Population mixing mediates the intestinal flora composition and facilitates invasiveness in a globally invasive fruit fly

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In the present study, we examined phenotypic differentiation, the intestinal microbiome composition and metabolism in the oriental fruit fly ( Bactrocera dorsalis ) by comparing an inbred (monophyletic) native population and an outbred (mixed) invasive population. Results The results showed that the outbred population of B. dorsalis had significantly higher biomass, adult longevity, and fecundity than the inbred population. Additionally, intestinal microflora analysis revealed that both Diutina rugosa and Komagataeibacter saccharivorans were significantly enriched in the outbred population with higher genetic heterozygosity. D. rugosa enrichment altered amino acid metabolism in the intestinal tract, and supplementing essential amino acids (e.g., histidine and glutamine) in the diet led to an increase in pupal weight of the outbred population. Additionally, transcriptome analysis revealed that the HSPA1S gene was significantly downregulated in the outbred population. HSPA1S was involved in activation of the JNK-MAPK pathway through negative regulation, caused the upregulation of juvenile hormone (JH), and led to an increase in biomass in outbred flies. Conclusion In conclusion, the outbred population had an altered intestinal microbe composition, mediating metabolism and transcriptional regulation, leading to phenotypic differentiation; this may be a potential mechanism driving the global invasion of B. dorsalis . Thus, multiple introductions could lead to invasiveness enhancement in B. dorsalis through population mixing, providing preliminary evidence that changes in the intestinal microbiome can promote biological invasion. Bactrocera dorsalis inbred outbred phenotype microbiome transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The successful invasion of an alien organism often leads to adaptive colonization, continuous reproduction and the expansion of invasive populations, which seriously threaten agriculture, forestry and animal husbandry worldwide and are extremely harmful to economic development[ 1 ]. The invasive alien species (IAS) could compete with local species or protected species for ecological niches and, in serious cases, lead directly or indirectly to a reduction in/loss of native biodiversity in the invaded area, adversely deteriorating ecosystem functions [ 2 ]. It is generally believed that high levels of genetic diversity facilitate the establishment and persistence of IAS in new habitats[ 3 ]. For example, high genetic diversity in Arabidopsis thaliana can speed up the initial emergence and flowering time of seedlings and increase biomass and reproductive capacity[ 4 , 5 ]. Furthermore, hybrid progeny may enhance their phenotype and invasiveness due to the high genetic diversity caused by increasing heterozygosity[ 6 ]. Locally grown Spartina maritima in the UK was crossed with introduced S. alterniflora to form allotetraploid S. anglica , which had greatly enhanced invasiveness, resulting in displacement of the parent strains in coastal areas and rapid colonization and spread to many new areas[ 7 , 8 ]. Given the strong link between species introductions and global trade and transport, it is not surprising that many IAS are introduced from several sources. The propagule pressure of IAS has been well explored and is related to the bottleneck effect, Allee effects, and founder effect. Multiple introductions (e.g., of the oriental fruit fly) from different geographical populations could provide opportunities for intraspecific hybridization and the evolution of new genotypes[ 3 ]. The genetic admixture of different geographical populations of IAS could directly affect the fitness of colonies through heterosis after intraspecific hybridization, which is an unexplored topic in invasion ecology[ 9 – 11 ]. It can also greatly enhance the adaptive potential of colonizing populations by subjecting new allelic combinations to natural selection[ 3 , 11 – 15 ]. For example, the invasive population of the pine wood nematode, Bursaphelenchus xylophilus , in China originated from many different countries[ 16 ]. Maintaining high genetic diversity during the invasion process is one of the invasion mechanisms of successful IAS[ 16 , 17 ]. Some mechanisms, such as gene recombination and invasion evolution acting on individual traits and trait combinations, have been shown to drive the production of key genetic characteristics after interspecific hybridization[ 13 , 18 ]. In the context of intraspecific hybridization, genetic admixture has been shown to greatly increase genetic phenotypic variation in invasive populations of plants[ 19 ]. For example, the grain weight per panicle and grain yield in a millet hybrid population were higher than those in their parents[ 20 ]. Heterosis in fecundity, growth traits and genetic diversity of Macrobrachium rosenbergii has been observed[ 21 ]. In recent years, gut microbes and their symbiotic functional genomes have been important mechanisms of enhanced invasiveness in hybrid offspring[ 22 , 23 ]. For example, all hybrid offspring of two carp species widely distributed in the United States, bighead carp, Hypophthalmichthys nobilis , and silver carp, H. molitrix , have high fertilization rates and high embryo viability[ 22 ]. The oriental fruit fly (OFF, Bactrocera dorsalis ) is one of the most important and notorious pests worldwide, with the widest host range; it causes major losses in the global fruit and vegetable industries. Previous research revealed that the invasive population of B. dorsalis originated from several geographical populations (e.g., North China). For example, the molecular marker mitochondrial CO1 clearly showed that the invasive population in North China was a mixed population originating from tropical (Southeast Asia) and temperate regions (South China)[ 24 ]. Therefore, the invasive population of B. dorsalis comprises hybrid offspring of individuals from several geographical populations; this could increase the invasiveness of B. dorsalis and cause a major threat to agriculture. We addressed two research topics in the present research. i) Phenotypic differences between originated (inbred) and invasive (outbred) populations of B. dorsalis were determined to reveal invasiveness traits by using phenotypic omics analyses. ii) Omics (phenomics, microbiomics, transcriptomics) technology was used to examine biotic interactions and explore the potential invasion mechanism of the invasive population. We hope that these findings will be helpful in guiding control of B. dorsalis in northern China. Methods Inbred and outbred populations Two geographical populations of B. dorsalis were collected from Fujian (F) Province, which has a subtropical climate, and Hainan (H) Province, which has a tropical climate (Fig. 1). For each geographical population, 10 pairs of adult B. dorsalis were selected for purification for 3 generations. After that, two inbred geographical populations (F and H) were separately established in the laboratory. To obtain an invasive population, we combined 100 males/females of the F population with 100 females/males of the H population and allowed them to hybridize, forming two outbred populations (F♀×H♂ and H♀×F♂: Fig. 1). Both the inbred and outbred populations of B. dorsalis were reared in an intelligent artificial climate chamber with a 14 h:10 h light:dark photoperiod, a temperature of 28±1°C and humidity at 65±5%. After eclosion, the adults of each population were placed in a mesh cage and fed an artificial diet (sucrose:soy peptone = 3:1)(Table S1). Water was continuously provided via a wetted cotton ball. Larva that hatched from the collected eggs were fed an artificial diet (Table S1)[25]. When the larvae reached the final larval stage, they were removed with forceps and placed in sterile sand with 60%~70% water content. After pupation, the larvae were placed into a cage for subsequent feeding. Life -history traits Eggs, larvae and pupae One hundred eggs were incubated on wet filter paper, and the hatch rates of eggs at 24 h, 36 h, 48 h and 72 h were observed. Then, hatched 1st instar larvae were individually fed in a 3.5 mm petri dish filled with feed. The developmental stage of each larva was observed every 24 h, and all larval deaths were recorded. The cephalopharyngeal skeleton of 3rd instar larvae was dissected to examine differences between the inbred and outbred groups. When the larvae were reached the final larval stage, 300 individuals were selected and allowed to pupate, and pupal weight and pupation and eclosion rates were measured on the sixth day. Adult sur vival and reproduction Fly populations (300 newly emerged adults) were maintained to record the number of surviving individuals every day for 60 days; these data were used to calculate the survival rate, sex ratio, and weight. Furthermore, 10 males/females were selected to examine their fecundity capacity. Eggs were collected from mango slices every 24 h for 20 days, and the cumulative number of eggs laid per female was counted. Additionally, the ovaries of female adults were dissected to determine their size under the microscope on days 5 and 15. Survival and reproduction data were used to calculate the parameters of the life table[26, 27]. Omics analyses Intestinal dissection and microbiome composition Final-stage larvae (30 individuals) of the inbred (H and F) and outbred (F♀×H♂ and H♀×F♂) populations were dissected to analyse the intestinal microbiome composition. All samples were stored on dry ice and sent to Shanghai Applied Protein Technology Co., Ltd. for metabolic analysis of amino acids and derivatives and to Shanghai Majorbio Biopharm Technology Co., Ltd. for microbial diversity sequencing analysis and transcriptome sequencing. Bacterial amplification was performed in intestinal samples