four DEGs ( PGRP-L4, PGRP-S5, CTL 11 and SCRAL 1 ) were up-regulated while ten DEGs (PGRP-S3, CTL 5/7/9/10/12, SCRAC 1/2, SCRB 7/9/13) were down-regulated. PGRP-S5 has been reported as the negative regulator of IMD pathway [53]. As expected, the IMD pathway was inhibited (Fig. 8 ), five genes ( IMD, Dredd , Table 2 , ikk β and Relish ) were down-regulated. The upstream of Toll pathway was up-regulation and the downstream was down-regulated (Fig. 7 ). Spz 1/3, Toll 3 − 2/3–3 and TRAF 3 were significantly up-regulated, Spz 2, Toll 6/9 − 2/10 − 2/10 − 3, Tube and TRAF 2 were significantly down-regulated. At this time, The Socs and Fos in JAK-STAT pathway were up-regulated, and Hem was down-regulated (Fig. 9 ).The PPO pathway at 3 dph was regulated by pathogens (Fig. 10 ). CLIP 5, SPN 8/10/12/15/16/18/21 and PPAE were significant down-regulated and CLIP 12/13, SPN 22 and Reeler 1 were up-regulated significantly. The Lysozyme was also significantly down-regulated. Similar to 2 dph, almost all AMPs were significant down-regulation but morLP-B1 ~ B5 were significant up-regulation at 3 dph. In general, compared with diapause silkworms, the immune-related genes, especially the AMPs of non-diapause silkworm larvae showed a fully induced immune responses in newly hatched larvae, and a lot of immune related genes in different steps were down-regulated, indicating that pathogens could regulate host immune pathways. Discussion N. bombycis has a complex and unique route to enter into the eggs and lead to congenital infection [54]. After that, N. bombycis starts to proliferate inside the eggs with embryo development. The histopathogical characteristics of N. bombycis proliferation in non-diapause silkworms had been investigated [12], here we provide the molecular proliferation characteristics of N. bombycis by Dual RNA-seq, our result is consistent with histopathological characteristics. For non-diapause silkworms, most of parasites were in meront stage at 1 dpo. From 2 dpo, many meronts enter into the sporogony stage, the corresponding molecular marker is SWP 1 ranking as the Top 1 expressed gene. We discovered that the big difference of the molecular proliferation characteristics between non-diapause silkworm and diapause silkworm resulted from the hot HCl treatment for diapause silkworm eggs, which can kill many parasites inside the eggs [36]. The hot HCl bath treatment interrupts the proliferation cycle of N. bombycis , and reduces the pathogen load in host. It has been reported that spore formation and parasite loads of Nosema muscidifuracis were reduced because of heat shock treatments in Musidifurax raptor eggs [55]. The pathogen load in non-diapause silkworm eggs and larvae was much higher than that of diapause silkworm eggs and larvae, which far influence the host immune responses in embryos and larvae. Compared previous results of larvae immune responses to N. bombycis infection [24, 56] with our current RNA-Seq analysis, immune responses to congenital infection in embryo stages were weaker than that of larva stages both in diapause and non-diapause silkworms. Four reasonable explanations were put forward. Firstly, the embryo gradually mounts a systemic and positive immunity and acquires its immune competence after or near hatching. Secondly, the proliferation of N. bombycis in embryos primarily centered around yolk granules and intestinal lumen [12]. The strategy adopted by N. bombycis may reduce the parasite’s direct stimulation on the embryo and weaken the priming of the host immune system. On the contrary, the parasites can cause a systemic and serious infection in larvae [57]. Thirdly, for diapause silkworm, the unique sequence mapped rates of N. bombycis were between 0.07% and 0.89% at embryo stages but increased to 3.35%~13.89% at larva stages. As a result, the parasite load difference between embryo and larva stages is another candidate factor. Finally, N. bombycis may actively regulate or escape the host immune response through its secreted proteins such as Serpin 6 [58], or abundant highly expressed genes while the function of these secretions is poorly understood [12, 59, 60]. It also has been reported that Encephalitozoon intestinalis may escape from host immune recognition through the modulation of dendritic cell differentiation and maturation [61]. The systematic immune response of B. mori to pathogen infection has been extensively analyzed [21, 22, 53]. However, there is limited knowledge regarding the equipping time of B. mori innate immunity. Here, we found the transcription of AMPs in infected groups significantly up-regulated at the 5 dpo in non-diapause silkworm embryo if we ignore the AMPs DEGs in “First day chaos”. At 7 dpo, the significant up-regulation of PPO 1 and PPO 2 marks the establishment of melanization pathway. In conclusion, for non-diapause silkworm, the differential expression of host genes proved that the congenital infection of N. bombycis in the embryo may lead to an earlier establishment of the host positive immune system. However, to elucidate the equipping time of silkworm innate immunity is a challenging task, only considering the transcription of immune-related genes is not enough to answer this question. We have analyzed the proteome composition in silkworm eggs with N. bombycis congenital infection and found there are several different AMPs and a lot of different immune related proteins, such as PRRs, CTL, SPNs, Spz 1, etc. (unpublished data), which suggest that maternal immunity may be also existed in insect eggs, especially the eggs with congenital infection [62]. In the larva stage, with the establishment of the immune system, the host immune response was more intense, and a large number of AMP genes were significantly up-regulated. However, at 2 dph, the upstream genes of PPO cascade was inhibited, the expression of intracellular signaling molecules in Toll pathway and IMD pathway were down-regulated, and the JAK-STAT pathway, which was related to survival and antibacterial activity, was also inhibited. On the whole, although the immune system of the host was completely established in the larva stage, the pathogen load increased and the physiological state of the host continued to deteriorate, the main antimicrobial peptides also showed a dynamic trend of up-regulation and then down-regulation, except for Moricins , which is the unique AMP family to Lepidoptera. Moricins maintained a very high expression level through the larva stage, its role in resistance to microsporidia infection needs further study. We obtained a large number (6,071) DEGs at 1 dpo for non-diapause silkworms, but at the following time points in embryo, the number of DEGs decreased to a lot, we call this phenomenon as “First Day Chaos” in non-diapause silkworm eggs. Why are there so many DEGs obtained at 1dpo? It is an intrigued question for future verification, we are wondering whether there is connection with different strains of silkworms. There may be different pathogen tolerance existed between the non-diapause silkworms and diapause silkworms. [63, 64] Notably, there are some limitations in our work. Although we found some genes with different expression patterns in immune responses to pathogen infection between the diapause silkworms and non-diapause silkworms, we should be cautious to face these differences, because their genetic backgrounds are not identical, some of our findings are just based on transcriptomic analyses and provide a clue for further research. It is necessary to adopt careful wet-lab work for future confirmation and verification. In summary, our study using deep transcriptional profiling of the silkworm hosts illustrates that gene expression characteristics of pathogen and host different immune responses in diapause and non-diapause silkworm embryo and larvae with N. bombycis congenital infection. Rapid proliferation of pathogen in host, weak immune responses in embryo stage and strong immune responses in larvae stage are the main findings here. Different pathogen loads in diapause and non-diapause silkworm are results from the hot HCL bath treatment for diapause silkworm eggs at 24 hours post oviposition, which has the strong killing effect for N. bombycis . In addition, the low pathogen load in diapause silkworm embryo and larvae is also connected with the weaker immune responses compared with non-diapause silkworm group. In general, here we not only developed a microsporidia-host system for analysis of microsporidia congenital infection and the host response, but also provide abundant molecular data for silkworm embryo innate immunity development anda sound basis for the complicated host-pathogen interactions. Declarations Ethics approval and consent to participate Silkworms are not a protected species and can be used as experimental materials without ethical approval. Consent for publication Not applicable. Availability of data and material The raw data generated was submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, http:// www. ncbi. nlm. nih. gov/ sra) with the BioProject accession number (PRJNA1215458). Competing interests Competing interests The authors declare no competing interests. Funding This study was funded by the National Natural Science Foundation of China (Grant No. 32272942 and 31470250, The Chongqing Modern Agricultural Industry Technology System (COMAITS202311), Chongqing elite, innovation and entrepreneurship demonstration team (CQYC202203091213), Fundamental Research Funds for the Central Universities (SWU-XDJH202322) and The Natural Science Foundation of Chongqing, China (cstc2021jcyj-msxmX1003). Authors' contributions TXL and YS: conceptualization and visualization; analyzed the data; experiments implementation; writing original draft; QY, YLT and ZSH: prepared the samples; QS, YLT, ZSH, ZGS and YBD: analyzed the data; TL and XZM: analyzed and uploaded the data; QS, JZZ and SL: prepared figures S1-S7; ZYZ: Conceptualization and supervision; JC and GQP: Supervision, review & editing the manuscript and Funding acquisition. Acknowledgements We appericiate Mulualem Lemma Kebede come from University of Gondar, Ethiopia for modification of the manuscript. References Vavra J, Lukes J: Microsporidia and 'the art of living together' . Adv Parasitol 2013, 82 :253-319. Didier ES: Microsporidiosis: an emerging and opportunistic infection in humans and animals . Acta Trop 2005, 94 (1):61-76. Stentiford GD, Becnel J, Weiss LM, Keeling PJ, Didier ES, Williams BP, Bjornson S, Kent ML, Freeman MA, Brown MJF et al : Microsporidia - Emergent Pathogens in the Global Food Chain . Trends in parasitology 2016, 32 (4):336-348. Wang ZD, Liu Q, Liu HH, Li S, Zhang L, Zhao YK, Zhu XQ: Prevalence of Cryptosporidium, microsporidia and Isospora infection in HIV-infected people: a global systematic review and meta-analysis . Parasites & vectors 2018, 11 (1):28. Han B, Pan G, Weiss LM: Microsporidiosis in Humans . Clin Microbiol Rev 2021, 34 (4):e0001020. Dunn AM, Smith JEJM, Infection: Microsporidian life cycles and diversity: the relationship between virulence and transmission . 2001, 3 (5):381-388. Raina SK, Das S, Rai MM, Khurad AM: Transovarial transmission of Nosema locustae (Microsporida: Nosematidae) in the migratory locust Locusta migratoria migratorioides . Parasitol Res 1995, 81 (1):38-44. Frankenhuyzen KV, Nystrom C, Liu YJJoIP: Vertical transmission of Nosema fumiferanae (Microsporidia: Nosematidae) and consequences for distribution, post-diapause emergence and dispersal of second-instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae) . 2007, 96 (2):173-182. Nageli: Uber die neue krankheit der seidenraupe und verwandte organismen . Bot Z 1857:15: 760-761. Pasteur L: Études sur la maladie des vers à soie : moyen pratique assuré de la combattre et d'en prévenir le retour : Paris :La-Villars, successeur de Mallet-Bachelier,1870; 1870. Meng XZ, Luo B, Tang XY, He Q, Xiong TR, Fang ZY, Pan G, Li T, Zhou ZY: Pathological analysis of silkworm infected by two microsporidia Nosema bombycis CQ1 and Vairimorpha necatrix BM . Journal of invertebrate pathology 2018, 153 :75-84. Song Y, Tang Y, Yang Q, Li T, He Z, Wu Y, He Q, Li T, Li C, Long M et al : Proliferation characteristics of the intracellular microsporidian pathogen Nosema bombycis in congenitally infected embryos . Journal of invertebrate pathology 2020, 169 :107310. Shen Z, Ke Z, Yang Q, Ghebremichael ST, Li T, Li T, Chen J, Meng X, Xiang H, Li C et al : Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae . BMC genomics 2024, 25 (1):321. Engstrom Y: Induction and regulation of antimicrobial peptides in Drosophila . Developmental and comparative immunology 1999, 23 (4-5):345-358. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA: Phylogenetic perspectives in innate immunity . Science 1999, 284 (5418):1313-1318. Hoffmann JA: The immune response of Drosophila . Nature 2003, 426 (6962):33-38. Hultmark D: Drosophila immunity: paths and patterns . Current opinion in immunology 2003, 15 (1):12-19. Lemaitre B, Hoffmann J: The host defense of Drosophila melanogaster . Annu Rev Immunol 2007, 25 :697-743. Yu XQ, Zhu YF, Ma C, Fabrick JA, Kanost MR: Pattern recognition proteins in Manduca sexta plasma . Insect biochemistry and molecular biology 2002, 32 (10):1287-1293. Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C et al : A draft sequence for the genome of the domesticated silkworm (Bombyx mori) . Science 2004, 306 (5703):1937-1940. Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T et al : A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori . Insect biochemistry and molecular biology 2008, 38 (12):1087-1110. Cheng T, Zhao P, Liu C, Xu P, Gao Z, Xia Q, Xiang Z: Structures, regulatory regions, and inductive expression patterns of antimicrobial peptide genes in the silkworm Bombyx mori . Genomics 2006, 87 (3):356-365. Cheng TC, Zhang YL, Liu C, Xu PZ, Gao ZH, Xia QY, Xiang ZH: Identification and analysis of Toll-related genes in the domesticated silkworm, Bombyx mori . Developmental and comparative immunology 2008, 32 (5):464-475. Ma Z, Li C, Pan G, Li Z, Han B, Xu J, Lan X, Chen J, Yang D, Chen Q et al : Genome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis . PLoS One 2013, 8 (12):e84137. Shen Z, Yang Q, Luo L, Li T, Ke Z, Li T, Chen J, Meng XZ, Xiang H, Li CJBG: Non-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection . 2023, 24 . Liu H, Pan G, Song S, Xu J, Li T, Deng Y, Zhou Z: Multiple rDNA units distributed on all chromosomes of Nosema bombycis . Journal of invertebrate pathology 2008, 99 (2):235-238. Kitta R, Yamahama Y, Yamamoto T, Mase K, Sawada H: Molecular Characterization and Tissue Distribution of Mitochondrial Ca(2+) -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori . Zoological science 2018, 35 (6):487-493. Tsurumaru S, Kawamori A, Mitsumasu K, Niimi T, Imai K, Yamashita O, Yaginuma T: Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause . Journal of insect physiology 2010, 56 (12):1721-1727. Wu S, Tong X, Peng C, Xiong G, Lu K, Hu H, Tan D, Li C, Han M, Lu C et al : Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori . Sci Rep 2016, 6 :26114. Kim D, Langmead B, Salzberg SLJNm: HISAT: a fast spliced aligner with low memory requirements . 2015, 12 (4):357-360. Lu F, Wei Z, Luo Y, Guo H, Zhang G, Xia Q, Wang Y: SilkDB 3.0: visualizing and exploring multiple levels of data for silkworm . 2020(1362-4962 (Electronic)). Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SLJNB: StringTie enables improved reconstruction of a transcriptome from RNA-seq reads . 2015, 33 (3):290-295. Kim, Daehwan, Leek, Jeffrey, T., Pertea, Geo, M., Salzberg, researchers SJNpef: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown . 2016, 11 (9):1650-1667. Love MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol 2014, 15 (12):550. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R: TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data . Mol Plant 2020, 13 (8):1194-1202. Lu Wang QY, Dongxu Xing, Zhengang Ma, Qingrong Li, Yang Xiao, Sentai Liao: Inactivation Effect of High Temperature Instant Acid Treatment on Nosema bombycis in Silkworm Eggs Detected by Ｒeal-time Fluorescent Quantitative PCR_In Chinese . Science of Sericulture 2019. Wu Z, Li Y, Pan G, Tan X, Xiang ZJP: Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia) . 2010, 8 (12):2447-2461. Han B, Takvorian P, Weiss LJEs: The Function and Structure of the Microsporidia Polar Tube . 2022, 114 :179-213. Chen Y, Wei E, Chen Y, He P, Wang R, Wang Q, Tang X, Zhang Y, Zhu F, Shen Z: Identification and subcellular localization analysis of membrane protein Ycf 1 in the microsporidian Nosema bombycis . 2022(2167-8359 (Print)). Dorey A, Cwiklinski K, Rooney J, Verissimo CDM, Lalor RJFiC, Microbiology I: Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1 . 2021, 11 :667272. He W, Sun X, Luo B, Liu M, Li L, Fan X, Ye J, Zhou B: Regulation of piglet T-cell immune responses by thioredoxin peroxidase from Cysticercus cellulosae excretory-secretory antigens . 2022, 13 . Bao J, Liu L, An Y, Ran M, Ni W, Chen J, Wei J, Li T, Pan G, Zhou Z: Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade . Journal of invertebrate pathology 2019, 168 :107260. Ran M, Bao J, Li B, Shi Y, Yang W, Meng X, Chen J, Wei J, Long M, Li T et al : Microsporidian Nosema bombycis secretes serine protease inhibitor to suppress host cell apoptosis via Caspase BmICE . PLOS Pathogens 2025, 21 (1):e1012373. Ran M, Shi Y, Li B, Xiang H, Tao M, Meng X, Li T, Li C, Bao J, Pan G et al : Genome-Wide Characterization and Comparative Genomic Analysis of the Serpin Gene Family in Microsporidian Nosema bombycis . 2023, 24 (1):550. Yoshida H, Ochiai M, Ashida M: β-1,3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system . 1986, 141 (3):0-1184. Rao XJ, Shahzad T, Liu S, Wu P, He YT, Sun WJ, Fan XY, Yang YF, Shi Q, Yu XQJD et al : Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori . 2015, 53 (2):328-338. Kinoshita, K.: Purification of a Peptidoglycan Recognition Protein from Hemolymph of the Silkworm, Bombyx mori . Journal of Biological Chemistry 1996, 271 (23):13854-13860. Yang J, Wang X, Tang S, Shen Z, Wu J: Peptidoglycan recognition protein S2 from silkworm integument: characterization, microbe-induced expression, and involvement in the immune-deficiency pathway . J Insect Sci 2015, 15 (1). Zhan MY, Yang PJ, Rao XJ: Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori . Comp Biochem Physiol B Biochem Mol Biol 2018, 215 :19-30. Yu B, Sang Q, Pan G, Li C, Zhou ZJI: A Toll-Sptzle Pathway in the Immune Response of Bombyx mori . 2020, 11 (9):586. Muhammad A, Sun C, Shao Y: The humoral immune response of the lepidopteran model insect, silkworm Bombyx mori L., to microbial pathogens . Current Research in Insect Science 2024, 6 :100097. Bao Y-Y, Xue J, Wu W-J, Wang Y, Lv Z-Y, Zhang C-X: An immune-induced Reeler protein is involved in the Bombyx mori melanization cascade . Insect biochemistry and molecular biology 2011, 41 (9):696-706. Chen K, Lu Z: Immune responses to bacterial and fungal infections in the silkworm, Bombyx mori . Developmental and comparative immunology 2018, 83 :3-11. Wang C, Yu B, Meng X, Xia D, Pei B, Tang X, Zhang G, Wei J, Long M, Chen J et al : Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission . PLOS Pathogens 2023, 19 (12):e1011859. Boohene CK, Geden CJ, Becnel JJ: Evaluation of Remediation Methods for Nosema Disease in Muscidifurax raptor (Hymenoptera: Pteromalidae) . Environmental Entomology 2003(5):1146-1153. Yue YJ, Tang XD, Xu L, Yan W, Li QL, Xiao SY, Fu XL, Wang W, Li N, Shen ZY: Early responses of silkworm midgut to microsporidium infection--A Digital Gene Expression analysis . Journal of invertebrate pathology 2015, 124 :6-14. Pan G, Bao J, Ma Z, Song Y, Han B, Ran M, Li C, Zhou Z: Invertebrate host responses to microsporidia infections . Developmental and comparative immunology 2018, 83 :104-113. Bao J, Liu L, An Y, Ran M, Ni W, Chen J, Wei J, Li T, Pan G, Zhou Z: Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade . J Invertebr Pathol 2019:107260. Weiss L: Microsporidia: pathogens of opportunity . John Wiley & Sons Inc 2015. Desjardins CA, Sanscrainte ND, Goldberg JM, Heiman D, Young S, Zeng Q, Madhani HD, Becnel JJ, Cuomo CA: Contrasting host-pathogen interactions and genome evolution in two generalist and specialist microsporidian pathogens of mosquitoes . Nature communications 2015, 6 :7121. Bernal CE, Zorro MM, Sierra J, Gilchrist K, Botero JH, Baena A, Ramirez-Pineda JR: Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism . Frontiers in cellular and infection microbiology 2016, 6 :4. Vilcinskas A: Mechanisms of transgenerational immune priming in insects . Developmental & Comparative Immunology 2021, 124 :104205. Shixian Liu YZ, Shaorong Ou: Genetic study of resistance to pébrine in Bombyx mori_ In Chinese . Guangdong Agricultural Sciences 1981. Yuanneng Zhang SL, Yongmei Huo, Shaorong Ou: Identification of Resistance of Several Silkworm Strains to Six Major Silkworm Diseases_In Chinese . Acta Sericologica Sinica 1982. Additional Declarations No competing interests reported. Supplementary Files S1DatasetTOP100ofN.bombycisinnondiapausesilkworm.zip S2DatasetDEGstesofN.bombycisinnondiapausesilkworm.zip S3DatasetDEGsetsofdiapausesilkworm.zip S4DatasetDEGsetsofnondiapausesilkworm.zip TableS1qRTPCRPrimersofDEGsinnondiapausesilkworm.docx TableS2ThegenesofN.bombycispredictedwithsignalpeptides.xlsx TableS4MappedreadsofNbandsilkworminnondiapausesilkworm.docx TableS3MappedreadsofNbandsilkwormindiapausesilkworm.docx Supplementarypictures.docx TableS5GeneIDsofthegenesinvolvedinimmuneresponseinsilkworms.docx TableS6GeneIDsoftheNbSerpingenes.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 20 May, 2025 Reviews received at journal 19 May, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers invited by journal 10 Apr, 2025 Submission checks completed at journal 09 Apr, 2025 First submitted to journal 09 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5908051","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441177321,"identity":"8eb96ae2-0249-49d1-aa1d-d713a6a093b0","order_by":0,"name":"Tangxin Li","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Tangxin","middleName":"","lastName":"Li","suffix":""},{"id":441177323,"identity":"8daff74f-09ab-4154-901e-09508a117df0","order_by":1,"name":"Yue Song","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Song","suffix":""},{"id":441177324,"identity":"05a09b2b-4756-4725-a885-9b9e08626df3","order_by":2,"name":"Quan Sun","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Quan","middleName":"","lastName":"Sun","suffix":""},{"id":441177326,"identity":"8a4fcc25-9598-48fd-b77c-e3343682db64","order_by":3,"name":"Qiong Yang","email":"","orcid":"","institution":"Guangdong Academy of Agricultural sciences","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Yang","suffix":""},{"id":441177328,"identity":"07de5871-b6d3-414b-9afa-c307767fc4d5","order_by":4,"name":"Yunlin Tang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yunlin","middleName":"","lastName":"Tang","suffix":""},{"id":441177330,"identity":"61404d0c-3938-4bc5-a463-ab6d16061d74","order_by":5,"name":"Zigang Shen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Zigang","middleName":"","lastName":"Shen","suffix":""},{"id":441177332,"identity":"70506111-c22b-4b20-9d63-caf4cd33da11","order_by":6,"name":"Zhangshuai He","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Zhangshuai","middleName":"","lastName":"He","suffix":""},{"id":441177333,"identity":"0cf6f6fd-6270-4a2b-aa31-91e4a93a00e8","order_by":7,"name":"Yabin Dou","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yabin","middleName":"","lastName":"Dou","suffix":""},{"id":441177334,"identity":"c4372b9d-49c3-4fd2-9ccd-88fc7e0be448","order_by":8,"name":"Junzheng Zhang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Junzheng","middleName":"","lastName":"Zhang","suffix":""},{"id":441177335,"identity":"d7ac5948-c5aa-4b55-8146-6d6a41ffe3c1","order_by":9,"name":"Sheng Li","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Li","suffix":""},{"id":441177336,"identity":"011cd336-d968-4cbc-b3fc-3b7f1f833ada","order_by":10,"name":"Tian Li","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Li","suffix":""},{"id":441177337,"identity":"f0be0b27-55a2-4589-9ae2-02d9fea4cef1","order_by":11,"name":"Xianzhi Meng","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xianzhi","middleName":"","lastName":"Meng","suffix":""},{"id":441177338,"identity":"83678f53-9bf1-4170-b0be-f13327735fea","order_by":12,"name":"Zeyang Zhou","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Zeyang","middleName":"","lastName":"Zhou","suffix":""},{"id":441177339,"identity":"7c974c3f-7aa9-4052-af3c-5d6b88c044bf","order_by":13,"name":"Jie Chen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Chen","suffix":""},{"id":441177340,"identity":"5e2c0ad9-6d2e-4c49-96d0-8f767c7e6426","order_by":14,"name":"Guoqing Pan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDADNgYGxgdAOoEkLcwGUC2MDUTrkiBKizn72QOMX9tsZPuk269V81TY5TFINx9/gE+LZU9eArNsW5pxm8yZsts8Z5KLGWSOJeK1xeBAjgGzZNvhxDaJnLTbvG3MiQ0SOYb4tZx/g9BSzPuvHqgl/yN+LTdyDBg/grWkH2PmbTgMsgW/9y1nAG1hOAf0i0QOs+ScY8eBetMMZ+DTYs4PtOVHmY3s/BnpDz+8qalO7JdIfvABr8MYGNh/84CjgseACcgAxSl+AIpwxh9gLewPQIxRMApGwSgYBRgAAHVxS1FKCBixAAAAAElFTkSuQmCC","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Guoqing","middleName":"","lastName":"Pan","suffix":""}],"badges":[],"createdAt":"2025-01-26 19:23:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5908051/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5908051/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11762-z","type":"published","date":"2025-07-01T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80364611,"identity":"f7860746-a778-4020-bbe7-be4481498246","added_by":"auto","created_at":"2025-04-11 05:00:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323836,"visible":true,"origin":"","legend":"\u003cp\u003eThe mapped rate of \u003cem\u003eN. bombycis\u003c/em\u003e reads to its genome in the transcriptome data of non-diapause silkworms (305) and diapause silkworms (Chun 5). dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.1MappedNbreadsintwostrian.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/86f593d48212dee330540397.jpg"},{"id":80364624,"identity":"3be31d28-064a-4e31-b164-4360cebf6076","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":519993,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenic transcripts dynamics in the transcriptome analysis of \u003cem\u003eN. bombycis\u003c/em\u003e infected non-diapause silkworm embryos and larvae. A: Principal component analysis of \u003cem\u003eN. bombycis\u003c/em\u003e RNA-Seq data. B: The transcription of \u003cem\u003eHistone H4\u003c/em\u003e (lower blue line chart) and spore wall protein \u003cem\u003eSWP 1\u003c/em\u003e (upper orange line chart) of \u003cem\u003eN. bombycis\u003c/em\u003e. The microsporidium in silkworms began to develop spore walls and mature progressively from 2 dpo. C: Numbers of significantly changed \u003cem\u003eN. bombycis\u003c/em\u003e transcripts. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.2ThemolecularcharacteristicofNbinnondiapausesilkworm.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/78274041fc3c2a396503436a.jpg"},{"id":80365088,"identity":"b5c940d9-d037-49bb-9981-91cfebc3e5f4","added_by":"auto","created_at":"2025-04-11 05:08:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":779977,"visible":true,"origin":"","legend":"\u003cp\u003eConsistently high expression genes in \u003cem\u003eN. bombycis\u003c/em\u003eduring proliferation in non-diapause silkworms. A: The transcription profiles of 41 genes which were among the top 100 highly expressed gene sets at each time point. Each column corresponds to a time-point, while each row represents an individual gene. B: Scatter plot showing the top 100 highly expressed gene sets of \u003cem\u003eN. bombycis\u003c/em\u003e, excluding \u003cem\u003ehistone proteins\u003c/em\u003e, \u003cem\u003eribosomal proteins\u003c/em\u003e and \u003cem\u003ehypothetical proteins\u003c/em\u003e. Red dots are \u003cem\u003eSWPs\u003c/em\u003e, blue dots are \u003cem\u003ePTPs\u003c/em\u003e, green dots are \u003cem\u003eTubulins\u003c/em\u003e and black dots are other genes in this sets.\u003c/p\u003e","description":"","filename":"Fig.3TheTOP100genesofNbinnondiapauseslikworm.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/4ea21e1d4e54c86a5b775801.jpg"},{"id":80365112,"identity":"dca321b2-01c9-4bdc-8a57-b0ed5d0c8d74","added_by":"auto","created_at":"2025-04-11 05:08:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1436191,"visible":true,"origin":"","legend":"\u003cp\u003eSecretome characteristics of \u003cem\u003eN. bombycis\u003c/em\u003e in non-diapause silkworms. A: The KEGG pathway of 264 \u003cem\u003eN. bombycis\u003c/em\u003e secreted protein genes predicted by SignalP 6.0 The global and overview maps contain a high-level comprehensive presentation of key metabolic pathways and functions in living organisms, including antibiotics biosynthesis, secondary metabolites biosynthesis, carbon metabolism, general metabolic processes and microbial metabolism across diverse environments. B: Clustering analysis of 66 core annotation genes with predicted signal peptide throughout the entire embryonic development. Each column corresponds to a time-point, and each row represents an individual gene. Gene expression variations at different time points are quantified relative to the 1 day post-oviposition (dpo) using log2 fold change (Log2FC). C: The numbers of DEGs which were predicted with signal peptide. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatch. D: Clustering analysis of \u003cem\u003eN. bombycis\u003c/em\u003e serine protease inhibitors \u003cem\u003e(Nbserpins)\u003c/em\u003e. Genes highlighted in red are predicted to encode signal peptides. The color gradient transitioning from white to red signifies a progressive increase in gene transcript levels. Each column represents a time-point, and each row represents a gene.\u003c/p\u003e","description":"","filename":"Fig.4SecretedproteingenesduringtheproliferationprocessoftheN.bombycis.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/d9956e2f03bb7040c974a7ff.jpg"},{"id":80364613,"identity":"dadbc7a1-1e62-4f23-b244-cf5caaa05605","added_by":"auto","created_at":"2025-04-11 05:00:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":806447,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome characterization of silkworm congenitally infected with \u003cem\u003eN. bombycis\u003c/em\u003e. A-B: Pairwise correlation coefficients (A) and principal component analysis (B) of diapause silkworm embryos with\u003cem\u003e N. bombycis\u003c/em\u003e congenital infection. C: Principal component analysis of non-diapause silkworm embryos and larvae with \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection. Un, healthy silkworm; In, \u003cem\u003eN. bombycis\u003c/em\u003e infected silkworm. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched. D: Comparison of qRT-PCR results with transcriptome expression trends.\u003c/p\u003e","description":"","filename":"Fig.5PCAofBminnondiapausesilkworm.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/58c29fe79d853f506a40e110.jpg"},{"id":80364649,"identity":"d107329b-94e5-45dd-8932-27de7c942f81","added_by":"auto","created_at":"2025-04-11 05:00:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":629805,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression of recognition protein genes were investigated in non-diapause (left) and diapause (right) silkworms. A positive sign (+) indicates significant up-regulation, whereas a negative sign (-) indicates significant down-regulation. Each column in the heatmap corresponds to a time-point, and each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.6Thedifferentexpressionofrecognitiongenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/6e75eb4ef0a588312bfd6d39.jpg"},{"id":80365093,"identity":"5d3ba52d-129d-431d-83e4-e05b2d6dec66","added_by":"auto","created_at":"2025-04-11 05:08:50","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":612939,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression analysis of Toll pathway related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.7ThedifferentexpressionofTollpathwaygenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/b34bd09f2d8d3dcdcb79f124.jpg"},{"id":80364629,"identity":"951a6b40-7bdc-49cd-95a2-555ee6dffdfd","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":375263,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression analysis of IMD pathway related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.8ThedifferentexpressionofIMDpathwaygenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/0583e7e29b2518c1047b6d17.jpg"},{"id":80364635,"identity":"ca395ba7-b815-4bfa-bb6b-50e9d3f91439","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":369760,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression analysis of JAK-STAT related pathway genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.9ThedifferentexpressionofJAKSTATpathwaygenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/371ea3fd6eb56192b689d096.jpg"},{"id":80364685,"identity":"f49ece80-117f-4605-ad75-813bbff49ad0","added_by":"auto","created_at":"2025-04-11 05:00:52","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":651065,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression analysis of melanization related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.10ThedifferentexpressionofPPOpathwaygenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/14629ea3c76b21252c18666d.jpg"},{"id":80364680,"identity":"62728be2-fe08-45ec-91d0-21b51528b32b","added_by":"auto","created_at":"2025-04-11 05:00:52","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":645854,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcriptional dynamics and differential expression analysis of antimicrobial peptides genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched.\u003c/p\u003e","description":"","filename":"Fig.11ThedifferentexpressionofAMPgenesinnondiapauseanddiapausesilkworm..tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/84f3e7d579059c39d76b1a1b.jpg"},{"id":86180828,"identity":"e24665d4-bc39-41fa-a048-1ea102f17c25","added_by":"auto","created_at":"2025-07-07 16:22:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11109662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/4ded8522-c583-4025-8b03-ebf523b42311.pdf"},{"id":80364631,"identity":"ce7bec1b-7bed-4e92-9fee-eb4cb374eda5","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":92197,"visible":true,"origin":"","legend":"","description":"","filename":"S1DatasetTOP100ofN.bombycisinnondiapausesilkworm.zip","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/16d47372425d028730a95711.zip"},{"id":80366151,"identity":"21fad8e5-19a0-4824-9e18-09c1088a3395","added_by":"auto","created_at":"2025-04-11 05:32:49","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":154164,"visible":true,"origin":"","legend":"","description":"","filename":"S2DatasetDEGstesofN.bombycisinnondiapausesilkworm.zip","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/d0e5ab5d9a2139c715af1747.zip"},{"id":80364619,"identity":"68d98040-4435-46f0-be25-d6d5231dd8e0","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":507039,"visible":true,"origin":"","legend":"","description":"","filename":"S3DatasetDEGsetsofdiapausesilkworm.zip","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/a8320b659fb447faf97547f7.zip"},{"id":80364615,"identity":"1e96a399-e45a-4cdf-b2ca-52a9ba9c4390","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"zip","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1327603,"visible":true,"origin":"","legend":"","description":"","filename":"S4DatasetDEGsetsofnondiapausesilkworm.zip","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/2f6157c4dcf6c9d9e5ca7cd7.zip"},{"id":80364620,"identity":"196aebbe-9d49-4fd5-953e-17200372670b","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16302,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1qRTPCRPrimersofDEGsinnondiapausesilkworm.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/96faf40cb6b14331ddacd34e.docx"},{"id":80366152,"identity":"923d2841-84c2-4215-a5a0-e2ead4d38705","added_by":"auto","created_at":"2025-04-11 05:32:50","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15749,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2ThegenesofN.bombycispredictedwithsignalpeptides.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/f1a369184a31d76bb4316141.xlsx"},{"id":80364622,"identity":"0a32e506-4440-4065-be77-c00e62eca5e8","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17357,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4MappedreadsofNbandsilkworminnondiapausesilkworm.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/97b2944949f80a9ca0eb9b24.docx"},{"id":80364630,"identity":"383afb42-44f0-4b0e-bccf-a18c80e6dc3a","added_by":"auto","created_at":"2025-04-11 05:00:50","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20845,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3MappedreadsofNbandsilkwormindiapausesilkworm.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/0b884d4034f4ece1d62ef0fb.docx"},{"id":80364650,"identity":"585a4985-d268-4a38-93c8-9ee3ab73bba5","added_by":"auto","created_at":"2025-04-11 05:00:51","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17690060,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarypictures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/aab49524c24082d617c384b5.docx"},{"id":80365101,"identity":"7765b2eb-0541-4927-8ce6-1bd8b7d6c362","added_by":"auto","created_at":"2025-04-11 05:08:51","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":20356,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5GeneIDsofthegenesinvolvedinimmuneresponseinsilkworms.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/d72ddcef132f0fa965c9fe17.docx"},{"id":80364651,"identity":"804006f4-8524-46e3-aaa4-ff47fde1d9af","added_by":"auto","created_at":"2025-04-11 05:00:51","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":15245,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6GeneIDsoftheNbSerpingenes.docx","url":"https://assets-eu.researchsquare.com/files/rs-5908051/v1/4503bfaeff0daadfba8e8027.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insights to Micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegarded as \u0026ldquo;the Master Parasites\u0026rdquo; [1], Microsporidia are a group of fungi-related obligate intracellular and opportunistic parasites that infect a broad range of hosts, even immunocompromised humans [2\u0026ndash;4]. To date, more than 1,700 species belonging to over 200 genera have been identified [5]. There are two modes of transmission of microsporidia, one is horizontal transmission and the other is vertical transmission [6, 7]. Vertical transmission is common in insect microsporidiosis. The insect microsporidia can reduce its virulence and can be transmitted to the next generation without affecting the ovarian development of the female host. \u003cem\u003eAntonospora locustae\u003c/em\u003e infects embryonic tissue through vertical transmission, resulting in low hatching rate and high embryo mortality [7]. Vertical transmission of the \u003cem\u003eNosema fumiferanae\u003c/em\u003e can result in congenital infection that delay the hatching of \u003cem\u003eChoristoneura fumiferana\u003c/em\u003e larvae [8]. As the first reported Microsporidia, \u003cem\u003eNosema bombycis\u003c/em\u003e is a major pathogen that causes a highly fatal silkworm disease, p\u0026eacute;brine [9, 10]. The infection of \u003cem\u003eN. bombycis\u003c/em\u003e in silkworm ovaries results in the parasite transovarial transmission, which causes congenital infection in silkworm embryos and larvae [11]. The congenital infection system composed of \u003cem\u003eN. bombycis\u003c/em\u003e and silkworms becomes a good model to explore the characteristics of pathogen proliferation and host immune responses. In our previous work, Song et al. found that congenital \u003cem\u003eN. bombycis\u003c/em\u003e proliferated mainly around yolk granules and in the intestinal lumen during the development of silkworm embryo, meanwhile, a small amount of infection presented in embryonic tissues [12]. Recently, Shen et al. investigated molecular proliferation in congenital infected embryo and larva of non-diapause silkworms, however, very limited data of only one time point (5 dpo) of embryo was obtained [13], the dynamic molecular proliferation characteristics of \u003cem\u003eN. bombycis\u003c/em\u003e during the whole process of embryo development are not known yet.\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003eN. bombycis\u003c/em\u003e proliferation pattern in congenital infection, the host immune responses are very vital to understand the pathogenesis of p\u0026eacute;brine disease. Despite the lack of adaptive immunity, invertebrates have an efficient innate immunity to recognize and eliminate invading pathogens [14, 15]. Innate immunity, including humoral and cellular responses, is activated during pathogen invasion and infection inside the host [16\u0026ndash;19]. Generally, innate immunity in insects mainly includes the Toll pathway, immune deficiency pathway (IMD pathway), Janus kinase-signal transducer and activator of transcription signaling pathway (JAK-STAT signaling pathway), and melanization cascade, etc. [18]. Considering its economic value and the availability of genetic information [20], \u003cem\u003eB. mori\u003c/em\u003e has become a valuable and well-characterized model system in Lepidoptera for studying insect innate immunity [21\u0026ndash;23]. In 2013, Ma et al. found that \u003cem\u003eN. bombycis\u003c/em\u003e oral infection induced a strong and complicated host response in silkworm larvae. Analysis of immune-related genes showed that the Toll pathway, JAK-STAT pathway, cellular immunity, and ROS response were induced, while the melanization of silkworm was inhibited [24]. For \u003cem\u003eN. bombycis\u003c/em\u003e congenital infected silkworms, Shen et al. found that the expression of immune related genes, such as \u003cem\u003eβGRP 2\u003c/em\u003e, \u003cem\u003eSpz 3\u003c/em\u003e and \u003cem\u003epro-phenol oxidase\u003c/em\u003e, decreased in 5-day embryos with \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection compared with uninfected samples. While most immune genes, such as \u003cem\u003epeptidoglycan recognition protein like\u003c/em\u003e (\u003cem\u003ePGRP-L\u003c/em\u003e), \u003cem\u003eToll-like receptor 3\u003c/em\u003e and antimicrobial peptide genes, were up-regulated in larvae exposed to the congenital \u003cem\u003eN. bombycis\u003c/em\u003e challenge compared with uninfected samples [25]. However, also because the limited data of only one time point (5 dpo) in the embryo immune responses in congenital infection are available, it is necessary to explore the dynamic host immune responses during the whole process of embryo development. What\u0026rsquo;s more, although studies on \u003cem\u003eB. mori\u003c/em\u003e innate immunity have progressed dramatically in post-genomics era, it remains unclear when silkworm establishes an immunocompetent immune system during the embryo development and how it responds to pathogen congenital infection.\u003c/p\u003e \u003cp\u003eThus, here we collected the silkworm eggs and larvae with \u003cem\u003eNosema bombycis\u003c/em\u003e congenital infection, dual RNA-seq was adopted to explore and compare the pathogen proliferation characteristics and host immune responses of the diapause and non-diapause silkworm embryos and larvae, we are also looking forward to investigate the temporal and spatial clues of innate immune system development and establishment in \u003cem\u003eBombyx mori\u003c/em\u003e.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003ePreparation of eggs congenitally infected by\u003c/b\u003e \u003cb\u003eN. bombycis\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eN. bombycis\u003c/em\u003e isolate CQ1 (Chongqing, China) was purified form infected silkworms and conserved in the China Veterinary Culture Collection Center (CVCC No. 102059). Spores were isolated from silkworm pupae that were challenged at the fourth instar stage by oral infection (approximately 10\u003csup\u003e4\u003c/sup\u003e spores per larvae) [26].\u003c/p\u003e \u003cp\u003eThe silkworms of strain Chun 5 (diapause silkworm) and 305 (non-diapause silkworm), were reared at 26℃ under natural lighting in a dedicated room. Fifth instar larvae were challenged with mature spores by oral inoculation (approximately 10\u003csup\u003e6\u003c/sup\u003e spores per larva). Surviving pupae closed at approximately 17 days post inoculation. Approximately 2 hours post mating, the majority of moths completed oviposition and the eggs were collected and mixed immediately as pooled samples. Eggs from infected females were transferred to a sterile climate incubator at 28 ℃. Eggs were collected from uninfected moths and were prepared under the same conditions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRNA preparation and Illumina RNA-sequencing\u003c/h2\u003e \u003cp\u003eFor diapause silkworm (strain: Chun 5), deep RNA sequencing from infected and uninfected eggs was performed at 1 day post-oviposition (dpo), 26 hours post-oviposition (hpo), and 2, 4, 6, 8 dpo and 1 days post-hatched (dph) from the same pooled samples. Those above newly hatched larvae (1 dph) were collected directly without feeding mulberry leaves. Silkworm larvae at 5 dph and 10 dph from another batch of congenitally infected pooled samples were collected as larva stages samples. Approximately 24 hpo, eggs were treated with an HCl solution (specific gravity 1.075) at 46℃ for 5 min to prevent eggs from diapausing [27, 28]. In our study, samples at 1 dpo represented eggs without HCl solution treatment, and samples at 26 hpo represented eggs treated with HCl solution for 5 min at 24 hours and collected 2 h later. Infected and uninfected samples were treated identically.