Dynamics of maternal gene expression in Rhodnius prolixus

Dynamics of maternal gene expression in Rhodnius prolixus | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Dynamics of maternal gene expression in Rhodnius prolixus Agustina Pascual , View ORCID Profile Rolando Rivera Pomar doi: https://doi.org/10.1101/2021.08.13.456198 Agustina Pascual 1 Centro de BioInvestigaciones (CeBio-CICBA), Universidad Nacional del Noroeste de la Provincia de Buenos Aires (UNNOBA) Avenida Presidente Frondizi 2650 (2700) , Pergamino, Buenos Aires, Argentina 2 Centro de Investigaciones y Transferencias del Noroeste de la provincia de Buenos Aires (CITNOBA- CONICET), UNNOBA-UNSAdA, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) , Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: rrivera{at}unnoba.edu.ar apascual{at}comunidad.unnoba.edu.ar Rolando Rivera Pomar 1 Centro de BioInvestigaciones (CeBio-CICBA), Universidad Nacional del Noroeste de la Provincia de Buenos Aires (UNNOBA) Avenida Presidente Frondizi 2650 (2700) , Pergamino, Buenos Aires, Argentina 2 Centro de Investigaciones y Transferencias del Noroeste de la provincia de Buenos Aires (CITNOBA- CONICET), UNNOBA-UNSAdA, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) , Buenos Aires, Argentina 3 Centro Regional de Estudios Genómicos. Facultad de Ciencias Exactas. Universidad Nacional de La Plata. Bvd 120 y 62 (1900) . La Plata. Buenos Aires. Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rolando Rivera Pomar For correspondence: rrivera{at}unnoba.edu.ar apascual{at}comunidad.unnoba.edu.ar Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The study of the developmental processes in Rhodnius prolixus has recently advanced with the sequencing of the genome. In this work, we study maternal gene expression driving oogenesis and early embryogenesis in R. prolixus . We analyze the transcriptional profile of mRNAs to establish the genes expressed across the ovary, unfertilized eggs and different embryonic stages of R. prolixus until the formation of the germ band anlage (0, 12, 24, and 48 hours post egg laying). We identified 81 putative maternal and ovary-related genes and validated their expression by qRT-PCR. Consistent with a role in oogenesis and early development of R. prolixus , we show that parental RNAi against Rp-BicD results in embryos that did not show any distinguishable embryonic structure. In this framework we propose three hierarchies of maternal genes that affect early and late oogenesis, and embryonic patterning. Introduction During insect embryogenesis, a sequential series of dynamic processes that include cell division, growth and fate specification take place to establish the necessary components to give rise to a complete organism, playing a fundamental role to support the developmental process of the whole life cycle 1 - 3 . There are three modes of insect embryogenesis: long, intermediate, and short germ embryogenesis 4 . Long germ embryogenesis is defined by the simultaneous establishment of all segmental fates at the blastoderm stage. This is a derived mode of embryogenesis, found in scattered species among the Holometabola, such as Drosophila melanogaster . These insects have polytrophic meroistic ovaries. In short or intermediate germ insects only the most anterior segments are specified before gastrulation, while the more posterior segments are generated and patterned progressively from a posterior region called the growth zone. This represents an ancestral type of insect embryogenesis, described in insect models such Oncopeltus fasciatus, Rhodnius prolixus, Bombyx mori, Tribolium castaneum , It corresponds to insects with telotrophic or panoistic ovaries 4 - 8 . A common feature across the different modes of embryogenesis is the loading of maternal mRNA transcripts and proteins in the egg during oogenesis 9 . In the last 20 years the rise of new models for comparative insect development provided a framework to understand the genetic basis of development and evolution 10 - 17 . The different mechanisms of insect embryogenesis are determined by specific spatiotemporal gene expression patterns derived from common genetic program, suggesting that the mechanisms are much more conserved than the diversity of germ types might suggest 4 , 18 - 22 . In addition, a detailed study of cell flow during germ band extension and the fate map of T. castaneum embryo led to the idea that short and long germ bands share many more common features than thought 23 . In the last decade the expanse of genomics and transcriptomic analysis provided an insight of the transcriptional basis of the embryonic development in non-model