from the inbred and outbred populations on the Illumina HiSeq platform using the 16S rDNA ITS1 variable region and 16S rDNA V3-V4 variable region, respectively. First, data quality was controlled and screened. Next, the diversity index was analysed according to operational taxonomic unit (OTU) cluster analysis and species taxonomic analysis. Biostatistical analysis was performed to understand the relative species abundance of bacteria and fungi in the samples, and linear discriminant analysis effect size (LEfSe) was used to evaluate the significance of species richness differences. Finally, the intestinal microbes with significant differences between the inbred and inbred groups were identified. The effects of different bacterial groups on pupal weight was verified by exchanging the feed provided to the inbred and outbred populations, and the effects of differential amino acids on pupal weight was verified by adding different amino acids to the diet of an inbred subpopulation (see Supplementary material). Transcriptome analysis Heterozygosity between the inbred and outbred populations was represented by the number of single nucleotide polymorphisms (SNPs) measured by transcriptomic analysis. Raw data were qualitatively controlled, optimized, and evaluated using SeqPrep, and gene/transcript expression levels were quantitatively analysed using RSEM. Then, DESeq2 was used to analyse the different expression levels of genes between samples to identify the differentially expressed genes to study the function of differentially expressed genes. The differentially expressed genes were divided into different gene sets for analysis according to the up- and downregulation of genes, and the obtained gene sets were subjected to functional enrichment analysis to obtain the main metabolic pathways. Statistical analysis SPSS 25.0 was used for data analysis. The Z score extreme value standardization method was used to process the original data, and extreme values with an absolute Z score higher than 2 were excluded. Then, the data were tested for homogeneity of variance and a normal distribution. If the P value of the test for homogeneity of variance was greater than or equal to 0.05 and the data were normally distributed, one-way ANOVA or the T test was used for biostatistical analysis of the data. If one of these criteria was not met, then nonparametric tests were used. Origin 2020 was used to perform nonlinear fitting for the mortality rate and accumulated offspring quantity of B. dorsalis in the different populations. The fitting result had a high fitting degree with the logistic curve, so the logistic curve was used to fit the mortality rate and accumulated offspring quantity in B. dorsalis to determine whether outbreeding affected survival and reproduction. Then, survival parameters, such as the death distribution and death expectation, and reproductive parameters, such as oviposition distribution and oviposition expectation, were calculated using the fitted data. Origin 2021 was used to draw all the resulting graphs. Results Phenotypic differences between inbred and outbred OFFs The outbred population had higher genetic heterozygosity (F 1,7 =4.324, p =0.33) and a larger number of SNPs (F 1,7 = 4.323, p = 0.33) than the inbred population (Fig. 2A and B). Additionally, the outbred population had a higher pupal weight (F 1,156 =591.78, p < 0.001) than the inbred population, with an increase of 7.32% (Fig. 2C). However, diet intake volume and the development rate did not differ between the two populations (F 1,156 =591.78, p < 0.001, Fig. S1). Additionally, adult female ovaries in the outbred population were 34.47% larger than those of the inbred population at 15 days post-emergence (F 1,87 =99.45, P<0.001) (Fig. 2D). The ovary length and width in the outbred population were 23.15% (F 1,87 =52.14, P<0.001) and 55.02% (F 1,87 = 117.74, p < 0.001) longer and wider than those in the inbred population, respectively. The survival curve of the outbred population was significantly lower than that of the inbred population (F 1,7 =64.73, P <0.05) (Fig. 2E). Regarding cohort fecundity, the outbred population laid a significantly larger number of eggs per female than the inbred population (F 1,7 =12.52, P<0.01), indicating that the reproductive potential was enhanced after population mixing (Fig. 2F). In total, the accumulated egg number in the outbred population was 26.12% higher than that in the inbred population (p<0.01). The innate rate decreased from 0.33±0.00 in the inbred population to 0.32±0.01 in the outbred population (p=0.257), while the doubling time in the outbred population was 2.82% lower than that in the inbred population (p=0.242) (Table S2). Pharyngeal bone length (p=0.174) and food intake (p=0.275) showed no significant differences between the inbred population and outbred population (Fig. S1 A and B). Microbiome differences between inbred and outbred OFFs In total, 367,190 and 367,190 high-quality fungal and bacterial sequences were obtained, respectively. The mean base pair lengths of each sequence were 227 bp and 417 bp, respectively. The microbiome OTU analysis revealed 113 species, 67 families, and 8 phyla. In the inbred population, intestinal fungi were mainly distributed in Eurotiales (43.03%), Saccharomycetales (35.35%), Trichosporonales (7.43%), Hypocreales (5.63%), Tremellales (1.05%), and others (7.51%). In the outbred population, fungi were mainly distributed in Saccharomycetales (87.95%), Trichosporonales (2.27%), Hypocreales (2.20%), Eurotiales (1.04%), and others (6.54%). The abundance of Saccharomycetales in the outbred population was 148.80% higher than that in the inbred population (P=0.046) (Fig. 3A). Regarding intestinal fungal LEfSe, Diutina rugosa (P=0.009) showed the most significant difference between the outbred and inbred groups (Fig. 3C). The number of intestinal bacteria OTUs was 2,571, with 1,651 species, 516 families, and 47 phyla. The intestinal bacteria in the inbred population mainly belonged to Lactobacillales (14.32%), Pseudomonadales (5.89%), Burkholderiales (4.62%), and Acetobacterales (0.68%). The intestinal bacteria in the outbred population mainly belonged to Lactobacillales (45.78%), Acetobacterales (16.44%), Burkholderiales (3.64%), Sphingomonadales (2.20%), and Pseudomonadales (2.17%). The abundance of Lactobacillales in the outbred population was 219.70% higher than that in the inbred population (P=0.023). (Fig. 3B). Regarding intestinal bacteria LEfSe between the outbred and inbred groups, Komagataeibacter saccharivorans (P=0.017) was the most significantly different between the two populations (Fig. 3D). When the outbred and inbred populations were fed each other's diet, the pupal weight in the inbred population was 1.06% higher than the original pupal weight (F 1,63 =0.472, P <0.05). The pupal weight in the outbred population was 6.28% lower than the original pupal weight (F 1,74 =0.853, P <0.001) (Fig. S1 C). The concentrations of histidine, arginine, glutamine, glutamate, isoleucine, and valine in the intestine in the outbred population were significantly higher than those in the intestine in the inbred population (Fig. 3E). The pupal weight in the inbred population was significantly increased after adding glutamic acid and histidine to diets. In contrast, there was no significant difference in pupal weight between the inbred subpopulation with no diet composition change and those fed diets supplemented with arginine, isoleucine and valine. The pupal weights in those fed diets supplemented with histidine and glutamic acid were 9.23% and 8.10% higher, respectively, than that in those fed a diet with no amino acid supplementation (F 1,202 =10.066, P <0.001; F 1,202 =10.037, P <0.001). The pupal weights in inbred subpopulations fed diets supplemented with valine and isoleucine were 14.76±0.04 mg and 14.63±0.05 mg, respectively, which was not significantly different from the pupal weight in those fed the normal diet (F 1,202 =28.818, P =0.904; F 1,202 =22.995, P =0.200) (Fig. S1 C). Transcriptomic differences between inbred and outbred OFFs Genomic material of the inbred and outbred populations were sequenced on the Illumina platform, and 46.83×10 6 , 49.30×10 6 , 50.33×10 6 , and 49.65×10 6 high-quality sequences were obtained. Each sample generated approximately 7 G of data, with the Q20 value reaching 98%. A total of 24,000 genes and 36,955 transcripts were obtained after assembly. The longest transcript was 16,079 bp, the shortest was 201 bp, and the average length was 1082.85 bp. Most of the transcripts were distributed between 200 and 500 bps, and the length of N50 was 2,156 bp. A false discovery rate (FDR) ≤0.05 and fold-change ≥ 2 were applied to compare unigenes between the inbred and outbred populations. The results showed that 784 unigenes were differentially expressed in the transcriptomes of the inbred and outbred populations, of which 637 unigenes were upregulated and 147 unigenes were downregulated (Fig. 4A). Thirty-four functional components and 147 downregulated unigenes in the outbred population were obtained based on 309 annotations. Among them, 95 annotations were attributed to molecular function, 110 to biological processes, and 104 to cellular components. In the molecular function category, binding (GO:0005488) (27.21%) and catalytic activity (GO:0003824) (22.45%) were the most enriched. Cellular process (GO:0009987) (19.05%) and metabolic process (GO:0005488) (14.97%) were the most involved biological processes. Cell part (GO:0008152) (23.13%) and membrane part (GO:0044425) (14.97%) were the most upregulated cellular components (Fig. 4B). Some downregulated differential genes that may affect phenotypic differentiation were screened considering p<0.05. The expression levels of the CRYAB gene in the inbred and outbred populations were 750.15±36.49 and 101.47±47.49, respectively. The expression levels of HSPA1S in the inbred and outbred populations were 617.63±336.22 