\u003c/p\u003e \u003cp\u003eFor non-diapause silkworm (strain: 305), the samples were collected at 1, 2, 3, 5, 7 dpo and 1, 2, 3 dph from the same pooled samples. Newly hatched larvae (1 dph) were collected immediately with no feeding of mulberry leaves. All samples were treated with liquid nitrogen and stored at -80℃. Three replicates were included at each time point, and each sample included 50 eggs or larvae.\u003c/p\u003e \u003cp\u003eIllumina RNA-seq for diapause silkworm was conducted by the Biomarker Technology Company, Beijing, China. And Illumina RNA-seq for non-diapause silkworm was conducted by the GeneDenovo Technology Company, Guangzhou, China. Total RNA was prepared from a mixture of 50 silkworm eggs or 50 larvae from each time point with three biological repeats using TRIzol\u0026reg; reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol. RNA purity and integrity, the generation of sequencing libraries and the clustering of index-coded samples were performed as previously described [29]. After total RNA was extracted, the host and parasite mRNA were enriched by Oligo(dT) beads. Then the enriched mRNAs were fragmented into short fragments and reverse transcribed into cDNAs with random primers. Subsequently, the cDNA fragments were purified, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were sequenced using Illumina HiSeq2500 platform and paired-end reads were generated. The rRNA removed reads of each sample were then individually mapped to the reference genome by HISAT2 (version 2. 4) [30]. \u003cem\u003eN. bombycis\u003c/em\u003e reference genome is download from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://silkpathdb.swu.edu.cn/\u003c/span\u003e\u003cspan address=\"https://silkpathdb.swu.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [20]. \u003cem\u003eB. mori\u003c/em\u003e reference genome is download from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://silkdb.bioinfotoolkits.net\u003c/span\u003e\u003cspan address=\"https://silkdb.bioinfotoolkits.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [31]. The alignment parameters were: (1) Maximum read mismatch is 2; (2) Maximum distance between mate-pair reads is 50 bp; (3) The error of distance between mate-pair reads is \u0026plusmn;\u0026thinsp;80 bp. Gene abundances were quantified by software StringTie (version 1.3.1) [32, 33] and normalized by using FPKM (Fragments Per Kilobase of transcript per Million mapped reads.\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\text{F}\\text{P}\\text{K}\\text{M}=\\frac{10E6C}{NL/10E3}\\)\u003c/span\u003e\u003c/span\u003e, E: scientific notation, C: number of fragments mapped to target gene, N: total number of fragments that mapped to reference genes, L: number of bases on target gene) method. Therefore, the calculated gene expression was directly used for comparing the difference of gene expression among samples.\u003c/p\u003e \u003cp\u003eThe sequence data generated in this study have been submitted to the NCBI Sequence Read Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the accession number PRJNA549766 (for diapause silkworms) and PRJNA1215458 (for non-diapause silkworms).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ecDNA preparation and qRT-PCR detection of differential expression genes\u003c/h3\u003e\n\u003cp\u003eThe RNA samples of non-diapause silkworm returned from the GeneDenovo Technology Company were reverse-transcribed by Hifair\u0026reg;Ⅱ 1st Strand cDNA Synthesis SuperMix for qRT-PCR (Yeasen Biotech Co., Ltd) to get the same concentration of cDNA. The obtained cDNA samples were used as a template for qRT-PCR. qRT-PCR were done by using Hieff\u0026reg; qPCR SYBR\u0026reg; Green Master Mix (Yeasen Biotech Co., Ltd), and the primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eData analysis methods\u003c/h3\u003e\n\u003cp\u003eRNAs differential expression analysis was performed by DESeq2 [34] software between two different groups. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 were considered differentially expressed genes. Principal component analysis (PCA) was performed with R package gmodels (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in this experience. PCA is a statistical procedure that converts hundreds of thousands of correlated variables (gene expression) into a set of values of linearly uncorrelated variables called principal components. PCA is largely used to reveal the relationship of the samples. All the heatmaps were done with TBtools [35], all phylogenetic tree were done with MEGA11. All the protein sequences of \u003cem\u003eN. bombycis\u003c/em\u003e were downloaded from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/taxonomy/27978/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/taxonomy/27978/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and all the protein sequences of \u003cem\u003eB. mori\u003c/em\u003e were downloaded from SilkDB 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://silkdb.bioinfotoolkits.net/main/species-info/-1\u003c/span\u003e\u003cspan address=\"https://silkdb.bioinfotoolkits.net/main/species-info/-1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Signal peptides prediction were all done by SignalP 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/SignalP-6.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/SignalP-6.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMolecular proliferation characteristics of\u003c/b\u003e \u003cb\u003eN. bombycis\u003c/b\u003e \u003cb\u003eduring the development process of non-diapause silkworm eggs with congenital infection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize parasite proliferation in non-diapause silkworm embryos and larvae from a molecular viewpoint, we obtained 645.02 Gb Clean Data from the non-diapause silkworm eggs and larvae, with 0.51\u0026ndash;5.20% of these reads mapped to the \u003cem\u003eN. bombycis\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), totally, we get 4,413 genes expressed of the 4,486 annotated genes in \u003cem\u003eN. bombycis\u003c/em\u003e. For diapause strain Chun 5, 300.42 Gb Clean Data from the infected eggs were obtained, with 0.07\u0026ndash;0.89% of these reads mapped to the \u003cem\u003eN. bombycis\u003c/em\u003e genome [12] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). It is obvious that for the diapause eggs, the 24 hrs\u0026rsquo; hot HCl treatment to lift the diapause has a big impact on the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e inside the eggs, there was a sharp down at 2 dpo for the detected mRNA of \u003cem\u003eN. bombycis\u003c/em\u003e. Hot HCl bath treatment killed many parasites in congenital infected eggs, finally contribute the big difference in pathogen load in newly hatched larva between the non-diapause strain 305 and the diapause strain Chun 5, similar results also were verified by qRT-PCR in 2019 [36].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, we performed principal component analysis (PCA) of \u003cem\u003eN. bombycis\u003c/em\u003e in non-diapause silkworm eggs based on the original RNA-seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The samples of 2 dpo, 3 dpo, 5 dpo, 7 dpo and 1 dph gather into a big cluster, among them, samples of 5 dpo, 7 dpo and 1 dph form a small and closer cluster. The samples of 1 dpo, 2 dph and 3 dph are isolated from each other. \u003cem\u003eHistone H4\u003c/em\u003e and \u003cem\u003eSpore wall protein 1\u003c/em\u003e were the signature genes for merongony and sporogony stages respectively in the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e, the expression level of \u003cem\u003eHistone H4\u003c/em\u003e was relatively high and stable, which suggest the pathogen proliferation rate was rapid. The expression level of \u003cem\u003eSWP 1\u003c/em\u003e increased sharply at 2 dpo, then decreased a little, and reached a peak at 3 dph (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), \u003cem\u003eSWP 1\u003c/em\u003e become the top 1 highest transcribed gene except the 2 dph as top 2 gene, which suggest the more moronts start to enter the sporogony state from 2 dpo. In addition, we listed the number of DEG sets at each time point of \u003cem\u003eN. bombycis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We found that there were more up-regulated genes at 2dpo-vs-3dpo and 3dpo-vs-5dpo, and more genes were down-regulated in the larval stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore transcriptional signatures of the life cycles of \u003cem\u003eN. bombycis\u003c/em\u003e, we analyzed the top 100 highly expressed genes of \u003cem\u003eN. bombycis\u003c/em\u003e from congenitally infected non-diapause silkworm samples (S1 Dataset). According to Venn diagrams from each time point, we identified 41 highly expressed core genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) including eleven ribosomal proteins, ten histone proteins, four hypothetical proteins, three tubulin proteins, three actin proteins and ten genes related to essential elements of cell biology, involved in transcription and translation, cellular component, protein transport. The identification of genes related to ribosome synthesis, DNA replication and translation factors showed \u003cem\u003eN. bombycis\u003c/em\u003e rapid proliferation in the embryo. After removing \u003cem\u003ehypothetical proteins, ribosomal proteins\u003c/em\u003e and \u003cem\u003ehistone\u003c/em\u003e related genes from the top 100 gene sets of all time points, we conducted continuous statistics on the transcription of \u003cem\u003eSWPs\u003c/em\u003e (\u003cem\u003espore wall proteins\u003c/em\u003e) [37], \u003cem\u003ePTPs\u003c/em\u003e (\u003cem\u003epolar tube proteins\u003c/em\u003e) [38] and \u003cem\u003etubulin proteins\u003c/em\u003e in each gene set (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At 1 dpo, cellular component \u003cem\u003eycf1\u003c/em\u003e (\u003cem\u003eyeast cadmium factor 1\u003c/em\u003e) [39] was the highest transcribed gene, however \u003cem\u003eSWP 1\u003c/em\u003e become the highest transcribed gene in the following time points except in 2 dph as the second highest transcribed gene. In the data of 1 dpo, we did not find \u003cem\u003eSWP 1\u003c/em\u003e in Top 100 highly expressed genes, but ranked 1083th. What\u0026rsquo;s more, we did not find \u003cem\u003ePTP 1, PTP 2, PTP 3\u003c/em\u003e in 1 dpo data either. Above results suggest that most of parasites in 1 dpo just start their meront proliferation. However, from 2 dpo, more and more parasites enter into sporogony stage with the high expressed marker gene \u003cem\u003eSWP 1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the sampling time between non-diapause silkworm 305 and diapause silkworm Chun 5, they share three common time points, 1 dpo, 2 dpo and 1 dph, which provide us an opportunity to compare \u003cem\u003eN. bombycis\u003c/em\u003e proliferation tendency between them. For the 1 dpo, we did not observe the marker genes of sporogony, such as \u003cem\u003eSWP 1, PTP 1, PTP 2, PTP 3\u003c/em\u003e in Top 100 highly expressed genes neither in diapause nor non-diapause samples, which suggest that for 1 dpo, the main parasites were in meront stage inside diapause and non-diapause eggs, which is in consistent with the IFA (indirect immunefluorescence assay) results. The IFA results revealed that only a few scattered early development stages \u003cem\u003eN. bombycis\u003c/em\u003e were situated around the yolk granules and germ band at 1 dpo, with no mature spores being found [12]. For 2 dpo, in non-diapause samples, sporogony marker genes, \u003cem\u003eSWP 1, SWP 2, PTP 1, PTP 2\u003c/em\u003e have been listed in Top 100 highly expressed genes, however for diapause samples, these genes were not listed in Top 100 highly expressed genes, for examples, \u003cem\u003eSWP 1\u003c/em\u003e was ranked 709th, two copies of \u003cem\u003ePTP 1, NBO_79g0015, NBO_943g004\u003c/em\u003e are ranked 785th and 1,861th respectively. These above results suggest Hot HCl treatment at 24 hrs (1 dpo) killed many parasites in the diapause silkworm eggs, Wang et al also verified the similar killing effect by qPCR method [36]. At 1dph, for the \u003cem\u003eN. bombycis\u003c/em\u003e Top 100 highly expressed genes, the diapause and non-diapause samples share 58 genes including \u003cem\u003eSWP 1, SWP 2, PTP 1, PTP 2\u003c/em\u003e, which indicates that the parasite proliferation tendency is similar in newly hatched larvae of diapause and non-diapause silkworms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression characteristics of\u003c/b\u003e \u003cb\u003eN. bombycis\u003c/b\u003e \u003cb\u003esecreteome during the process of non-diapause silkworm egg development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs the intracellular parasite, \u003cem\u003eN. bombycis\u003c/em\u003e secreteome plays an important role in interaction between pathogen and host, especially some secreted effectors behaving in host manipulation. Here, a novel secreteome of \u003cem\u003eN. bombycis\u003c/em\u003e was collected by SignalP6.0 prediction. Totally, 277 of 4,486 annotated proteins are predicted with signal peptides (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). 264 of 277 secreteome genes were detected with transcription, 194 of 264 coding genes were annotated as hypothetical proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). 