insect species 24 . However, the complete repertory of genes involved in oogenesis and early embryogenesis has been reported in detail only in D. melanogaste r 25 - 29 and T. castaneum 15 , 30 , 31 , remaining an open question in other model organisms. The blood-feeding insect Rhodnius prolixus is one vectors of Trypanosoma cruzi , the etiologic agent of Chagas disease 32 , 33 . In addition to its medical interest, it has been a classical model for physiology and biochemistry 34 - 37 . The embryonic development of R. prolixus has been described from fertilization to hatching 7 , and the process of oogenesis studied in detail 38 - 44 . The genome was recently sequenced 45 and since then, R. prolixus is an emerging model for developmental biology 46 - 50 . Several transcriptome analyses were reported, focusing in the gene expression of the follicular epithelium, the early previtellogenic stage of oogenesis; as well, in the impact of the nutritional state on regulatory pathways associated with reproductive performance 51 - 53 . Very recently, a thorough study on previtellogenic ovaries and unfertilized eggs discovered a large number of unannotated genes in the R. prolixus genome and unveiled a large set of maternal genes 54 . With all this knowledge in place, we have moved forward to the understanding of the genetic and molecular mechanisms driving oogenesis and the maternal contribution to embryo patterning in R. prolixus . Here, we present a transcriptome profiling approach to identify the genetic basis underlying oogenesis and early embryogenesis of R. prolixus until the onset of gastrulation, with a focus on genes related to embryonic patterning and egg formation. We provide novel insight into the molecular basis of early embryo formation and show the dynamic of mRNA expression during early embryo development in R. prolixus . Our study provides maternal and early embryonic transcriptomes of this hemimetabolous insect. We present a comprehensive qualitative data about genes related to segmentation, dorsal ventral axis and oogenesis, validate gene expression by qRT-PCR and show the phenotype of Bicaudal D ( BicD ) homolog likely related to early steps of the maternal cascade that leads to patterning. Materials and methods Insect rearing A colony of R. prolixus was maintained in our laboratory in a 12:12 hour light/dark period at 28 °C and 80% relative humidity in controlled environment incubators. In these conditions, embryogenesis takes 14 ± 1 days. Insects were regularly fed on chickens, which were housed, cared, fed and handled in accordance with resolution 1047/2005 (Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET) regarding the bioethical framework for biomedical research with laboratory, farm, and nature collected/wild animals. This framework is in accordance with international standard procedures. Biosecurity considerations agree with CONICET resolution 1619/2008, which is in accordance with the WHO Biosecurity Handbook (ISBN 92 4 354 6503). Sample collection, RNA isolation and sequencing Adult mated insects 6 th days after the feeding regimen were used to collect fertilized eggs at specific points in developmental time – 0 (zygote), 12 (blastoderm), 24 (cellular blastoderm) and 48 (onset of germ band formation) hours post egg laying ( hPL ). Virgin female adults were used to collect unfertilized eggs, which were immediately frozen and stored in liquid nitrogen. At the same time, female ovaries were dissected in the vitellogenic stage and placed in a cryotube containing Trizol (Invitrogen), flash frozen and stored in liquid nitrogen until use. For the transcriptome profiling, RNA was extracted from 150 embryos for each developmental time and 30 vitellogenic ovaries. For qRT-PCR analysis, independent experiments were carried out using 75 embryos from each specific time and 10 vitellogenic ovaries. Total RNA was isolated using Trizol (Invitrogen) as recommended by the manufacturer. RNA integrity was determined by agarose electrophoresis and concentration measured using Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies, Invitrogen). cDNA libraries were synthetized from 1 µg of total RNA and sequenced using a HiSeq-3000 platform (Illumina) to obtain the 50 base pairs (bp) (single-end) or 150 bp (paired-end) reads. The RNA-seq data has been submitted to the NCBI SRA database, available under accession code PRJNA694974. Quality control, alignment and transcriptome assembly Raw data were processed with FASTX-toolkit software ( http://hannonlab.cshl.edu/fastx_toolkit/ ), to remove adapter sequences, reads with unknown bases and reads with quality scores lower than