and 14.04±2.09, respectively. The expression levels of the HSPA5 gene in the inbred and outbred populations were 136.90±51.12 and 22.25±0.98, respectively. The expression levels of the CYP6 gene in the inbred and outbred populations were 22.76±3.58 and 4.18±0.09, respectively. The expression levels of CYP18A1 in the inbred and outbred populations were 4.96±33.13 and 0.02±0.00, respectively. The expression levels of FOXA2 in the inbred and outbred populations were 3.57±0.05 and 0.64±0.01, respectively (Fig. 4C). Based on the KEGG database of 122 metabolic pathways, 60 downregulated genes were obtained, among which 10 pathways were significantly enriched in the outbred population (FDR≤0.05). The immune system pathway, including the antigen processing and presentation pathway, had the most significant differences between the two populations (P=0.0019). Additionally, the longevity regulating pathway-multiple species pathway (P=0.0021) and protein processing in the endoplasmic reticulum pathway (P=0.0022) and the MAPK signalling pathway (P=0.0033) showed significant differences. Thus, the expression of the HSPA1S gene in the MAPK pathway was downregulated, which led to upregulation of the JNK pathway. Additionally, the CYP18A1 gene, which regulates the insect hormone synthesis pathway, namely, the ecdysone pathway, was significantly downregulated, but the pathway was not significantly enriched (Table 1, Fig. 4D). Table 1 KEGG enrichment pathways and descriptions Pathway ID Description P value map04612 Antigen processing and presentation 0.0019 map04213 Lifespan regulation pathway - multiple species 0.0021 map04141 Protein processing in endoplasmic reticulum 0.0022 map03040 spliceosome 0.039 map04010 MAPK signalling pathway 0.045 Discussion Effects of genetic diversity on the phenotype of the OFF Heterosis refers to the phenomenon that the offspring of a hybrid are superior to their parents in terms of body size, reproductive survival time and reproductive capacity[ 28 ]. The pupal weight, adult biomass and reproductive capacity in offspring of silkworm hybrids were significantly higher than those of those with the parental phenotype, and the rate of body weight increase in F 1 hybrids was also higher than that in their parents[ 29 ]. A Hong Kong oyster indoor seeding study revealed that the hybrid population had a significant survival advantage over the self-crossing population[ 30 ]. Heterosis exists in the hybrid offspring of horse-donkey pairs, and the offspring are characterized by strong disease resistance, higher coarse feed tolerance and a longer lifespan[ 31 ]. After crossing purple scallops and Gulf scallops, the fertilization rate of the hybrid scallops was higher than that of the self-breeding population[ 32 ]. In this study, it was found that the outbred population of B. dorsalis had advantages such as a higher pupal weight, survival rate and egg quantity, which was consistent with the results of previous studies. It was also found that hybridization had no significant effect on the development duration of larvae and pupae. Effects of the intestinal microbiota on OFFs Changes in amino acid compositions led to changes in the microbiome composition[ 33 ]. Therefore, differences in intestinal microbiota compositions between the outbred and inbred populations of OFFs was due to differences in amino acid concentrations. Among the outbred and inbred populations, the fungal species with the highest degree of differentiation was D. rugosa in Saccharomycetales, and the bacterial species with the highest degree of differentiation was K. saccharivorans in Lactobacillus. Moreover, the concentrations of histidine, glutamic acid and arginine in the intestinal tract in the outbred population were significantly higher than those in the intestinal tract in the inbred population. After feeding diets supplemented with amino acids, the intestinal microbiota composition changed in the populations. The intestinal microbiota compositions in the inbred and outbred populations were also altered by exchanging diets between populations. After diet exchange, the pupal weight in the inbred population increased, and that in the outbred population decreased. The results also showed that the increase in pupal weight in the outbred population of the OFF was caused by a change in the microbial environment after the conversion of amino acids in the intestine. Effects of the insect transcriptome on genetic diversity MAPK, short for mitogen-activated protein kinases, is a group of evolutionarily conserved silk/threonine protein kinases that are activated by a series of extracellular stimulation signals and mediate signal transmission from the cell membrane to the nucleus. They regulate many physiological activities, such as inflammation, apoptosis, carcinogenesis, and the metastasis of tumour cells[ 34 ]. JNK, also known as stress-activated protein kinase (SAPK), is another subclass of the MAPK (mitogen-activated protein kinase) signalling pathway in mammalian cells. The MAPK-JNK pathway can be activated by various environmental stressors, inflammatory cytokines, growth factors, and CPCR agonists. Stress response signals are transmitted to the targets of this cascade by small molecule GTp-enzymes in the RHO family. JNK transport into the nucleus can regulate the activity of a variety of transcription factors and then mediate the transcriptional activation of JUN, ELK1, P53, etc., which play important roles in various physiological and pathological processes, such as the cell cycle, reproduction, apoptosis, and cell stress[ 35 , 36 ]. In this study, it was found that the HSPA1S gene was significantly downregulated and enriched in the MAPK pathway. Combined with the observation results from the differential phenotypes, it is believed that downregulated HSPA1S upregulated the JNK gene, which then affected the growth and development of the outbred population. Therefore, the pupal weight in the outbred population increased. Increases in fecundity and other indicators were regulated by the JNK-MAPK pathway, consistent with the results of previous studies[ 37 ]; moreover, the HSPA1S gene was also enriched in the pathway of immune function. In this study, it was also found that the CYP18A1 gene was significantly downregulated and enriched in insect synthetic hormones, and the decrease in insect ecdysone could have caused the increase in insect body weight[ 38 ]. Mechanisms by which hybridization promotes invasion Population mixing has the potential to promote species invasion by creating unique opportunities for positive genetic interactions between previously isolated alleles and the adaptive evolution of new genotypes, leading to improved fitness in hybrid populations. These mechanisms directly contribute to colonization by nonnative species and the evolution of particularly invasive new genotypes in the case of interspecies hybridization, and the potential for interspecies hybridization to yield these benefits is widespread[ 12 ]. There are several nonmutually exclusive mechanisms that may produce the following positive fitness effects due to either genetic interactions between new combinations of alleles within individuals or increased genetic diversity resulting from different population combinations[ 12 , 39 , 40 ]: the genetic remedial effect, superdominant effect, epistatic effect, complementary effect and evolutionary remedial effect. The continued spatial expansion of the OFF due to the domestic trade of fruits and vegetables increases the risk of outbreeding among individuals originating from multiple populations during invasion processes. Additionally, some studies have shown that microbes in organisms may be a newly discovered mechanism promoting hybrid invasion[ 23 ]. In this study, a novel mechanism of invasiveness in hybridized OFFs was proposed, that is, intestinal microbial-mediated hybridization induced phenotypic changes and promoted the invasion of B. dorsalis . Conclusion Intraspecific hybridization of IAS has the potential to contribute to enhanced invasiveness and genetic resiliency to rapidly adapt to newly invaded habitats. Our study revealed difference in the life history traits of outbred and inbred populations of the OFF. Both the survival curves and oviposition rates of outbred populations were increased by population mixing. We also found that composition changes in the gut microbiota caused an increase in essential amino acid concentrations, which led to a shift in HSPA1S transcription. We found that downregulation of the HSPA1S gene in the intestinal tract of outbred populations of OFFs may affect longevity and body weight gain in outbred populations through upregulation of the JNK-MAPK pathway. It can also affect the cell apoptosis and reproductive functions in insects to enhance survival and fecundity in outbred populations. We highlight that these intraspecific hybrids have the potential to produce a variety of unique genetic combinations that could result in greater success among outbred populations in their invaded range, potentially facilitating the colonization of invasive species[ 41 ]. We conducted an exploratory study on OFF invasiveness, revealed phenotypic differentiation between inbred and outbred populations, and proposed a physiological mechanism of intestinal microbiota-mediated outbreeding success, promoting the invasion of the oriental fruit fly. The physiological mechanism of hybrid invasion promotion provides a new avenue for the study of hybrid invasion. Declarations Availability of data and materials The sequencing dataset for transcriptome has been submitted to the National Center for Biotechnology Information, accession are SRR21206475 - SRR21206478 (SRP394084). The sequencing dataset for gut microbes has been submitted to the National Center for Biotechnology Information, accession are SRR21275060-SRR21275070 (SRP394415). A cknowledgements We acknowledge graduates, Mr. Hao Li and Ms. Yan Zhao for experimental assistance of insect feeding and data collection. The authors declare no competing interests. Funds This research is supported by the National Key R&D Program of China (No. 2021YFC2600401). Author information Authors and Affiliations Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China. Yidan Wang, Zhihong Li& Zihua Zhao Contributions The experimental deign and idea of the paper were conceived by ZZ and ZL; data collection and arrangements was conducted by YW and ZZ; statistical analyses were performed by YW; YW analysis were performed using the online platform of Majorbio Cloud Platform (www.majorbio.com). YW wrote the first manuscript with substantial contributions from ZZ; all authors contributed to revisions of the draft. Corresponding author Correspondence to Zihua Zhao Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Yang XY. Hazards of invasive alien organisms and countermeasures for prevention and control. Jiangxi Agriculture. 