66 annotated genes were detected expression at each time-points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We also listed the number of DEG sets of secreteome genes between two adjacent time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Among these genes with signal peptides, a part of genes are coding some structural proteins, such as \u003cem\u003ePTP 1\u0026thinsp;~\u0026thinsp;3, SWP 1, SWP 5, ycf1\u003c/em\u003e, most of them are belonging to highly expressed genes. What\u0026rsquo; more, there are some enzymes with signal peptides, such as \u003cem\u003epeptidase\u003c/em\u003e (NBO_65g0001), \u003cem\u003ethreonyl-tRNA synthetase\u003c/em\u003e (NBO_76gi004), \u003cem\u003eIsoleucyl-tRNA synthetase\u003c/em\u003e (NBO_71g0001), \u003cem\u003eHexokinase-2\u003c/em\u003e (NBO_1320g0001), \u003cem\u003eTrehalase\u003c/em\u003e (NBO_10g0113), \u003cem\u003epolysaccharide deactylase\u003c/em\u003e (NBO_53g0005), \u003cem\u003eAcidic endochitinase sp2\u003c/em\u003e (NBO_41g0043), \u003cem\u003eThioredoxin\u003c/em\u003e (NBO_10g0070). Here we carried out comparative genomics of \u003cem\u003eThioredoxin\u003c/em\u003e in microsporidia and found that \u003cem\u003eThioredoxin\u003c/em\u003e (with the signal peptide) is conserved in nearly all microsporidia, all orthologs in other microsporidia are with the predicted signal peptides (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Specifically, the thioredoxin in \u003cem\u003eN. bombycis\u003c/em\u003e may be secreted and modulate the host's redox balance, and create favorable conditions for the pathogen's survival and proliferation [40, 41]. Thus, microsporidia thioredoxin\u0026rsquo;s localization and function are worthy of being investigated further. Now, we have confirmed that the \u003cem\u003eN. bombycis\u003c/em\u003e secreted effectors, such as serpin 6 involved in host immunity regulation, serpin 14 inhibiting host cell apoptosis [42, 43]. It is believed that there are more secreted effectors in the secreteome that would be the arsenals of \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSerpin family is very unique in \u003cem\u003eNosema\u003c/em\u003e genus, 19 \u003cem\u003eNbSerpin\u003c/em\u003e members were annotated in the \u003cem\u003eN. bombycis\u003c/em\u003e genome [44]. Among them, ten were predicted with the signal peptide. Here, the transcription of \u003cem\u003eNbSerpins\u003c/em\u003e were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). On the whole, the expression of \u003cem\u003eNbSerpins\u003c/em\u003e in silkworm eggs were relatively low, and the transcription of \u003cem\u003eserpin 9\u003c/em\u003e and \u003cem\u003eserpin 14\u003c/em\u003e were not detected. \u003cem\u003eSerpin 2\u003c/em\u003e, \u003cem\u003eserpin 3\u003c/em\u003e, \u003cem\u003eserpin 4\u003c/em\u003e, \u003cem\u003eserpin 6\u003c/em\u003e and \u003cem\u003eserpin 10\u003c/em\u003e were expressed relatively higher than other \u003cem\u003eserpins\u003c/em\u003e, coincidentally, these five \u003cem\u003eserpins\u003c/em\u003e are all with predicted signal peptides. Meanwhile, these results are very similar to the \u003cem\u003eNbserpins\u003c/em\u003e expressions in diapause silkworm samples [12]. At 1 dpo, the transcription of \u003cem\u003eSerpin 2\u003c/em\u003e, \u003cem\u003eserpin 8\u003c/em\u003e, \u003cem\u003eserpin 12, serpin 15, serpin 16\u003c/em\u003e, \u003cem\u003eserpin 18\u003c/em\u003e and \u003cem\u003eserpin 19\u003c/em\u003e was not detected, \u003cem\u003eserpin 4, serpin 5, serpin 6, serpin 10\u003c/em\u003e were expressed relatively higher. For the newly hatched larva at 1 dph (9 dpo), only \u003cem\u003eserpin 7\u003c/em\u003e transcription was not detected. The function of \u003cem\u003eNbSerpins\u003c/em\u003e family during the embryo stage still need more verification in future.\u003c/p\u003e\n\u003ch3\u003eDifferential expression genes screening in host responses to microsporidia congenital infection\u003c/h3\u003e\n\u003cp\u003eIn this study, we extracted total RNA from three replicates of uninfected and infected silkworm eggs or larvae at different time points. For diapause silkworm strain, nine time-points including 1 dpo, 26 hpo, 2, 4, 6, 8 dpo in embryo stages, 1 dph (9 dpo) and 5 dph, 10 dph of larva stages were covered. For non-diapause silkworm eggs or larvae, eight time-points (1, 2, 3, 5, 7 dpo and 1, 2, 3 dph) were designed. Then we utilized the HiSeq Illumina platform to obtain a global and continuous profile of transcriptome related to embryo and larva response to \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection. After the sequence analysis, we obtained 684.03 Gb Clean data from diapause silkworm and 645.02 Gb Clean Data from non-diapause silkworm. All of the clean reads were mapped with \u003cem\u003eB. mori\u003c/em\u003e reference genome, the individual mapped rate from all samples was ranged from 54.11\u0026ndash;86.05% in diapause silkworms (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and from 79.43\u0026ndash;87.72% in non-diapause silkworms (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In addition, we also obtained information for 13,727 transcribed genes (93.8%) in diapause silkworm and 14,372 transcribed genes (98.28%) in non-diapause silkworm, compared with the total 14,623 annotated genes in \u003cem\u003eB. mori\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePairwise correlation coefficients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and principal component analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) of transcripts across all diapause silkworm egg samples illustrated that the reproducibility between samples was high. For both diapause silkworms and non-diapause silkworms, both uninfected groups and infected groups at the same time-point were clustered together (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that the congenital infection of \u003cem\u003eN. bombycis\u003c/em\u003e did not significantly impact embryonic development in silkworm. For non-diapause silkworm data, we chose 32 differentially expressed genes randomly for relative qRT-PCR to verify the accuracy of data. The qRT-PCR results of 25 genes were consistent with the trend of transcriptome differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), which proved that the differential genes screened by RNA-seq data were reliable. For the accuracy of diapause silkworm data, it had been verified by our previous work [12].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of DEGs (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, Fold change\u0026thinsp;\u0026ge;\u0026thinsp;2, S2 Dataset) at different host stages provides a number of potential clues to host stage-specific response against the \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In general, the congenital infection of \u003cem\u003eN. bombycis\u003c/em\u003e in diapause silkworm induces a weak embryo response with a total of 527 DEGs, including 342 up-regulated and 145 down-regulated in embryo stages (6 time-points), compared to a strong larva response with a total of 2,671 DEGs including 1,171 up-regulated and 1,500 down-regulated in larva stages (3 time-points) (Database S3). For non-diapause silkworm, we found 7,311 DEGs from eggs and 7,431 DEGs from larvae (Database S4). The surprising result is that there were so many DEGs (3,447 up-regulated and 2,624 down-regulated) at 1 dpo, we call it \u0026ldquo;First day Chaos\u0026rdquo;, the reason is not still known. We repeated the RNA-seq with same samples of 1dpo, including the infected and uninfected sample, we obtained similar results. In the remaining seven time points of the DEGs, 372 genes were up-regulated and 868 genes were down-regulated in all four time points of embryo stages, 1,827 genes up-regulated and 5,604 genes were down-regulated in all three time-points of larva stages.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eA The number of DEGs in diapause silkworms (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, Fold change\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 hpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10 dph\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e576\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e745\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eB The number of DEGs in non-diapause silkworms (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, Fold change\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3 dph\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3,447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1,176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2,624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1,652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3,546\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6,071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1,886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4,722\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDue to limited length of this paper, it is difficult to cover all host dynamic responses such as cell division, tissue and organ development, host metabolism and other responses according to the DEGs collected. Here we mainly analyze the host immune responses to the congenital infection in diapause and non-diapause silkworms. Besides the analysis of DEGs, we have screened most innate immunity-related genes involved in pathogen recognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), classic immune pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e) and AMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e) in embryo and larva stages. The numbers of DEGs related to immune response in diapause silkworms and non-diapause silkworms were listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, IDs of genes involved in immune response were listed in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eA The number of DEGs involved in immune response in diapause silkworms (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, Fold change\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 hpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10 dph\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eB The number of DEGs involved in immune response in non-diapause silkworms (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05, Fold change\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7 dpo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 dph\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3 dph\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eComparison of immune responses in embryo stage between diapause and non-diapause silkworms\u003c/h2\u003e \u003cp\u003eBased on the heat map, most of the immune-related genes in the diapause silkworms were expressed at a low level in embryo stages, among them, only a few genes that belonged to DEGs were significantly changed. For immune recognition genes, \u003cem\u003eβGRP 1\u003c/em\u003e (\u003cem\u003eβ\u003c/em\u003e-\u003cem\u003eGlucan recognition protein 1\u003c/em\u003e) [21, 45] was down-regulated at 1 dpo, \u003cem\u003eCTL 16\u003c/em\u003e (\u003cem\u003eC-type lectin 16\u003c/em\u003e) [46] and \u003cem\u003eSCRC\u003c/em\u003e (\u003cem\u003eScavenger receptor-C\u003c/em\u003e) [21] were down-regulated at 26 hpo. \u003cem\u003eCTL 15\u003c/em\u003e was up-regulated at 8 dpo in diapause silkworm eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor non-diapause silkworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), a number of recognition genes were up-regulated at 1 dpo, such as \u003cem\u003ePGRP-L5\u003c/em\u003e (\u003cem\u003epeptidoglycan recognition protein-L5\u003c/em\u003e) [47], \u003cem\u003ePGRP-S2, CTL 2/7/9/10/13/15/16/19, SCRB 1/5/6/8/9/13\u003c/em\u003e and \u003cem\u003eSCRC\u003c/em\u003e. Meanwhile, the \u003cem\u003eβGRP 1/3, PGRP-L6, PGRP-S1,CTL 4/5, SCRAC 1, SCRB 3\u003c/em\u003e and \u003cem\u003eSCRB 12\u003c/em\u003e were down-regulated at 1 dpo. Different from diapause silkworms, more recognition genes were significantly differentially expressed in the embryonic stage of non-diapause silkworms. However, at 2 dpo, only three genes were down-regulated, respectively were \u003cem\u003eβGRP 2\u003c/em\u003e, \u003cem\u003eCTL 5\u003c/em\u003e, \u003cem\u003eSCRB 7\u003c/em\u003e. At 3 dpo, \u003cem\u003eCTL 5\u003c/em\u003e, \u003cem\u003eSCRB 8\u003c/em\u003e were down-regulated. There were no up-regulated DEGs observed at 2 dpo and 3 dpo. At 5 dpo, \u003cem\u003ePGRP-L1\u003c/em\u003e, \u003cem\u003eSCRB 13\u003c/em\u003e were up-regulated while \u003cem\u003ePGRP-S2\u003c/em\u003e and \u003cem\u003eCTL 20\u003c/em\u003e were down-regulated. In silkworm, PGRP-S2 had been reported to be involved in the activation of IMD pathway [48], and PGRP-L1 has no interaction with IMD [49]. At 7 dpo, \u003cem\u003ePGRP-S1\u003c/em\u003ewas up-regulated, it had been reported that PGRP-S1 can bind to PGN resulting the activation of the PPO cascade [47]. More analyses of \u003cem\u003ePGRPs, CTLs\u003c/em\u003e and \u003cem\u003eSCRs\u003c/em\u003e with signal peptides and domain information were showed in Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e/4/5.\u003c/p\u003e \u003cp\u003eAfter the pathogens were recognized by pattern recognition receptors (PRRs), it would be presented to the signaling molecules of the immune pathways. For diapause silkworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), there were no significantly expressed genes in Toll pathway in the embryonic stage. Notably, a universal adapter protein used by almost all \u003cem\u003etolls\u003c/em\u003e, \u003cem\u003eMyd88\u003c/em\u003e, was not expressed at embryonic stages. For non-diapause silkworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), at 1 dpo, \u003cem\u003eSpz 1\u003c/em\u003e (\u003cem\u003eSp\u0026auml;tzle 1\u003c/em\u003e) [50], \u003cem\u003eSpz 2, Toll receptor 8/9\u0026thinsp;\u0026minus;\u0026thinsp;2/10\u0026thinsp;\u0026minus;\u0026thinsp;2/10\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/em\u003e [23, 50]and \u003cem\u003ePelle\u003c/em\u003e were up-regulated, only \u003cem\u003eToll 3\u0026ndash;3\u003c/em\u003e and \u003cem\u003eTRAF 2\u003c/em\u003e were down-regulated. For the rest time points of the embryonic stage, only \u003cem\u003eSpz 3\u003c/em\u003e was significantly down-regulated at 7 dpo. It is interesting that in non-diapause silkworms, \u003cem\u003eMyd88\u003c/em\u003e expression was detected in all time points but at a relatively low level.\u003c/p\u003e \u003cp\u003eIn diapause silkworms, genes of IMD pathway were not induced significantly yet in the embryonic stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In non-diapause silkworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e), there were five DEGs (\u003cem\u003eFadd\u003c/em\u003e was up-regulated, \u003cem\u003eIAP 2, Tak 1, ikk β\u003c/em\u003e and \u003cem\u003eRelish\u003c/em\u003e were down-regulated) in the embryonic stage, they were all belong to 1 dpo DEGs sets. For JAK-STAT pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e), there were no DEGs in diapause silkworm in the embryos, and two down-regulated DEGs (\u003cem\u003eSocs\u003c/em\u003e and \u003cem\u003eFos\u003c/em\u003e) in non-diapause silkworms at 1 dpo, however, no DEG in the following time points in embryonic stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs to the PPO pathway, because now most CLIPs and SPNs function are not identified with detail, here we temporarily classify all \u003cem\u003eCLIPs\u003c/em\u003e and \u003cem\u003eSPNs\u003c/em\u003e into PPO pathway though some CLIPs are also behave in Toll and other pathways [51]. For diapause silkworms, only \u003cem\u003eSPN 24\u003c/em\u003e was down-regulated at 1 dpo and \u003cem\u003eSPN 20\u003c/em\u003e was down-regulated at 2 dpo, \u003cem\u003eCLIP 2/4/8/9\u003c/em\u003e were down-regulated at 2 dpo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). In non-diapause silkworm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), at 1dpo, \u003cem\u003eCLIP 1/3/5/15\u003c/em\u003e were up-regulated, \u003cem\u003eCLIP 2/4/9\u003c/em\u003e were down regulated. \u003cem\u003eSPN 1/18/20/21/24\u003c/em\u003e were up-regulated, \u003cem\u003eSPN 3/5/6/7/22/26\u003c/em\u003e were down regulated. We also found \u003cem\u003ePPAE\u003c/em\u003e was up-regulated in 1 dpo. A putative defense protein precursor named \u003cem\u003eReeler 1\u003c/em\u003e (BGIBMGA014360) was listed as up-regulated DEG at 1 dpo, which was verified as the reeler protein involved in regulating the PPO activity in PPO pathway [52].After 1 dpo, DEGs decreased a lot, there was only one DEG, \u003cem\u003eSPN 9\u003c/em\u003e (down-regulated) at 2 dpo, \u003cem\u003eCLIP 4\u003c/em\u003e was down regulated at 3 dpo, \u003cem\u003eCLIP 7\u003c/em\u003e, \u003cem\u003eCLIP 15\u003c/em\u003e, \u003cem\u003eLysozyme\u003c/em\u003e and \u003cem\u003eReeler 1\u003c/em\u003e were up-regulated at 5 dpo. At 7 dpo, there are three up-regulated gene (\u003cem\u003eSPN 22, PPO 1, PPO 2\u003c/em\u003e) and five down-regulated genes (\u003cem\u003eCLIP 5/6/14, SPN 18/21\u003c/em\u003e). More analyses of \u003cem\u003eCLIPs\u003c/em\u003e and \u003cem\u003eSPNs\u003c/em\u003e with signal peptides and domain information were showed in Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e and S7.