2017(01):99. Ju RT, Li H, Shi ZN, Li B. Research progress of biological invasions in China in recent ten years. Biodiversity. 2012;20(05):581-611. 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Shen JB, Liu MH, Wang Q, Zhang TQ. Preliminary study on some rules of heterosis intensity in interspecific and intraspecific hybridization of Carp. Journal of Fisheries. 1993(01):1-8. Wang J, Gaughan S, Lamer JT, Deng C, Hu W, Wachholtz M, et al. Resolving the genetic paradox of invasions: Preadapted genomes and postintroduction hybridization of bigheaded carps in the Mississippi River Basin. Evol Appl. 2020;13(2):263-77. Zhu L, Zhang Z, Chen H, Lamer JT, Wang J, Wei W, et al. Gut microbiomes of bigheaded carps and hybrids provide insights into invasion: A hologenome perspective. Evol Appl. 2021;14(3):735-45. Zhao Y, Duan YH, Zhao ZH, Wang YN, Wu Q, Ding JY, et al. Overwintering study on experimental population of Bactrocera dorsalis in Beijing area. China Plant Protection Guide. 2019;39(10):68-71. Liu B, Li B, Zhan G, Zha T, Wang Y, Ma C. Forced hot-air treatment against Bactrocera papayae (Diptera: Tephritidae) in papaya. Appl Entomol Zool. 2017;52(4):531-41. Pang XF, Liang GW, You MS, Wu WJ. Equation of state for the study of insect population life systems. Journal of South China Agricultural University. 1988(02):1-10. Zhao ZH. The concept and application of Bio Demography. Journal of Plant Protection. 2020;47(04):904-11. Lou DY. Fish breeding science. Beijing: China Agriculture Press; 1999. Cai MW. Studies on genetic Relationship and heterosis of Bombyx mori by RAPD and SSR. In. Master: Suzhou University; 2009; 92. Guan JL, Zhang YH, Su JQ, Xiao S, Zhang M, Li QZ, et al. Studies on the heterosis of early growth and development of hybrid progeny of Crassostrea hongkongensis. Oceanologia et Limnologia Sinica. 2016;47(01):182-7. Han HM. Structural characteristics of the genome of horse×donkey hybrid. In. Doctor: Inner Mongolia University; 2020; 236. Nan LH, Zhang JS, Feng W, Liu X, Li ZX, Wang CD. A preliminary study on interspecific hybridization between Scallop purple and Scallop gulf. Chinese Agricultural Science Bulletin. 2012;28(20):131-5. Liu Z, Yang SB, Song XM, Nian JX, Zhang H. Effects of amino acid nutrition on microbial growth and activity. Heilongjiang Agric Sci. 2010(05):13-5. Luo SQ, Jiang Y. Molecular basis and functional regulation of cell signal transduction. Beijing: Science Press; 2005. Sabapathy K. Role of the JNK pathway in human diseases. Prog Mol Biol Transl Sci. 2012(106):145-69. Zhang PJ, Zhu LX, Geng XP. Research status of p38MAPK signaling pathway and its inhibitors. Anhui Med Pharm J. 2010;5(14):596-8. Guo YJ, Zheng YL, Zhang CX, Ren YS, Zhang CX. Research progress of MAPK signaling pathway in animal reproduction. China Animal Husbandry and Veterinary Medicine. 2015;05(42):1268-73. Guittard E, Blais C, Maria A, Parvy J, Pasricha S, Lumb C, et al. CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis. Dev Biol. 2011;349(1):35-45. Hufbauer RA. Admixture is a driver rather than a passenger in experimental invasions. J Anim Ecol. 2017;86(1):4-6. Lynch M. The genetic interpretation of inbreeding depression and outbreeding depression. Evolution. 1991;45(3):622-9. Coulter AA, Brey MK, Lamer JT, Whitledge GW, Garvey JE. Early generation hybrids may drive range expansion of two invasive fishes. Freshwater Biol. 2019;65(4):716-30. Additional Declarations No competing interests reported. Supplementary Files supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 28 Sep, 2023 Read the published version in Microbiome → Version 1 posted Editorial decision: Major revision 09 Jan, 2023 Reviewers agreed at journal 27 Dec, 2022 Reviews received at journal 21 Dec, 2022 Reviewers agreed at journal 30 Nov, 2022 Reviewers invited by journal 18 Oct, 2022 Editor assigned by journal 29 Aug, 2022 Submission checks completed at journal 28 Aug, 2022 First submitted to journal 17 Aug, 2022 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. 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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-1971748","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":132325707,"identity":"532e3d33-b9dd-4457-953c-4c69405ec4f7","order_by":0,"name":"Yidan Wang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yidan","middleName":"","lastName":"Wang","suffix":""},{"id":132325709,"identity":"50f4d952-2a95-4d75-a57a-59e61084786e","order_by":1,"name":"Zhihong Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Li","suffix":""},{"id":132325710,"identity":"f28007d6-6b73-4ab0-8cad-192e084ae180","order_by":2,"name":"Zihua Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDACCQY2IGkD4fCQoCWNdC2HSdAiP7v92YMfFefl+WckMD5428Ygb05IC+OcA+mGPWduG864kcBsOLeNwXBnAwEtzBIJxyR4224nMNxIYJPmbWNIMDhAQAubRGKb5N+2cwnyNxLYfxOlhUciGWT4gQQDoC3MRGmRkEhjk5Y5k2y48czDZsk55yQMNxDSIj8j/Znkmwo7ebnjyQc/vCmzkSdoCxJgbADZSrz6UTAKRsEoGAW4AQC4dzpuFy3KlQAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zihua","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2022-08-17 15:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1971748/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1971748/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40168-023-01664-1","type":"published","date":"2023-09-28T15:01:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":25896131,"identity":"54b241db-2ffe-45c2-9321-5a84b13575da","added_by":"auto","created_at":"2022-08-31 16:12:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":607861,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for the inbred and outbred populations\u003c/p\u003e","description":"","filename":"Figurefile1.png","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/e07fd8eeee09dea5e0cfe6cc.png"},{"id":25896129,"identity":"767c9963-15fe-4bb9-b6a2-93ea0f3eff25","added_by":"auto","created_at":"2022-08-31 16:12:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":680333,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic differences between the inbred and outbred populations. A Heterozygosity. B Number of SNPs. C Pupal weights; the pictures of pupae were taken under a microscope. D Ovary size, calculated by multiplying the length of the ovary by the width; the pictures of ovaries were taken under a microscope. E Adult mortality rate. The small figure shows the distribution of deaths. F Cumulative number of eggs laid by a single female. The small area shows the reproduction distribution.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figurefile2.png","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/aabeafcc69a1de83b36f545d.png"},{"id":25896130,"identity":"e1405bce-03d8-4c6f-b596-674bfc53daa7","added_by":"auto","created_at":"2022-08-31 16:12:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2244283,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies compositions of the microbiomes of the inbred and outbred populations. A, B diagram of intestinal fungal and bacterial species abundance in B. dorsalis. The inner circle is the inbred population, and the outer circle is the outbred population. C, D LEfSe diagram of intestinal fungi and bacteria in B. dorsalis. Red indicates that the abundance of the microbe group in the inbred population was significantly higher than that in the outbred population, and the opposite is shown in blue. E Concentrations of amino acids and their derivatives in the intestinal tract of B. dorsalis. EAA stands for essential amino acids, NAA for nonessential amino acids, and AAD for amino acid derivatives.\u003c/p\u003e","description":"","filename":"Figurefile3.png","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/acbce56e94bcb491949767bd.png"},{"id":25896932,"identity":"5fafd89f-0c57-4749-b3b7-43619b1b1898","added_by":"auto","created_at":"2022-08-31 16:17:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":775677,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic analyses of the inbred and outbred populations. Red represents a significantly upregulated gene in the outbred population, blue represents a significantly downregulated gene in the outbred population, and grey represents no significant difference. B. The GO functions of significantly different genes were enriched in the outbred population of B. dorsalis. MF stands for molecular function, BP for biological processes and CC for cell components. C Significantly downregulated gene expression in outbred populations. D Intestinal transcriptome metabolic pathway map.\u003c/p\u003e","description":"","filename":"Figurefile4.png","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/9e2d4fb3028356b205c52eda.png"},{"id":43974648,"identity":"366cf96b-eb1c-4130-8dd3-19bc17313c4a","added_by":"auto","created_at":"2023-10-02 15:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1340509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/2356572c-6255-4e36-89ab-c0486cae4768.pdf"},{"id":25896133,"identity":"4a2793aa-a1c1-46d3-bb6e-8dd33f327960","added_by":"auto","created_at":"2022-08-31 16:12:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":344796,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-1971748/v1/f19a8d56662ac1ba85145c7f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Population mixing mediates the intestinal flora composition and facilitates invasiveness in a globally invasive fruit fly","fulltext":[{"header":"Background","content":"\u003cp\u003eThe successful invasion of an alien organism often leads to adaptive colonization, continuous reproduction and the expansion of invasive populations, which seriously threaten agriculture, forestry and animal husbandry worldwide and are extremely harmful to economic development[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The invasive alien species (IAS) could compete with local species or protected species for ecological niches and, in serious cases, lead directly or indirectly to a reduction in/loss of native biodiversity in the invaded area, adversely deteriorating ecosystem functions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is generally believed that high levels of genetic diversity facilitate the establishment and persistence of IAS in new habitats[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For example, high genetic diversity in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e can speed up the initial emergence and flowering time of seedlings and increase biomass and reproductive capacity[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, hybrid progeny may enhance their phenotype and invasiveness due to the high genetic diversity caused by increasing heterozygosity[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Locally grown \u003cem\u003eSpartina maritima\u003c/em\u003e in the UK was crossed with introduced \u003cem\u003eS. alterniflora\u003c/em\u003e to form allotetraploid \u003cem\u003eS. anglica\u003c/em\u003e, which had greatly enhanced invasiveness, resulting in displacement of the parent strains in coastal areas and rapid colonization and spread to many new areas[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the strong link between species introductions and global trade and transport, it is not surprising that many IAS are introduced from several sources. The propagule pressure of IAS has been well explored and is related to the bottleneck effect, Allee effects, and founder effect. Multiple introductions (e.g., of the oriental fruit fly) from different geographical populations could provide opportunities for intraspecific hybridization and the evolution of new genotypes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The genetic admixture of different geographical populations of IAS could directly affect the fitness of colonies through heterosis after intraspecific hybridization, which is an unexplored topic in invasion ecology[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It can also greatly enhance the adaptive potential of colonizing populations by subjecting new allelic combinations to natural selection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For example, the invasive population of the pine wood nematode, \u003cem\u003eBursaphelenchus xylophilus\u003c/em\u003e, in China originated from many different countries[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Maintaining high genetic diversity during the invasion process is one of the invasion mechanisms of successful IAS[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome mechanisms, such as gene recombination and invasion evolution acting on individual traits and trait combinations, have been shown to drive the production of key genetic characteristics after interspecific hybridization[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the context of intraspecific hybridization, genetic admixture has been shown to greatly increase genetic phenotypic variation in invasive populations of plants[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For example, the grain weight per panicle and grain yield in a millet hybrid population were higher than those in their parents[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Heterosis in fecundity, growth traits and genetic diversity of \u003cem\u003eMacrobrachium rosenbergii\u003c/em\u003e has been observed[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In recent years, gut microbes and their symbiotic functional genomes have been important mechanisms of enhanced invasiveness in hybrid offspring[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, all hybrid offspring of two carp species widely distributed in the United States, bighead carp, \u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e, and silver carp, \u003cem\u003eH. molitrix\u003c/em\u003e, have high fertilization rates and high embryo viability[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe oriental fruit fly (OFF, \u003cem\u003eBactrocera dorsalis\u003c/em\u003e) is one of the most important and notorious pests worldwide, with the widest host range; it causes major losses in the global fruit and vegetable industries. Previous research revealed that the invasive population of \u003cem\u003eB. dorsalis\u003c/em\u003e originated from several geographical populations (e.g., North China). For example, the molecular marker mitochondrial CO1 clearly showed that the invasive population in North China was a mixed population originating from tropical (Southeast Asia) and temperate regions (South China)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, the invasive population of \u003cem\u003eB. dorsalis\u003c/em\u003e comprises hybrid offspring of individuals from several geographical populations; this could increase the invasiveness of \u003cem\u003eB. dorsalis\u003c/em\u003e and cause a major threat to agriculture. We addressed two research topics in the present research. i) Phenotypic differences between originated (inbred) and invasive (outbred) populations of \u003cem\u003eB. dorsalis\u003c/em\u003e were determined to reveal invasiveness traits by using phenotypic omics analyses. ii) Omics (phenomics, microbiomics, transcriptomics) technology was used to examine biotic interactions and explore the potential invasion mechanism of the invasive population. We hope that these findings will be helpful in guiding control of \u003cem\u003eB. dorsalis\u003c/em\u003e in northern China.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eInbred and outbred populations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo geographical populations of \u003cem\u003eB. dorsalis\u003c/em\u003e were collected from Fujian (F) Province, which has a subtropical climate, and Hainan (H) Province, which has a tropical climate (Fig. 1). For each geographical population, 10 pairs of adult \u003cem\u003eB. dorsalis\u003c/em\u003e were selected for purification for 3 generations. After that, two inbred geographical populations (F and H) were separately established in the laboratory. To obtain an invasive population, we combined 100 males/females of the F population with 100 females/males of the H population and allowed them to hybridize, forming two outbred populations (F♀\u0026times;H♂ and H♀\u0026times;F♂: Fig. 1).\u003c/p\u003e\n\u003cp\u003eBoth the inbred and outbred populations of \u003cem\u003eB. dorsalis\u003c/em\u003e were reared in an intelligent artificial climate chamber with a 14 h:10 h light:dark photoperiod, a temperature of 28\u0026plusmn;1\u0026deg;C and humidity at 65\u0026plusmn;5%. After eclosion, the adults of each population were placed in a mesh cage and fed an artificial diet (sucrose:soy peptone = 3:1)(Table S1). Water was continuously provided via a wetted cotton ball. Larva that hatched from the collected eggs were fed an artificial diet (Table S1)[25]. When the larvae reached the final larval stage, they were removed with forceps and placed in sterile sand with 60%~70% water content. After pupation, the larvae were placed into a cage for subsequent feeding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLife\u003c/strong\u003e\u003cstrong\u003e-history traits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEggs, larvae and pupae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne hundred eggs were incubated on wet filter paper, and the hatch rates of eggs at 24 h, 36 h, 48 h and 72 h were observed. Then, hatched 1st instar larvae were individually fed in a 3.5 mm petri dish filled with feed. The developmental stage of each larva was observed every 24 h, and all larval deaths were recorded. The cephalopharyngeal skeleton of 3rd instar larvae was dissected to examine differences between the inbred and outbred groups. When the larvae were reached the final larval stage, 300 individuals were selected and allowed to pupate, and pupal weight and pupation and eclosion rates were measured on the sixth day.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdult \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003esur\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evival and reproduction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFly populations (300 newly emerged adults) were maintained to record the number of surviving individuals every day for 60 days; these data were used to calculate the survival rate, sex ratio, and weight. Furthermore, 10 males/females were selected to examine their fecundity capacity. Eggs were collected from mango slices every 24 h for 20 days, and the cumulative number of eggs laid per female was counted. Additionally, the ovaries of female adults were dissected to determine their size under the microscope on days 5 and 15. Survival and reproduction data were used to calculate the parameters of the life table[26, 27].