\u003c/p\u003e \u003cp\u003eAs to the antimicrobial peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e), there was no AMP gene listed as DEG in diapause silkworms. On the contrary, in non-diapause silkworms, the earliest AMPs listed in DEGs were up-regulated \u003cem\u003egloverin 2, gloverin 3, gloverin 4\u003c/em\u003e and \u003cem\u003edefensin\u003c/em\u003e at 1 dpo. \u003cem\u003eLebocin\u003c/em\u003e was up-regulated at 5 dpo. At 7 dpo, \u003cem\u003emorLP-B1\u003c/em\u003e, \u003cem\u003emorLP-B4\u003c/em\u003e were up-regulated. Although some genes exhibited with big changes, but not listed as DEGs because FDR value was not \u0026le;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison of immune responses in larva stage between diapause and non-diapause silkworms\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmune responses to congenital infection in larva stage of diapause silkworms\u003c/h2\u003e \u003cp\u003eFor diapause silkworms, compared with embryonic stage, it was evident that immune responses of larvae were induced strongly by \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection, mainly reflected by the significant expression changes of host immune-related genes. At 1 dph, there was only one DEG, \u003cem\u003eReeler 1\u003c/em\u003e involved in regulating the PPO activity in PPO pathway. Although the expression of \u003cem\u003ececropin A1\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e, \u003cem\u003ececropin B3\u003c/em\u003e, and \u003cem\u003egloverin 2\u003c/em\u003e were higher in the infected group than the expression in the uninfected group but not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e ), suggesting the congenital infection of \u003cem\u003eN. bombycis\u003c/em\u003e induced the innate immunity.\u003c/p\u003e \u003cp\u003eBy contrast, as shown in the heat map at 5 dph, a dozen of recognition related genes such as \u003cem\u003eβGRP 2\u003c/em\u003e, \u003cem\u003ePGRP-S1\u003c/em\u003e/\u003cem\u003eS3\u003c/em\u003e/\u003cem\u003eS5\u003c/em\u003e/\u003cem\u003eS6\u003c/em\u003e, \u003cem\u003eCTL 11\u003c/em\u003e, \u003cem\u003eCTL 19, SCRAL1, SCRB 6\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e/\u003cem\u003e13\u003c/em\u003e were up-regulated, but \u003cem\u003eCTL 1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e12\u003c/em\u003e/\u003cem\u003e16\u003c/em\u003e/\u003cem\u003e18\u003c/em\u003e and \u003cem\u003eSCRASP 4\u003c/em\u003e were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the Toll pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), \u003cem\u003eSpz 2/3/4/6\u003c/em\u003e were down-regulated but only \u003cem\u003eSpz 4/6\u003c/em\u003e were listed as DEGs, \u003cem\u003eToll 9\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/em\u003e and \u003cem\u003eTRAF 3\u003c/em\u003e were up-regulated significantly. Interestingly, the \u003cem\u003eCactus\u003c/em\u003e, a negative regulatory factor in the Toll pathway, was significantly up-regulated. In addition, there was no DEG in IMD pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and two up-regulated DEGs (\u003cem\u003eDome\u003c/em\u003e and \u003cem\u003eFos\u003c/em\u003e) in JAK-STAT pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e). However, the PPO pathway was another sight (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), \u003cem\u003eCLIP 4/11\u003c/em\u003e/\u003cem\u003e12\u003c/em\u003e, \u003cem\u003eSPN 5\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e/\u003cem\u003e/19\u003c/em\u003e/\u003cem\u003e22\u003c/em\u003e and \u003cem\u003eReeler 1\u003c/em\u003ewere up-regulated; however, \u003cem\u003eCLIP 5\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e, \u003cem\u003eSPN 10\u003c/em\u003e/\u003cem\u003e12\u003c/em\u003e/\u003cem\u003e15\u003c/em\u003e/\u003cem\u003e17\u003c/em\u003e/\u003cem\u003e20\u003c/em\u003e/\u003cem\u003e21\u003c/em\u003e were down-regulated. \u003cem\u003eLysozyme\u003c/em\u003e and \u003cem\u003eLysozyme like protein 2\u003c/em\u003e were significantly up-regulated.\u003c/p\u003e \u003cp\u003eEffectors related genes including \u003cem\u003eCec B1\u0026thinsp;~\u0026thinsp;B6\u003c/em\u003e, \u003cem\u003egloverin 2\u003c/em\u003e, \u003cem\u003edefensin\u003c/em\u003e, \u003cem\u003emoricin\u003c/em\u003e, \u003cem\u003emorLP-B2\u003c/em\u003e, and \u003cem\u003emorLP-B5\u003c/em\u003e were all up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e). All results above showed that the innate immune system has been fully initiated by the congenital infection, and the pathogens also behave a lot to manipulate the host immunity through regulating the immune recognition and classic immune pathways.\u003c/p\u003e \u003cp\u003eAt 10 dph, the congenital infection became more serious, the infected silkworms were very weak and near to die. At this time point, recognition related genes such as \u003cem\u003eCTL 1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e/\u003cem\u003e9\u003c/em\u003e and \u003cem\u003eSCRB 8\u003c/em\u003e were significantly down-regulated, \u003cem\u003eβGRP 1\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e and \u003cem\u003ePGRP-L2\u003c/em\u003e/\u003cem\u003eS3\u003c/em\u003e/\u003cem\u003eS5\u003c/em\u003e/\u003cem\u003eS6\u003c/em\u003e, \u003cem\u003eCTL 3\u003c/em\u003e were up-regulated but not significant. In modulation related genes, \u003cem\u003eCLIP 5\u003c/em\u003e/\u003cem\u003e9\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e and \u003cem\u003eSPN 6\u003c/em\u003e/\u003cem\u003e20\u003c/em\u003e were significantly down-regulated. At this time, most of Toll pathway genes did not show significant differences in expression, only \u003cem\u003eSpz 2\u003c/em\u003e, \u003cem\u003eSpz 4\u003c/em\u003e and \u003cem\u003eSpz 6\u003c/em\u003e were significantly down-regulated, \u003cem\u003eSpz 3\u003c/em\u003e was also down-regulated but not significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the PPO pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), only \u003cem\u003eReeler 1\u003c/em\u003e was significantly up-regulated, the other genes listed as DEGs were all down-regulated, they were \u003cem\u003eCLIP 5/9/10\u003c/em\u003e, \u003cem\u003eSPN 6/20\u003c/em\u003e and \u003cem\u003ePPO 1/2\u003c/em\u003e, suggesting that the PPO pathway was inhibited by pathogens at this time. Moreover, effectors related genes such as \u003cem\u003eenb 1, enb 2, gloverin 1, gloverin 2, leb\u003c/em\u003e and \u003cem\u003emorLP-B1\u003c/em\u003e were up-regulated but only \u003cem\u003egloverin 3\u003c/em\u003e was significant. Notably, although the expression of \u003cem\u003eCec B1\u0026thinsp;~\u0026thinsp;B6\u003c/em\u003e were down-regulated, but not significantly, which formed an obvious contrast with the data of 5 dph.\u003c/p\u003e \u003cp\u003eDEGs aforementioned implied that the host (larva stage) significantly enhanced the immunity against \u003cem\u003eN. bombycis\u003c/em\u003e infection. Simultaneously, the pathogen may evade or manipulate the host\u0026rsquo;s innate immunity supported by these regulated genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmune responses to congenital infection in larva stage of non-diapause silkworms\u003c/h2\u003e \u003cp\u003eSimilar to dispause silkworms, the immune response in the larva stage of non-diapause silkworm was stronger than embryo stage. At 1 dph, immune recognition genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), \u003cem\u003ePGRP-L1/S1/S3\u003c/em\u003e and \u003cem\u003eSCRB 13\u003c/em\u003e were up-regulated, \u003cem\u003ePGRP-L4\u003c/em\u003e, \u003cem\u003eSCRB 10\u003c/em\u003e and \u003cem\u003eSCRB 11\u003c/em\u003e were down-regulated. The signal transduction genes in Toll pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), IMD pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and JAK-STAT pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e) had no DEG at 1dph. In PPO pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), there seven genes up-regulated but only three genes (\u003cem\u003eSPN 17\u003c/em\u003e, \u003cem\u003eLysozyme\u003c/em\u003e and \u003cem\u003eReeler 1\u003c/em\u003e) were significant. Most of antimicrobial peptides genes were up-regulated, \u003cem\u003ecec A1\u003c/em\u003e, \u003cem\u003ecec B1\u0026thinsp;~\u0026thinsp;B6\u003c/em\u003e, \u003cem\u003egloverin 2/4\u003c/em\u003e and \u003cem\u003emorLP-B1\u0026thinsp;~\u0026thinsp;B5\u003c/em\u003e significantly up-regulated with big changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt 2 dph, there were two up-regulation DEGs (\u003cem\u003eCTL 7\u003c/em\u003e and \u003cem\u003eSCRC\u003c/em\u003e) and six down-regulation DEGs (\u003cem\u003ePGRP-S4\u003c/em\u003e, \u003cem\u003eSCRAC 2\u003c/em\u003e, \u003cem\u003eSCRASP 2\u003c/em\u003e, \u003cem\u003eSCRB 7\u003c/em\u003e, \u003cem\u003eSCRB 8\u003c/em\u003e and \u003cem\u003eSCRB 13\u003c/em\u003e) listed in the recognition related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). There were six \u003cem\u003eTolls\u003c/em\u003e (\u003cem\u003eToll 6/7\u0026thinsp;\u0026minus;\u0026thinsp;2/7\u0026thinsp;\u0026minus;\u0026thinsp;3/8/10\u0026thinsp;\u0026minus;\u0026thinsp;2/12\u003c/em\u003e) were down-regulated while \u003cem\u003eToll 3\u0026ndash;3\u003c/em\u003e was up-regulated, implied that the Toll pathway was inhibited at this time. In JAK-STAT pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e), it was the similar case, two genes (\u003cem\u003eDome\u003c/em\u003e and \u003cem\u003eHem\u003c/em\u003e) were significantly down-regulated. Inhibition of JAK-STAT pathway resulted in decreased survival rate and antibacterial activity of silkworm, we also observed that the larvae began to die at 2 dph. According to the transcriptome data, the surviving larvae at this time were also weak and the function of the immune system was deteriorated.\u003c/p\u003e \u003cp\u003eAlthough the positive regulator of PPO pathway \u003cem\u003ePGRP-S4\u003c/em\u003e was down-regulated at this time point, the PPO pathway seemed as activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). \u003cem\u003eCLIP 3/13\u003c/em\u003e, \u003cem\u003eSPN 16 PPO 1/2\u003c/em\u003e and \u003cem\u003eReeler 1\u003c/em\u003e were up-regulated, \u003cem\u003eCLIP 8\u003c/em\u003e and \u003cem\u003eSPN 10/20/21\u003c/em\u003e were down-regulated. In addition, \u003cem\u003eSPN 15\u003c/em\u003e was up-regulated which was the negative regulator of PPO pathway. At this time point, it was interesting that the \u003cem\u003emorLP-B1\u0026thinsp;~\u0026thinsp;B5\u003c/em\u003e were up-regulated, which same as 1 dph, while the other \u003cem\u003eAMPs\u003c/em\u003e were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt 3 dph, most of immune-related DEGs were down-regulation. The recognition related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), only four DEGs (\u003cem\u003ePGRP-L4, PGRP-S5, CTL 11\u003c/em\u003e and \u003cem\u003eSCRAL 1\u003c/em\u003e) were up-regulated while ten DEGs (PGRP-S3, CTL 5/7/9/10/12, SCRAC 1/2, SCRB 7/9/13) were down-regulated. PGRP-S5 has been reported as the negative regulator of IMD pathway [53]. As expected, the IMD pathway was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e), five genes (\u003cem\u003eIMD, Dredd\u003c/em\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cem\u003eikk β\u003c/em\u003e and \u003cem\u003eRelish\u003c/em\u003e) were down-regulated. The upstream of Toll pathway was up-regulation and the downstream was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). \u003cem\u003eSpz 1/3, Toll 3\u0026thinsp;\u0026minus;\u0026thinsp;2/3\u0026ndash;3\u003c/em\u003e and \u003cem\u003eTRAF 3\u003c/em\u003e were significantly up-regulated, \u003cem\u003eSpz 2, Toll 6/9\u0026thinsp;\u0026minus;\u0026thinsp;2/10\u0026thinsp;\u0026minus;\u0026thinsp;2/10\u0026thinsp;\u0026minus;\u0026thinsp;3, Tube\u003c/em\u003e and \u003cem\u003eTRAF 2\u003c/em\u003e were significantly down-regulated. At this time, The \u003cem\u003eSocs\u003c/em\u003e and \u003cem\u003eFos\u003c/em\u003e in JAK-STAT pathway were up-regulated, and \u003cem\u003eHem\u003c/em\u003e was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e).The PPO pathway at 3 dph was regulated by pathogens (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). \u003cem\u003eCLIP 5, SPN 8/10/12/15/16/18/21\u003c/em\u003e and \u003cem\u003ePPAE\u003c/em\u003e were significant down-regulated and \u003cem\u003eCLIP 12/13, SPN 22\u003c/em\u003e and \u003cem\u003eReeler 1\u003c/em\u003e were up-regulated significantly. The \u003cem\u003eLysozyme\u003c/em\u003e was also significantly down-regulated. Similar to 2 dph, almost all \u003cem\u003eAMPs\u003c/em\u003e were significant down-regulation but \u003cem\u003emorLP-B1\u0026thinsp;~\u0026thinsp;B5\u003c/em\u003e were significant up-regulation at 3 dph.\u003c/p\u003e \u003cp\u003eIn general, compared with diapause silkworms, the immune-related genes, especially the \u003cem\u003eAMPs\u003c/em\u003e of non-diapause silkworm larvae showed a fully induced immune responses in newly hatched larvae, and a lot of immune related genes in different steps were down-regulated, indicating that pathogens could regulate host immune pathways.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eN. bombycis\u003c/em\u003e has a complex and unique route to enter into the eggs and lead to congenital infection [54]. After that, \u003cem\u003eN. bombycis\u003c/em\u003e starts to proliferate inside the eggs with embryo development. The histopathogical characteristics of \u003cem\u003eN. bombycis\u003c/em\u003e proliferation in non-diapause silkworms had been investigated [12], here we provide the molecular proliferation characteristics of \u003cem\u003eN. bombycis\u003c/em\u003e by Dual RNA-seq, our result is consistent with histopathological characteristics. For non-diapause silkworms, most of parasites were in meront stage at 1 dpo. From 2 dpo, many meronts enter into the sporogony stage, the corresponding molecular marker is \u003cem\u003eSWP 1\u003c/em\u003e ranking as the Top 1 expressed gene. We discovered that the big difference of the molecular proliferation characteristics between non-diapause silkworm and diapause silkworm resulted from the hot HCl treatment for diapause silkworm eggs, which can kill many parasites inside the eggs [36]. The hot HCl bath treatment interrupts the proliferation cycle of \u003cem\u003eN. bombycis\u003c/em\u003e, and reduces the pathogen load in host. It has been reported that spore formation and parasite loads of \u003cem\u003eNosema muscidifuracis\u003c/em\u003e were reduced because of heat shock treatments in \u003cem\u003eMusidifurax raptor\u003c/em\u003e eggs [55]. The pathogen load in non-diapause silkworm eggs and larvae was much higher than that of diapause silkworm eggs and larvae, which far influence the host immune responses in embryos and larvae.