\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eOmics\u003cem\u003e \u003c/em\u003eanalyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIntestinal dissection \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eand microbiome composition\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinal-stage larvae (30 individuals) of the inbred (H and F) and outbred (F♀\u0026times;H♂ and H♀\u0026times;F♂) populations were dissected to analyse the intestinal microbiome composition. All samples were stored on dry ice and sent to Shanghai Applied Protein Technology Co., Ltd. for metabolic analysis of amino acids and derivatives and to Shanghai Majorbio Biopharm Technology Co., Ltd. for microbial diversity sequencing analysis and transcriptome sequencing.\u003c/p\u003e\n\u003cp\u003eBacterial amplification was performed in intestinal samples from the inbred and outbred populations on the Illumina HiSeq platform using the 16S rDNA ITS1 variable region and 16S rDNA V3-V4 variable region, respectively. First, data quality was controlled and screened. Next, the diversity index was analysed according to operational taxonomic unit (OTU) cluster analysis and species taxonomic analysis. Biostatistical analysis was performed to understand the relative species abundance of bacteria and fungi in the samples, and linear discriminant analysis effect size (LEfSe) was used to evaluate the significance of species richness differences. Finally, the intestinal microbes with significant differences between the inbred and inbred groups were identified. The effects of different bacterial groups on pupal weight was verified by exchanging the feed provided to the inbred and outbred populations, and the effects of differential amino acids on pupal weight was verified by adding different amino acids to the diet of an inbred subpopulation (see Supplementary material).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranscriptome analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeterozygosity between the inbred and outbred populations was represented by the number of single nucleotide polymorphisms (SNPs) measured by transcriptomic analysis. Raw data were qualitatively controlled, optimized, and evaluated using SeqPrep, and gene/transcript expression levels were quantitatively analysed using RSEM. Then, DESeq2 was used to analyse the different expression levels of genes between samples to identify the differentially expressed genes to study the function of differentially expressed genes. The differentially expressed genes were divided into different gene sets for analysis according to the up- and downregulation of genes, and the obtained gene sets were subjected to functional enrichment analysis to obtain the main metabolic pathways.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS 25.0 was used for data analysis. The Z score extreme value standardization method was used to process the original data, and extreme values with an absolute Z score higher than 2 were excluded. Then, the data were tested for homogeneity of variance and a normal distribution. If the P value of the test for homogeneity of variance was greater than or equal to 0.05 and the data were normally distributed, one-way ANOVA or the T test was used for biostatistical analysis of the data. If one of these criteria was not met, then nonparametric tests were used.\u003c/p\u003e\n\u003cp\u003eOrigin 2020 was used to perform nonlinear fitting for the mortality rate and accumulated offspring quantity of \u003cem\u003eB. dorsalis\u003c/em\u003e in the different populations. The fitting result had a high fitting degree with the logistic curve, so the logistic curve was used to fit the mortality rate and accumulated offspring quantity in \u003cem\u003eB. dorsalis\u003c/em\u003e to determine whether outbreeding affected survival and reproduction. Then, survival parameters, such as the death distribution and death expectation, and reproductive parameters, such as oviposition distribution and oviposition expectation, were calculated using the fitted data. Origin 2021 was used to draw all the resulting graphs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhenotypic differences between inbred and outbred OFFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe outbred population had higher genetic heterozygosity\u0026nbsp;(F\u003csub\u003e1,7\u003c/sub\u003e=4.324, p =0.33)\u0026nbsp;and a larger number of SNPs (F\u003csub\u003e1,7\u003c/sub\u003e = 4.323, p = 0.33) than the inbred population\u0026nbsp;(Fig. 2A and B). Additionally,\u0026nbsp;the outbred population had a higher pupal weight (F\u003csub\u003e1,156\u003c/sub\u003e =591.78, p \u0026lt; 0.001) than the inbred population, with an increase of\u0026nbsp;7.32% (Fig.\u0026nbsp;2C). However, diet intake volume and the development rate did not differ between the two\u0026nbsp;populations (F\u003csub\u003e1,156\u003c/sub\u003e =591.78, p \u0026lt; 0.001,\u0026nbsp;Fig.\u0026nbsp;S1). Additionally, adult female ovaries in the\u0026nbsp;outbred\u0026nbsp;population were\u0026nbsp;34.47% larger than those of the inbred population\u0026nbsp;at\u0026nbsp;15 days post-emergence (F\u003csub\u003e1,87\u003c/sub\u003e=99.45, P\u0026lt;0.001) (Fig.\u0026nbsp;2D). The ovary length and width in the outbred population were 23.15% (F\u003csub\u003e1,87\u003c/sub\u003e=52.14, P\u0026lt;0.001) and 55.02% (F\u003csub\u003e1,87\u003c/sub\u003e = 117.74, p \u0026lt; 0.001) longer and wider than those in the inbred population, respectively.\u003c/p\u003e\n\u003cp\u003eThe survival curve of the outbred population was significantly lower than that of the inbred population (F\u003csub\u003e1,7\u003c/sub\u003e=64.73, P \u0026lt;0.05) (Fig. 2E). Regarding cohort fecundity, the outbred population laid a significantly larger number of eggs per female than the inbred population\u0026nbsp;(F\u003csub\u003e1,7\u003c/sub\u003e=12.52, P\u0026lt;0.01), indicating that the reproductive potential was enhanced after population mixing (Fig. 2F). In total, the accumulated egg number in the outbred population was 26.12% higher than that in the inbred population\u0026nbsp;(p\u0026lt;0.01).\u0026nbsp;The innate rate decreased from 0.33\u0026plusmn;0.00 in the inbred population to 0.32\u0026plusmn;0.01 in the outbred population\u0026nbsp;(p=0.257),\u0026nbsp;while the doubling time in the outbred population was\u0026nbsp;2.82% lower than that in the inbred population\u0026nbsp;(p=0.242)\u0026nbsp;(Table S2).\u0026nbsp;Pharyngeal bone length (p=0.174) and food intake (p=0.275) showed no significant differences between the inbred population and outbred population (Fig. S1\u0026nbsp;A and B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobiome differences between inbred and outbred OFFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 367,190 and 367,190 high-quality fungal and bacterial sequences were obtained, respectively. The mean base pair lengths of each sequence were 227 bp and 417 bp, respectively. The microbiome OTU analysis revealed 113 species, 67 families, and 8 phyla. In the inbred population, intestinal fungi were mainly distributed in Eurotiales (43.03%), Saccharomycetales (35.35%), Trichosporonales (7.43%), Hypocreales (5.63%), Tremellales (1.05%), and others (7.51%). In the outbred population, fungi were mainly distributed in Saccharomycetales (87.95%), Trichosporonales (2.27%), Hypocreales (2.20%), Eurotiales (1.04%), and others (6.54%). The abundance of Saccharomycetales in the outbred population was 148.80% higher than that in the inbred population (P=0.046) (Fig.\u0026nbsp;3A). Regarding intestinal fungal LEfSe, \u003cem\u003eDiutina rugosa\u003c/em\u003e (P=0.009) showed the most significant difference between the outbred and inbred groups (Fig.\u0026nbsp;3C).\u003c/p\u003e\n\u003cp\u003eThe number of intestinal bacteria OTUs was 2,571, with 1,651 species, 516 families, and 47 phyla. The intestinal bacteria in the inbred population mainly belonged to Lactobacillales (14.32%), Pseudomonadales (5.89%), Burkholderiales (4.62%), and Acetobacterales (0.68%). The intestinal bacteria in the outbred population mainly belonged to Lactobacillales (45.78%), Acetobacterales (16.44%), Burkholderiales (3.64%), Sphingomonadales (2.20%), and Pseudomonadales (2.17%).\u0026nbsp;The abundance of Lactobacillales in the outbred population was 219.70% higher than that in the inbred population (P=0.023). (Fig.\u0026nbsp;3B). Regarding intestinal bacteria LEfSe between the outbred and inbred groups, \u003cem\u003eKomagataeibacter saccharivorans\u003c/em\u003e (P=0.017) was the most significantly different between the two populations (Fig.\u0026nbsp;3D).\u003c/p\u003e\n\u003cp\u003eWhen the outbred and inbred populations were fed each other\u0026apos;s diet,\u0026nbsp;the pupal weight in the inbred population was 1.06% higher than the original pupal weight (F\u003csub\u003e1,63\u003c/sub\u003e=0.472,\u0026nbsp;P \u0026lt;0.05). The pupal weight in the outbred population was\u0026nbsp;6.28% lower than the original pupal weight\u0026nbsp;(F\u003csub\u003e1,74\u003c/sub\u003e=0.853, P \u0026lt;0.001)\u0026nbsp;(Fig.\u0026nbsp;S1 C). The concentrations of histidine, arginine, glutamine, glutamate, isoleucine, and valine in the intestine in the outbred population were significantly higher than those in the intestine in the inbred population (Fig. 3E).\u003c/p\u003e\n\u003cp\u003eThe pupal weight in the inbred population was significantly increased after adding glutamic acid and histidine to diets. In contrast, there was no significant difference in pupal weight between the inbred subpopulation with no diet composition change and those fed diets supplemented with arginine, isoleucine and valine. The pupal weights in those fed diets supplemented with histidine and glutamic acid were 9.23% and 8.10% higher, respectively, than that in those fed a diet with no amino acid supplementation (F\u003csub\u003e1,202\u003c/sub\u003e=10.066, P \u0026lt;0.001; F\u003csub\u003e1,202\u003c/sub\u003e=10.037, P \u0026lt;0.001). The pupal weights in inbred subpopulations fed diets supplemented with valine and isoleucine were 14.76\u0026plusmn;0.04 mg and 14.63\u0026plusmn;0.05 mg, respectively, which was not significantly different from the pupal weight in those fed the normal diet (F\u003csub\u003e1,202\u003c/sub\u003e=28.818, P =0.904; F\u003csub\u003e1,202\u003c/sub\u003e=22.995, P =0.200) (Fig. S1 C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic differences between inbred and outbred OFFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic material of the inbred and outbred populations were sequenced on the Illumina platform, and 46.83\u0026times;10\u003csup\u003e6\u003c/sup\u003e, 49.30\u0026times;10\u003csup\u003e6\u003c/sup\u003e, 50.33\u0026times;10\u003csup\u003e6\u003c/sup\u003e, and 49.65\u0026times;10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ehigh-quality sequences were obtained. Each sample generated approximately 7 G of data, with the Q20 value reaching 98%. A total of 24,000 genes and 36,955 transcripts were obtained after assembly. The longest transcript was 16,079 bp, the shortest was 201 bp, and the average length was 1082.85 bp. Most of the transcripts were distributed between 200 and 500 bps, and the length of N50 was 2,156 bp.\u003c/p\u003e\n\u003cp\u003eA false discovery rate (FDR) \u0026le;0.05 and fold-change \u0026ge; 2 were applied to compare unigenes between the inbred and outbred populations. The results showed that 784 unigenes were differentially expressed in the transcriptomes of the inbred and outbred populations, of which 637 unigenes were upregulated and 147 unigenes were downregulated (Fig.\u0026nbsp;4A).\u003c/p\u003e\n\u003cp\u003eThirty-four functional components and 147 downregulated unigenes in the outbred population were obtained based on 309 annotations. Among them, 95 annotations were attributed to molecular function, 110 to biological processes, and 104 to cellular components. In the molecular function category, binding (GO:0005488) (27.21%) and catalytic activity (GO:0003824) (22.45%) were the most enriched. Cellular process (GO:0009987) (19.05%) and metabolic process (GO:0005488) (14.97%) were the most involved biological processes. Cell part (GO:0008152) (23.13%) and membrane part (GO:0044425) (14.97%) were the most upregulated cellular components (Fig.\u0026nbsp;4B).\u003c/p\u003e\n\u003cp\u003eSome downregulated differential genes that may affect phenotypic differentiation were screened considering p\u0026lt;0.05.\u0026nbsp;The expression levels of the CRYAB gene in the inbred and outbred populations were 750.15\u0026plusmn;36.49 and 101.47\u0026plusmn;47.49, respectively. The expression levels of HSPA1S in the inbred and outbred populations were 617.63\u0026plusmn;336.22 and 14.04\u0026plusmn;2.09, respectively. The expression levels of the HSPA5 gene in the inbred and outbred populations were 136.90\u0026plusmn;51.12 and 22.25\u0026plusmn;0.98, respectively. The expression levels of the CYP6 gene in the inbred and outbred populations were 22.76\u0026plusmn;3.58 and 4.18\u0026plusmn;0.09, respectively. The expression levels of CYP18A1 in the inbred and outbred populations were 4.96\u0026plusmn;33.13 and 0.02\u0026plusmn;0.00, respectively. The expression levels of FOXA2 in the inbred and outbred populations were 3.57\u0026plusmn;0.05 and 0.64\u0026plusmn;0.01, respectively (Fig.\u0026nbsp;4C).\u003c/p\u003e\n\u003cp\u003eBased on the KEGG database of 122 metabolic pathways, 60 downregulated genes were obtained, among which 10 pathways were significantly enriched in the outbred population (FDR\u0026le;0.05). The immune system pathway, including the antigen processing and presentation pathway, had the most significant differences between the two populations (P=0.0019). Additionally, the longevity regulating pathway-multiple species pathway (P=0.0021) and protein processing in the endoplasmic reticulum pathway (P=0.0022) and the MAPK signalling pathway (P=0.0033) showed significant differences. Thus, the expression of the HSPA1S gene in the MAPK pathway was downregulated, which led to upregulation of the JNK pathway. Additionally, the CYP18A1 gene, which regulates the insect hormone synthesis pathway, namely, the ecdysone pathway, was significantly downregulated, but the pathway was not significantly enriched (Table\u0026nbsp;1, Fig. 4D).\u003c/p\u003e\n\u003cp\u003eTable 1\u0026nbsp;KEGG enrichment pathways and descriptions\u003c/p\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"67%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003ePathway ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003emap04612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003eAntigen processing and presentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003e0.0019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003emap04213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003eLifespan regulation pathway - multiple species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003e0.0021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003emap04141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003eProtein processing in endoplasmic reticulum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003emap03040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003espliceosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003emap04010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.67676767676768%\"\u003e\n \u003cp\u003eMAPK signalling pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.141414141414142%\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of genetic diversity on the phenotype of the OFF\u003c/h2\u003e \u003cp\u003eHeterosis refers to the phenomenon that the offspring of a hybrid are superior to their parents in terms of body size, reproductive survival time and reproductive capacity[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The pupal weight, adult biomass and reproductive capacity in offspring of silkworm hybrids were significantly higher than those of those with the parental phenotype, and the rate of body weight increase in F\u003csub\u003e1\u003c/sub\u003e hybrids was also higher than that in their parents[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A Hong Kong oyster indoor seeding study revealed that the hybrid population had a significant survival advantage over the self-crossing population[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Heterosis exists in the hybrid offspring of horse-donkey pairs, and the offspring are characterized by strong disease resistance, higher coarse feed tolerance and a longer lifespan[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. After crossing purple scallops and Gulf scallops, the fertilization rate of the hybrid scallops was higher than that of the self-breeding population[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, it was found that the outbred population of \u003cem\u003eB. dorsalis\u003c/em\u003e had advantages such as a higher pupal weight, survival rate and egg quantity, which was consistent with the results of previous studies. It was also found that hybridization had no significant effect on the development duration of larvae and pupae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of the intestinal microbiota on OFFs\u003c/h2\u003e \u003cp\u003eChanges in amino acid compositions led to changes in the microbiome composition[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, differences in intestinal microbiota compositions between the outbred and inbred populations of OFFs was due to differences in amino acid concentrations. Among the outbred and inbred populations, the fungal species with the highest degree of differentiation was \u003cem\u003eD. rugosa\u003c/em\u003e in Saccharomycetales, and the bacterial species with the highest degree of differentiation was \u003cem\u003eK. saccharivorans\u003c/em\u003e in Lactobacillus. Moreover, the concentrations of histidine, glutamic acid and arginine in the intestinal tract in the outbred population were significantly higher than those in the intestinal tract in the inbred population. After feeding diets supplemented with amino acids, the intestinal microbiota composition changed in the populations. The intestinal microbiota compositions in the inbred and outbred populations were also altered by exchanging diets between populations. After diet exchange, the pupal weight in the inbred population increased, and that in the outbred population decreased. The results also showed that the increase in pupal weight in the outbred population of the OFF was caused by a change in the microbial environment after the conversion of amino acids in the intestine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of the insect transcriptome on genetic diversity\u003c/h2\u003e \u003cp\u003eMAPK, short for mitogen-activated protein kinases, is a group of evolutionarily conserved silk/threonine protein kinases that are activated by a series of extracellular stimulation signals and mediate signal transmission from the cell membrane to the nucleus. They regulate many physiological activities, such as inflammation, apoptosis, carcinogenesis, and the metastasis of tumour cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. JNK, also known as stress-activated protein kinase (SAPK), is another subclass of the MAPK (mitogen-activated protein kinase) signalling pathway in mammalian cells. The MAPK-JNK pathway can be activated by various environmental stressors, inflammatory cytokines, growth factors, and CPCR agonists. Stress response signals are transmitted to the targets of this cascade by small molecule GTp-enzymes in the RHO family. JNK transport into the nucleus can regulate the activity of a variety of transcription factors and then mediate the transcriptional activation of JUN, ELK1, P53, etc., which play important roles in various physiological and pathological processes, such as the cell cycle, reproduction, apoptosis, and cell stress[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, it was found that the HSPA1S gene was significantly downregulated and enriched in the MAPK pathway. Combined with the observation results from the differential phenotypes, it is believed