\u003c/p\u003e \u003cp\u003eCompared previous results of larvae immune responses to \u003cem\u003eN. bombycis\u003c/em\u003e infection [24, 56] with our current RNA-Seq analysis, immune responses to congenital infection in embryo stages were weaker than that of larva stages both in diapause and non-diapause silkworms. Four reasonable explanations were put forward. Firstly, the embryo gradually mounts a systemic and positive immunity and acquires its immune competence after or near hatching. Secondly, the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e in embryos primarily centered around yolk granules and intestinal lumen [12]. The strategy adopted by \u003cem\u003eN. bombycis\u003c/em\u003e may reduce the parasite\u0026rsquo;s direct stimulation on the embryo and weaken the priming of the host immune system. On the contrary, the parasites can cause a systemic and serious infection in larvae [57]. Thirdly, for diapause silkworm, the unique sequence mapped rates of \u003cem\u003eN. bombycis\u003c/em\u003e were between 0.07% and 0.89% at embryo stages but increased to 3.35%~13.89% at larva stages. As a result, the parasite load difference between embryo and larva stages is another candidate factor. Finally, \u003cem\u003eN. bombycis\u003c/em\u003e may actively regulate or escape the host immune response through its secreted proteins such as Serpin 6 [58], or abundant highly expressed genes while the function of these secretions is poorly understood [12, 59, 60]. It also has been reported that \u003cem\u003eEncephalitozoon intestinalis\u003c/em\u003e may escape from host immune recognition through the modulation of dendritic cell differentiation and maturation [61].\u003c/p\u003e \u003cp\u003eThe systematic immune response of \u003cem\u003eB. mori\u003c/em\u003e to pathogen infection has been extensively analyzed [21, 22, 53]. However, there is limited knowledge regarding the equipping time of \u003cem\u003eB. mori\u003c/em\u003e innate immunity. Here, we found the transcription of \u003cem\u003eAMPs\u003c/em\u003e in infected groups significantly up-regulated at the 5 dpo in non-diapause silkworm embryo if we ignore the \u003cem\u003eAMPs\u003c/em\u003e DEGs in \u0026ldquo;First day chaos\u0026rdquo;. At 7 dpo, the significant up-regulation of PPO 1 and PPO 2 marks the establishment of melanization pathway. In conclusion, for non-diapause silkworm, the differential expression of host genes proved that the congenital infection of \u003cem\u003eN. bombycis\u003c/em\u003e in the embryo may lead to an earlier establishment of the host positive immune system. However, to elucidate the equipping time of silkworm innate immunity is a challenging task, only considering the transcription of immune-related genes is not enough to answer this question. We have analyzed the proteome composition in silkworm eggs with \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection and found there are several different AMPs and a lot of different immune related proteins, such as PRRs, CTL, SPNs, Spz 1, etc. (unpublished data), which suggest that maternal immunity may be also existed in insect eggs, especially the eggs with congenital infection [62]. In the larva stage, with the establishment of the immune system, the host immune response was more intense, and a large number of AMP genes were significantly up-regulated. However, at 2 dph, the upstream genes of PPO cascade was inhibited, the expression of intracellular signaling molecules in Toll pathway and IMD pathway were down-regulated, and the JAK-STAT pathway, which was related to survival and antibacterial activity, was also inhibited. On the whole, although the immune system of the host was completely established in the larva stage, the pathogen load increased and the physiological state of the host continued to deteriorate, the main antimicrobial peptides also showed a dynamic trend of up-regulation and then down-regulation, except for \u003cem\u003eMoricins\u003c/em\u003e, which is the unique AMP family to Lepidoptera. \u003cem\u003eMoricins\u003c/em\u003e maintained a very high expression level through the larva stage, its role in resistance to microsporidia infection needs further study.\u003c/p\u003e \u003cp\u003eWe obtained a large number (6,071) DEGs at 1 dpo for non-diapause silkworms, but at the following time points in embryo, the number of DEGs decreased to a lot, we call this phenomenon as \u0026ldquo;First Day Chaos\u0026rdquo; in non-diapause silkworm eggs. Why are there so many DEGs obtained at 1dpo? It is an intrigued question for future verification, we are wondering whether there is connection with different strains of silkworms. There may be different pathogen tolerance existed between the non-diapause silkworms and diapause silkworms. [63, 64]\u003c/p\u003e \u003cp\u003eNotably, there are some limitations in our work. Although we found some genes with different expression patterns in immune responses to pathogen infection between the diapause silkworms and non-diapause silkworms, we should be cautious to face these differences, because their genetic backgrounds are not identical, some of our findings are just based on transcriptomic analyses and provide a clue for further research. It is necessary to adopt careful wet-lab work for future confirmation and verification.\u003c/p\u003e \u003cp\u003eIn summary, our study using deep transcriptional profiling of the silkworm hosts illustrates that gene expression characteristics of pathogen and host different immune responses in diapause and non-diapause silkworm embryo and larvae with \u003cem\u003eN. bombycis\u003c/em\u003e congenital infection. Rapid proliferation of pathogen in host, weak immune responses in embryo stage and strong immune responses in larvae stage are the main findings here. Different pathogen loads in diapause and non-diapause silkworm are results from the hot HCL bath treatment for diapause silkworm eggs at 24 hours post oviposition, which has the strong killing effect for \u003cem\u003eN. bombycis\u003c/em\u003e. In addition, the low pathogen load in diapause silkworm embryo and larvae is also connected with the weaker immune responses compared with non-diapause silkworm group. In general, here we not only developed a microsporidia-host system for analysis of microsporidia congenital infection and the host response, but also provide abundant molecular data for silkworm embryo innate immunity development anda sound basis for the complicated host-pathogen interactions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSilkworms are not a protected species and can be used as experimental materials without ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data generated was submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, http:// www. ncbi. nlm. nih. gov/ sra) with the BioProject accession number (PRJNA1215458).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (Grant No. 32272942 and 31470250, The Chongqing Modern Agricultural Industry Technology System (COMAITS202311), Chongqing elite, innovation and entrepreneurship demonstration team (CQYC202203091213), Fundamental Research Funds for the Central Universities (SWU-XDJH202322) and The Natural Science Foundation of Chongqing, China (cstc2021jcyj-msxmX1003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTXL and YS: conceptualization and visualization; analyzed the data; experiments implementation; writing original draft; QY, YLT and ZSH: prepared the samples; QS, YLT, ZSH, ZGS and YBD: analyzed the data; TL and XZM: \u0026nbsp;analyzed and uploaded the data; QS, JZZ and SL: prepared figures S1-S7; ZYZ: Conceptualization and supervision; JC and GQP: \u0026nbsp;Supervision, review \u0026amp; editing the manuscript and Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appericiate Mulualem Lemma Kebede come from University of Gondar, Ethiopia for \u0026nbsp;modification of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVavra J, Lukes J: \u003cstrong\u003eMicrosporidia and \u0026apos;the art of living together\u0026apos;\u003c/strong\u003e. \u003cem\u003eAdv Parasitol \u003c/em\u003e2013, \u003cstrong\u003e82\u003c/strong\u003e:253-319.\u003c/li\u003e\n\u003cli\u003eDidier ES: \u003cstrong\u003eMicrosporidiosis: an emerging and opportunistic infection in humans and animals\u003c/strong\u003e. \u003cem\u003eActa Trop \u003c/em\u003e2005, \u003cstrong\u003e94\u003c/strong\u003e(1):61-76.\u003c/li\u003e\n\u003cli\u003eStentiford GD, Becnel J, Weiss LM, Keeling PJ, Didier ES, Williams BP, Bjornson S, Kent ML, Freeman MA, Brown MJF\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMicrosporidia - Emergent Pathogens in the Global Food Chain\u003c/strong\u003e. \u003cem\u003eTrends in parasitology \u003c/em\u003e2016, \u003cstrong\u003e32\u003c/strong\u003e(4):336-348.\u003c/li\u003e\n\u003cli\u003eWang ZD, Liu Q, Liu HH, Li S, Zhang L, Zhao YK, Zhu XQ: \u003cstrong\u003ePrevalence of Cryptosporidium, microsporidia and Isospora infection in HIV-infected people: a global systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003eParasites \u0026amp; vectors \u003c/em\u003e2018, \u003cstrong\u003e11\u003c/strong\u003e(1):28.\u003c/li\u003e\n\u003cli\u003eHan B, Pan G, Weiss LM: \u003cstrong\u003eMicrosporidiosis in Humans\u003c/strong\u003e. \u003cem\u003eClin Microbiol Rev \u003c/em\u003e2021, \u003cstrong\u003e34\u003c/strong\u003e(4):e0001020.\u003c/li\u003e\n\u003cli\u003eDunn AM, Smith JEJM, Infection: \u003cstrong\u003eMicrosporidian life cycles and diversity: the relationship between virulence and transmission\u003c/strong\u003e. 2001, \u003cstrong\u003e3\u003c/strong\u003e(5):381-388.\u003c/li\u003e\n\u003cli\u003eRaina SK, Das S, Rai MM, Khurad AM: \u003cstrong\u003eTransovarial transmission of Nosema locustae (Microsporida: Nosematidae) in the migratory locust Locusta migratoria migratorioides\u003c/strong\u003e. \u003cem\u003eParasitol Res \u003c/em\u003e1995, \u003cstrong\u003e81\u003c/strong\u003e(1):38-44.\u003c/li\u003e\n\u003cli\u003eFrankenhuyzen KV, Nystrom C, Liu YJJoIP: \u003cstrong\u003eVertical transmission of Nosema fumiferanae (Microsporidia: Nosematidae) and consequences for distribution, post-diapause emergence and dispersal of second-instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)\u003c/strong\u003e. 2007, \u003cstrong\u003e96\u003c/strong\u003e(2):173-182.\u003c/li\u003e\n\u003cli\u003eNageli: \u003cstrong\u003eUber die neue krankheit der seidenraupe und verwandte organismen\u003c/strong\u003e. \u003cem\u003eBot Z \u003c/em\u003e1857:15: 760-761.\u003c/li\u003e\n\u003cli\u003ePasteur L: \u003cstrong\u003e\u0026Eacute;tudes sur la maladie des vers \u0026agrave; soie : moyen pratique assur\u0026eacute; de la combattre et d\u0026apos;en pr\u0026eacute;venir le retour\u003c/strong\u003e: Paris :La-Villars, successeur de Mallet-Bachelier,1870; 1870.\u003c/li\u003e\n\u003cli\u003eMeng XZ, Luo B, Tang XY, He Q, Xiong TR, Fang ZY, Pan G, Li T, Zhou ZY: \u003cstrong\u003ePathological analysis of silkworm infected by two microsporidia Nosema bombycis CQ1 and Vairimorpha necatrix BM\u003c/strong\u003e. \u003cem\u003eJournal of invertebrate pathology \u003c/em\u003e2018, \u003cstrong\u003e153\u003c/strong\u003e:75-84.\u003c/li\u003e\n\u003cli\u003eSong Y, Tang Y, Yang Q, Li T, He Z, Wu Y, He Q, Li T, Li C, Long M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eProliferation characteristics of the intracellular microsporidian pathogen Nosema bombycis in congenitally infected embryos\u003c/strong\u003e. \u003cem\u003eJournal of invertebrate pathology \u003c/em\u003e2020, \u003cstrong\u003e169\u003c/strong\u003e:107310.\u003c/li\u003e\n\u003cli\u003eShen Z, Ke Z, Yang Q, Ghebremichael ST, Li T, Li T, Chen J, Meng X, Xiang H, Li C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eTranscriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae\u003c/strong\u003e. \u003cem\u003eBMC genomics \u003c/em\u003e2024, \u003cstrong\u003e25\u003c/strong\u003e(1):321.\u003c/li\u003e\n\u003cli\u003eEngstrom Y: \u003cstrong\u003eInduction and regulation of antimicrobial peptides in Drosophila\u003c/strong\u003e. \u003cem\u003eDevelopmental and comparative immunology \u003c/em\u003e1999, \u003cstrong\u003e23\u003c/strong\u003e(4-5):345-358.\u003c/li\u003e\n\u003cli\u003eHoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA: \u003cstrong\u003ePhylogenetic perspectives in innate immunity\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e1999, \u003cstrong\u003e284\u003c/strong\u003e(5418):1313-1318.\u003c/li\u003e\n\u003cli\u003eHoffmann JA: \u003cstrong\u003eThe immune response of Drosophila\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e2003, \u003cstrong\u003e426\u003c/strong\u003e(6962):33-38.\u003c/li\u003e\n\u003cli\u003eHultmark D: \u003cstrong\u003eDrosophila immunity: paths and patterns\u003c/strong\u003e. \u003cem\u003eCurrent opinion in immunology \u003c/em\u003e2003, \u003cstrong\u003e15\u003c/strong\u003e(1):12-19.\u003c/li\u003e\n\u003cli\u003eLemaitre B, Hoffmann J: \u003cstrong\u003eThe host defense of Drosophila melanogaster\u003c/strong\u003e. \u003cem\u003eAnnu Rev Immunol \u003c/em\u003e2007, \u003cstrong\u003e25\u003c/strong\u003e:697-743.\u003c/li\u003e\n\u003cli\u003eYu XQ, Zhu YF, Ma C, Fabrick JA, Kanost MR: \u003cstrong\u003ePattern recognition proteins in Manduca sexta plasma\u003c/strong\u003e. \u003cem\u003eInsect biochemistry and molecular biology \u003c/em\u003e2002, \u003cstrong\u003e32\u003c/strong\u003e(10):1287-1293.\u003c/li\u003e\n\u003cli\u003eXia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA draft sequence for the genome of the domesticated silkworm (Bombyx mori)\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2004, \u003cstrong\u003e306\u003c/strong\u003e(5703):1937-1940.\u003c/li\u003e\n\u003cli\u003eTanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori\u003c/strong\u003e. \u003cem\u003eInsect biochemistry and molecular biology \u003c/em\u003e2008, \u003cstrong\u003e38\u003c/strong\u003e(12):1087-1110.\u003c/li\u003e\n\u003cli\u003eCheng T, Zhao P, Liu C, Xu P, Gao Z, Xia Q, Xiang Z: \u003cstrong\u003eStructures, regulatory regions, and inductive expression patterns of antimicrobial peptide genes in the silkworm Bombyx mori\u003c/strong\u003e. \u003cem\u003eGenomics \u003c/em\u003e2006, \u003cstrong\u003e87\u003c/strong\u003e(3):356-365.\u003c/li\u003e\n\u003cli\u003eCheng TC, Zhang YL, Liu C, Xu PZ, Gao ZH, Xia QY, Xiang ZH: \u003cstrong\u003eIdentification and analysis of Toll-related genes in the domesticated silkworm, Bombyx mori\u003c/strong\u003e. \u003cem\u003eDevelopmental and comparative immunology \u003c/em\u003e2008, \u003cstrong\u003e32\u003c/strong\u003e(5):464-475.\u003c/li\u003e\n\u003cli\u003eMa Z, Li C, Pan G, Li Z, Han B, Xu J, Lan X, Chen J, Yang D, Chen Q\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2013, \u003cstrong\u003e8\u003c/strong\u003e(12):e84137.\u003c/li\u003e\n\u003cli\u003eShen Z, Yang Q, Luo L, Li T, Ke Z, Li T, Chen J, Meng XZ, Xiang H, Li CJBG: \u003cstrong\u003eNon-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection\u003c/strong\u003e. 2023, \u003cstrong\u003e24\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eLiu H, Pan G, Song S, Xu J, Li T, Deng Y, Zhou Z: \u003cstrong\u003eMultiple rDNA units distributed on all chromosomes of Nosema bombycis\u003c/strong\u003e. \u003cem\u003eJournal of invertebrate pathology \u003c/em\u003e2008, \u003cstrong\u003e99\u003c/strong\u003e(2):235-238.\u003c/li\u003e\n\u003cli\u003eKitta R, Yamahama Y, Yamamoto T, Mase K, Sawada H: \u003cstrong\u003eMolecular Characterization and Tissue Distribution of Mitochondrial Ca(2+) -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori\u003c/strong\u003e. \u003cem\u003eZoological science \u003c/em\u003e2018, \u003cstrong\u003e35\u003c/strong\u003e(6):487-493.\u003c/li\u003e\n\u003cli\u003eTsurumaru S, Kawamori A, Mitsumasu K, Niimi T, Imai K, Yamashita O, Yaginuma T: \u003cstrong\u003eDisappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause\u003c/strong\u003e. \u003cem\u003eJournal of insect physiology \u003c/em\u003e2010, \u003cstrong\u003e56\u003c/strong\u003e(12):1721-1727.\u003c/li\u003e\n\u003cli\u003eWu S, Tong X, Peng C, Xiong G, Lu K, Hu H, Tan D, Li C, Han M, Lu C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eComparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori\u003c/strong\u003e. \u003cem\u003eSci Rep \u003c/em\u003e2016, \u003cstrong\u003e6\u003c/strong\u003e:26114.\u003c/li\u003e\n\u003cli\u003eKim D, Langmead B, Salzberg SLJNm: \u003cstrong\u003eHISAT: a fast spliced aligner with low memory requirements\u003c/strong\u003e. 2015, \u003cstrong\u003e12\u003c/strong\u003e(4):357-360.\u003c/li\u003e\n\u003cli\u003eLu F, Wei Z, Luo Y, Guo H, Zhang G, Xia Q, Wang Y: \u003cstrong\u003eSilkDB 3.0: visualizing and exploring multiple levels of data for silkworm\u003c/strong\u003e. 2020(1362-4962 (Electronic)).\u003c/li\u003e\n\u003cli\u003ePertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SLJNB: \u003cstrong\u003eStringTie enables improved reconstruction of a transcriptome from RNA-seq reads\u003c/strong\u003e. 2015, \u003cstrong\u003e33\u003c/strong\u003e(3):290-295.\u003c/li\u003e\n\u003cli\u003eKim, Daehwan, Leek, Jeffrey, T., Pertea, Geo, M., Salzberg, researchers SJNpef: \u003cstrong\u003eTranscript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown\u003c/strong\u003e. 2016, \u003cstrong\u003e11\u003c/strong\u003e(9):1650-1667.\u003c/li\u003e\n\u003cli\u003eLove MI, Huber W, Anders S: \u003cstrong\u003eModerated estimation of fold change and dispersion for RNA-seq data with DESeq2\u003c/strong\u003e. \u003cem\u003eGenome Biol \u003c/em\u003e2014, \u003cstrong\u003e15\u003c/strong\u003e(12):550.\u003c/li\u003e\n\u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R: \u003cstrong\u003eTBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data\u003c/strong\u003e. \u003cem\u003eMol Plant \u003c/em\u003e2020, \u003cstrong\u003e13\u003c/strong\u003e(8):1194-1202.\u003c/li\u003e\n\u003cli\u003eLu Wang QY, Dongxu Xing, Zhengang Ma, Qingrong Li, Yang Xiao, Sentai Liao: \u003cstrong\u003eInactivation Effect of High Temperature Instant Acid Treatment on Nosema bombycis in Silkworm Eggs Detected by \u003c/strong\u003e\u003cstrong\u003eＲeal-time Fluorescent Quantitative PCR_In Chinese\u003c/strong\u003e. \u003cem\u003eScience of Sericulture \u003c/em\u003e2019.\u003c/li\u003e\n\u003cli\u003eWu Z, Li Y, Pan G, Tan X, Xiang ZJP: \u003cstrong\u003eProteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)\u003c/strong\u003e. 2010, \u003cstrong\u003e8\u003c/strong\u003e(12):2447-2461.\u003c/li\u003e\n\u003cli\u003eHan B, Takvorian P, Weiss LJEs: \u003cstrong\u003eThe Function and Structure of the Microsporidia Polar Tube\u003c/strong\u003e. 2022, \u003cstrong\u003e114\u003c/strong\u003e:179-213.\u003c/li\u003e\n\u003cli\u003eChen Y, Wei E, Chen Y, He P, Wang R, Wang Q, Tang X, Zhang Y, Zhu F, Shen Z: \u003cstrong\u003eIdentification and subcellular localization analysis of membrane protein Ycf 1 in the microsporidian Nosema bombycis\u003c/strong\u003e. 2022(2167-8359 (Print)).\u003c/li\u003e\n\u003cli\u003eDorey A, Cwiklinski K, Rooney J, Verissimo CDM, Lalor RJFiC, Microbiology I: \u003cstrong\u003eAutonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1\u003c/strong\u003e. 2021, \u003cstrong\u003e11\u003c/strong\u003e:667272.\u003c/li\u003e\n\u003cli\u003eHe W, Sun X, Luo B, Liu M, Li L, Fan X, Ye J, Zhou B: \u003cstrong\u003eRegulation of piglet T-cell immune responses by thioredoxin peroxidase from Cysticercus cellulosae excretory-secretory antigens\u003c/strong\u003e. 2022, \u003cstrong\u003e13\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eBao J, Liu L, An Y, Ran M, Ni W, Chen J, Wei J, Li T, Pan G, Zhou Z: \u003cstrong\u003eNosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade\u003c/strong\u003e. \u003cem\u003eJournal of invertebrate pathology \u003c/em\u003e2019, \u003cstrong\u003e168\u003c/strong\u003e:107260.\u003c/li\u003e\n\u003cli\u003eRan M, Bao J, Li B, Shi Y, Yang W, Meng X, Chen J, Wei J, Long M, Li T\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMicrosporidian Nosema bombycis secretes serine protease inhibitor to suppress host cell apoptosis via Caspase BmICE\u003c/strong\u003e. \u003cem\u003ePLOS Pathogens \u003c/em\u003e2025, \u003cstrong\u003e21\u003c/strong\u003e(1):e1012373.\u003c/li\u003e\n\u003cli\u003eRan M, Shi Y, Li B, Xiang H, Tao M, Meng X, Li T, Li C, Bao J, Pan G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-Wide Characterization and Comparative Genomic Analysis of the Serpin Gene Family in Microsporidian Nosema bombycis\u003c/strong\u003e. 2023, \u003cstrong\u003e24\u003c/strong\u003e(1):550.\u003c/li\u003e\n\u003cli\u003eYoshida H, Ochiai M, Ashida M: \u003cstrong\u003e\u0026beta;-1,3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system\u003c/strong\u003e. 1986, \u003cstrong\u003e141\u003c/strong\u003e(3):0-1184.\u003c/li\u003e\n\u003cli\u003eRao XJ, Shahzad T, Liu S, Wu P, He YT, Sun WJ, Fan XY, Yang YF, Shi Q, Yu XQJD\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eIdentification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori\u003c/strong\u003e. 2015, \u003cstrong\u003e53\u003c/strong\u003e(2):328-338.\u003c/li\u003e\n\u003cli\u003eKinoshita, K.: \u003cstrong\u003ePurification of a Peptidoglycan Recognition Protein from Hemolymph of the Silkworm, Bombyx mori\u003c/strong\u003e. \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e1996, \u003cstrong\u003e271\u003c/strong\u003e(23):13854-13860.\u003c/li\u003e\n\u003cli\u003eYang J, Wang X, Tang S, Shen Z, Wu J: \u003cstrong\u003ePeptidoglycan recognition protein S2 from silkworm integument: characterization, microbe-induced expression, and involvement in the immune-deficiency pathway\u003c/strong\u003e. \u003cem\u003eJ Insect Sci \u003c/em\u003e2015, \u003cstrong\u003e15\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eZhan MY, Yang PJ, Rao XJ: \u003cstrong\u003eMolecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori\u003c/strong\u003e. \u003cem\u003eComp Biochem Physiol B Biochem Mol Biol \u003c/em\u003e2018, \u003cstrong\u003e215\u003c/strong\u003e:19-30.\u003c/li\u003e\n\u003cli\u003eYu B, Sang Q, Pan G, Li C, Zhou ZJI: \u003cstrong\u003eA Toll-Sptzle Pathway in the Immune Response of Bombyx mori\u003c/strong\u003e. 2020, \u003cstrong\u003e11\u003c/strong\u003e(9):586.\u003c/li\u003e\n\u003cli\u003eMuhammad A, Sun C, Shao Y: \u003cstrong\u003eThe humoral immune response of the lepidopteran model insect, silkworm Bombyx mori L., to microbial pathogens\u003c/strong\u003e. \u003cem\u003eCurrent Research in Insect Science \u003c/em\u003e2024, \u003cstrong\u003e6\u003c/strong\u003e:100097.\u003c/li\u003e\n\u003cli\u003eBao Y-Y, Xue J, Wu W-J, Wang Y, Lv Z-Y, Zhang C-X: \u003cstrong\u003eAn immune-induced Reeler protein is involved in the Bombyx mori melanization cascade\u003c/strong\u003e. \u003cem\u003eInsect biochemistry and molecular biology \u003c/em\u003e2011, \u003cstrong\u003e41\u003c/strong\u003e(9):696-706.\u003c/li\u003e\n\u003cli\u003eChen K, Lu Z: \u003cstrong\u003eImmune responses to bacterial and fungal infections in the silkworm, Bombyx mori\u003c/strong\u003e. \u003cem\u003eDevelopmental and comparative immunology \u003c/em\u003e2018, \u003cstrong\u003e83\u003c/strong\u003e:3-11.\u003c/li\u003e\n\u003cli\u003eWang C, Yu B, Meng X, Xia D, Pei B, Tang X, Zhang G, Wei J, Long M, Chen J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMicrosporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission\u003c/strong\u003e. \u003cem\u003ePLOS Pathogens \u003c/em\u003e2023, \u003cstrong\u003e19\u003c/strong\u003e(12):e1011859.\u003c/li\u003e\n\u003cli\u003eBoohene CK, Geden CJ, Becnel JJ: \u003cstrong\u003eEvaluation of Remediation Methods for Nosema Disease in Muscidifurax raptor (Hymenoptera: Pteromalidae)\u003c/strong\u003e. \u003cem\u003eEnvironmental Entomology \u003c/em\u003e2003(5):1146-1153.\u003c/li\u003e\n\u003cli\u003eYue YJ, Tang XD, Xu L, Yan W, Li QL, Xiao SY, Fu XL, Wang W, Li N, Shen ZY: \u003cstrong\u003eEarly responses of silkworm midgut to microsporidium infection--A Digital Gene Expression analysis\u003c/strong\u003e. \u003cem\u003eJournal of invertebrate pathology \u003c/em\u003e2015, \u003cstrong\u003e124\u003c/strong\u003e:6-14.\u003c/li\u003e\n\u003cli\u003ePan G, Bao J, Ma Z, Song Y, Han B, Ran M, Li C, Zhou Z: \u003cstrong\u003eInvertebrate host responses to microsporidia infections\u003c/strong\u003e. \u003cem\u003eDevelopmental and comparative immunology \u003c/em\u003e2018, \u003cstrong\u003e83\u003c/strong\u003e:104-113.\u003c/li\u003e\n\u003cli\u003eBao J, Liu L, An Y, Ran M, Ni W, Chen J, Wei J, Li T, Pan G, Zhou Z: \u003cstrong\u003eNosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade\u003c/strong\u003e. \u003cem\u003eJ Invertebr Pathol \u003c/em\u003e2019:107260.\u003c/li\u003e\n\u003cli\u003eWeiss L: \u003cstrong\u003eMicrosporidia: pathogens of opportunity\u003c/strong\u003e. \u003cem\u003eJohn Wiley \u0026amp; Sons Inc \u003c/em\u003e2015.\u003c/li\u003e\n\u003cli\u003eDesjardins CA, Sanscrainte ND, Goldberg JM, Heiman D, Young S, Zeng Q, Madhani HD, Becnel JJ, Cuomo CA: \u003cstrong\u003eContrasting host-pathogen interactions and genome evolution in two generalist and specialist microsporidian pathogens of mosquitoes\u003c/strong\u003e. \u003cem\u003eNature communications \u003c/em\u003e2015, \u003cstrong\u003e6\u003c/strong\u003e:7121.\u003c/li\u003e\n\u003cli\u003eBernal CE, Zorro MM, Sierra J, Gilchrist K, Botero JH, Baena A, Ramirez-Pineda JR: \u003cstrong\u003eEncephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism\u003c/strong\u003e. \u003cem\u003eFrontiers in cellular and infection microbiology \u003c/em\u003e2016, \u003cstrong\u003e6\u003c/strong\u003e:4.\u003c/li\u003e\n\u003cli\u003eVilcinskas A: \u003cstrong\u003eMechanisms of transgenerational immune priming in insects\u003c/strong\u003e. \u003cem\u003eDevelopmental \u0026amp; Comparative Immunology \u003c/em\u003e2021, \u003cstrong\u003e124\u003c/strong\u003e:104205.\u003c/li\u003e\n\u003cli\u003eShixian Liu YZ, Shaorong Ou: \u003cstrong\u003eGenetic study of resistance to p\u0026eacute;brine in Bombyx mori_ In Chinese\u003c/strong\u003e. \u003cem\u003eGuangdong Agricultural Sciences \u003c/em\u003e1981.\u003c/li\u003e\n\u003cli\u003eYuanneng Zhang SL, Yongmei Huo, Shaorong Ou: \u003cstrong\u003eIdentification of Resistance of Several Silkworm Strains to Six Major Silkworm Diseases_In Chinese\u003c/strong\u003e. \u003cem\u003eActa Sericologica Sinica \u003c/em\u003e1982.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bombyx mori, microsporidia, Nosema bombycis, congenital infection, immune response, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-5908051/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5908051/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrosporidia are a group of intracellular and unicellular eukaryotic parasites, which can nearly infect all animals, including human beings. As the first identified microsporidia, \u003cem\u003eNosema bombycis\u003c/em\u003e is a world-wide threat for silkworm eggs production, it can cause the congenital infection \u003cem\u003evia\u003c/em\u003e transovarial transmission. It is important for pathogenesis elucidation to unravel the molecular characteristics of \u003cem\u003eN. bombycis\u003c/em\u003e proliferation and host immune responses to the congenital infection in embryo and larva stage. Here, we adopted dual RNA-seq approach to investigate and compare the dynamic molecular pattern of pathogen proliferation and host immune responses between diapause and non-diapause silkworm eggs. Our results showed the \u003cem\u003eN. bombycis\u003c/em\u003e proliferation in non-diapause silkworm eggs is a continuous process, many parasites enter the sporogony stage at 2 days post-oviposition (dpo). For newly hatched larva (1dph), the abundance of pathogen mRNA sequences is up to 2.32% in non-diapause strain, far higher than 0.34% of diapause strain, the main reason is the hot HCl bath treatment at 24 hours post-oviposition for diapause silkworm eggs with the aim to free the egg diapause. As to immune responses, whatever for diapause strain or non-diapause strain, there is stronger immune responses to congenital infection in larva stage than that of embryo stage, however, the host immune responses to congenital infection are fairly different between non-diapause and diapause strains of silkworms, especially in embryo stage. We found the surprising \u0026ldquo;First day Chaos\u0026rdquo; that there are 6,071 differential expressed genes (DEGs) at 1 dpo for non-diapause strain between infection group and uninfected group, but decreases dramatically to 109 DEGs at 2 dpo. In non-diapause strain, the earliest DEGs of antimicrobial peptides were up-regulated at 1 dpo, then is 5 dpo with up-regulated \u003cem\u003elebocin\u003c/em\u003e, 7 dpo with \u003cem\u003emorLP-B1, morLP-B4.\u003c/em\u003e For non-diapause strain, the well-established immune responses were observed in newly hatched larvae. On the contrast, for diapause strain, the earliest DEGs of AMPs appear at 5 dph, the mature immune responses are well established at 5 dph too. In non-diapause silkworms, we observed obvious pathogen\u0026rsquo;s regulation in the main immune pathways including Toll, IMD, JAK-STAT and melanization at the different steps such as immune recognition, signal modulation and transduction, effectors. Taken together, our results for the first time provide a global molecular view of microsporidia proliferation and innate immunity responses in a congenital infection system and provide some new insights into immune development and establishment in the embryo and early larva stage of \u003cem\u003eBombyx mori\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Insights to Micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 05:00:44","doi":"10.21203/rs.3.rs-5908051/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-20T19:13:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T17:26:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101265366400744195439122342036055406488","date":"2025-04-11T03:37:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T05:56:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-10T02:13:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-04-09T10:40:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e647755e-c42b-47a8-8519-b7e65377424b","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:20:23+00:00","versionOfRecord":{"articleIdentity":"rs-5908051","link":"https://doi.org/10.1186/s12864-025-11762-z","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-07-01 15:58:09","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-11 05:00:44","video":"","vorDoi":"10.1186/s12864-025-11762-z","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11762-z","workflowStages":[]},"version":"v1","identity":"rs-5908051","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5908051","identity":"rs-5908051","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}