that downregulated HSPA1S upregulated the JNK gene, which then affected the growth and development of the outbred population. Therefore, the pupal weight in the outbred population increased. Increases in fecundity and other indicators were regulated by the JNK-MAPK pathway, consistent with the results of previous studies[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]; moreover, the HSPA1S gene was also enriched in the pathway of immune function. In this study, it was also found that the CYP18A1 gene was significantly downregulated and enriched in insect synthetic hormones, and the decrease in insect ecdysone could have caused the increase in insect body weight[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMechanisms by which hybridization promotes invasion\u003c/h2\u003e \u003cp\u003ePopulation mixing has the potential to promote species invasion by creating unique opportunities for positive genetic interactions between previously isolated alleles and the adaptive evolution of new genotypes, leading to improved fitness in hybrid populations. These mechanisms directly contribute to colonization by nonnative species and the evolution of particularly invasive new genotypes in the case of interspecies hybridization, and the potential for interspecies hybridization to yield these benefits is widespread[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There are several nonmutually exclusive mechanisms that may produce the following positive fitness effects due to either genetic interactions between new combinations of alleles within individuals or increased genetic diversity resulting from different population combinations[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]: the genetic remedial effect, superdominant effect, epistatic effect, complementary effect and evolutionary remedial effect. The continued spatial expansion of the OFF due to the domestic trade of fruits and vegetables increases the risk of outbreeding among individuals originating from multiple populations during invasion processes. Additionally, some studies have shown that microbes in organisms may be a newly discovered mechanism promoting hybrid invasion[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, a novel mechanism of invasiveness in hybridized OFFs was proposed, that is, intestinal microbial-mediated hybridization induced phenotypic changes and promoted the invasion of \u003cem\u003eB. dorsalis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIntraspecific hybridization of IAS has the potential to contribute to enhanced invasiveness and genetic resiliency to rapidly adapt to newly invaded habitats. Our study revealed difference in the life history traits of outbred and inbred populations of the OFF. Both the survival curves and oviposition rates of outbred populations were increased by population mixing. We also found that composition changes in the gut microbiota caused an increase in essential amino acid concentrations, which led to a shift in HSPA1S transcription. We found that downregulation of the HSPA1S gene in the intestinal tract of outbred populations of OFFs may affect longevity and body weight gain in outbred populations through upregulation of the JNK-MAPK pathway. It can also affect the cell apoptosis and reproductive functions in insects to enhance survival and fecundity in outbred populations. We highlight that these intraspecific hybrids have the potential to produce a variety of unique genetic combinations that could result in greater success among outbred populations in their invaded range, potentially facilitating the colonization of invasive species[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We conducted an exploratory study on OFF invasiveness, revealed phenotypic differentiation between inbred and outbred populations, and proposed a physiological mechanism of intestinal microbiota-mediated outbreeding success, promoting the invasion of the oriental fruit fly. The physiological mechanism of hybrid invasion promotion provides a new avenue for the study of hybrid invasion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing dataset for transcriptome has been submitted to the National Center for Biotechnology Information, accession are SRR21206475 - SRR21206478 (SRP394084). The sequencing dataset for gut microbes has been submitted to the National Center for Biotechnology Information, accession are SRR21275060-SRR21275070 (SRP394415).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ecknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge graduates, Mr. Hao Li and Ms. Yan Zhao for experimental assistance of insect feeding and data collection. The authors declare no competing interests. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by the National Key R\u0026amp;D Program of China (No. 2021YFC2600401). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors and Affiliations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China.\u003c/p\u003e\n\u003cp\u003eYidan Wang, Zhihong Li\u0026amp; Zihua Zhao\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eContributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental deign and idea of the paper were conceived by ZZ and ZL; data collection and arrangements was conducted by YW and ZZ; statistical analyses were performed by YW; YW analysis were performed using the online platform of Majorbio Cloud Platform (www.majorbio.com). YW wrote the first manuscript with substantial contributions from ZZ; all authors contributed to revisions of the draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorresponding author\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Zihua Zhao\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang XY. 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Freshwater Biol. 2019;65(4):716-30.\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":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bactrocera dorsalis, inbred, outbred, phenotype, microbiome, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-1971748/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1971748/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChanges in population heterozygosity and genetic diversity play important roles in mediating life history traits of organisms; these changes often lead to phenotypic evolution in offspring, which become superior to their parents. In the present study, we examined phenotypic differentiation, the intestinal microbiome composition and metabolism in the oriental fruit fly (\u003cem\u003eBactrocera dorsalis\u003c/em\u003e) by comparing an inbred (monophyletic) native population and an outbred (mixed) invasive population.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that the outbred population of \u003cem\u003eB. dorsalis\u003c/em\u003e had significantly higher biomass, adult longevity, and fecundity than the inbred population. Additionally, intestinal microflora analysis revealed that both \u003cem\u003eDiutina rugosa\u003c/em\u003e and \u003cem\u003eKomagataeibacter saccharivorans\u003c/em\u003e were significantly enriched in the outbred population with higher genetic heterozygosity. \u003cem\u003eD. rugosa\u003c/em\u003e enrichment altered amino acid metabolism in the intestinal tract, and supplementing essential amino acids (e.g., histidine and glutamine) in the diet led to an increase in pupal weight of the outbred population. Additionally, transcriptome analysis revealed that the HSPA1S gene was significantly downregulated in the outbred population. HSPA1S was involved in activation of the JNK-MAPK pathway through negative regulation, caused the upregulation of juvenile hormone (JH), and led to an increase in biomass in outbred flies.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, the outbred population had an altered intestinal microbe composition, mediating metabolism and transcriptional regulation, leading to phenotypic differentiation; this may be a potential mechanism driving the global invasion of \u003cem\u003eB. dorsalis\u003c/em\u003e. Thus, multiple introductions could lead to invasiveness enhancement in \u003cem\u003eB. dorsalis\u003c/em\u003e through population mixing, providing preliminary evidence that changes in the intestinal microbiome can promote biological invasion.\u003c/p\u003e","manuscriptTitle":"Population mixing mediates the intestinal flora composition and facilitates invasiveness in a globally invasive fruit fly","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-31 16:12:47","doi":"10.21203/rs.3.rs-1971748/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-01-09T12:33:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"07b5b487-d598-4046-a9a3-6c163a2f4c88","date":"2022-12-27T18:25:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-22T02:51:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22a72100-cbc2-4f6b-9b8d-ed708cc1c79a","date":"2022-11-30T23:35:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-18T13:38:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-08-29T06:32:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-08-29T00:28:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbiome","date":"2022-08-17T15:32:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fcc7229-3cf5-4c26-a851-1a1e3de11a8e","owner":[],"postedDate":"August 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-02T15:06:38+00:00","versionOfRecord":{"articleIdentity":"rs-1971748","link":"https://doi.org/10.1186/s40168-023-01664-1","journal":{"identity":"microbiome","isVorOnly":false,"title":"Microbiome"},"publishedOn":"2023-09-28 15:01:38","publishedOnDateReadable":"September 28th, 2023"},"versionCreatedAt":"2022-08-31 16:12:47","video":"","vorDoi":"10.1186/s40168-023-01664-1","vorDoiUrl":"https://doi.org/10.1186/s40168-023-01664-1","workflowStages":[]},"version":"v1","identity":"rs-1971748","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1971748","identity":"rs-1971748","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}