Q30, showed by the FastQC report ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). To avoid contaminants, the removal of adaptor sequences was ruled out using BLASTn 55 and the UniVec database ( ftp://ftp.ncbi.nlm.nih.gov/pub/UniVec/ ) from NCBI. Additionally, remove rRNA sequences the SILVA database was used 56 . The remaining reads were defined as clean reads and used for subsequent bioinformatics analyses. Tophat2 57 was used to map clean reads to the ab initio annotations of R. prolixus , genome dataset version RproC3.3 45 , 58 . The mapping statistics by the RNA-seq reads were calculated by using bam_stat.py implemented in the RSeQC package 59 and the advanced statistics of coverage analysis were performed by the Qualimap application 60 . After Tophat alignment, transcripts were assembled using Cufflinks 61 , 62 . Assembly quality was assessed for each assembly using BUSCO analysis 63 , with the reference gene set of arthropod (2.676 proteins) with default parameters. Fasta Statistics was used to display summary statistics from each transcriptome generated 64 . The eggNOG 5.0 database was used for functional annotation of the transcripts with common denominators or functional categories (i.e., derived from the original COG categories). Also, predicted protein-coding transcripts 65 were functionally annotated. For each protein sequence protein signatures were assigned, using InterProScan search Version 5.0.0, 66 through the PfamA and SuperFamily databases. Proteins annotated by signatures were assigned into GO (Gene Ontology) categories, including biological processes (BP), molecular functions (MF) and cellular components (CC). To statistically analyze GO-term enrichment, topGO package 67 was implemented, using Fisher’s exact test and the false discovery rate (FDR) adjusted method. A q-value smaller than 0,05 were considered as significant. The reference set of gene-to-GO mappings was available from VectorBase ( https://www.vectorbase.org/ ). Oogenesis and early embryogenesis gene identification Gene identification was performed using local BLAST 55 on the six transcriptome assemblies. The BLAST algorithm used was BLASTx. The search was limited to 84 protein sequences derived from FlyBase (Version FB2020_03, https://flybase.org/ ), comprising genes related to oogenesis and early embryogenesis, with an e-value threshold of 10 −5 . Transcript with blast hit to Drosophila were then manually checked by BLASTx against all Arthropoda protein sequences (NCBI non-redundant protein (nr) database, assessed January 2018) to establish orthology. BLAST results were classified into the known D. melanogaster developmental process. For the maternal gene search, a database was generated from different resources containing 10.277 specific protein sequences (Additional file 1) 15 , 25 - 29 . Quantitative real-time PCR Total RNA was isolated using Trizol reagent (Invitrogen) and treated with DNAse (QIAGEN). cDNA was synthesized using SuperScript™ VILO™ MasterMix kit (Invitrogen) following the manufacturer’s instructions. PCR was performed in technical triplicates (3 wells/cDNA sample), in a 10 μl final volume as follows: (i) 95 °C for 10 min; (ii) 95 °C for 15 sec; (iii) 55 °C for 30 sec; (iv) 72 °C for 45 sec; (v) steps (ii) to (iv) for 35 cycles. Gene expression level was quantified using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in an Applied Biosystems 7.500 Real-Time PCR System (Thermo Fisher Scientific). A control without a template was included in all batches and α-tubulin was used as reference, after a screen of several housekeeping gene candidates, as it provided consistent results on the embryonic stages analyzed. All primer pairs (Additional file 2) were tested for dimerization, efficiency, and amplification of a single product. The Ct value was averaged for the technical triplicate experiments and subtracted from the average Ct of the reference gene, to yield the expression difference (dCt) for each biological replicate. The results were analyzed according to 68 . To test whether the expression of a given gene was significantly different across developmental times, a one-way ANOVA was carried out followed by post-hoc test using GraphPad Prism v6.0 software (GraphPad Software, CA, USA, www.graphpad.com ). In situ Hybridization and Parental RNAi DNA templates used to synthesize in situ hybridization probes were obtained by PCR using oligonucleotides carrying T7 promoter sequences at the 5’-end. The templates were in vitro transcribed using DIG RNA labeling kit (Roche). In situ hybridization was performed as described in Pascual, et al. 50 . For parental RNAi, dsRNA was produced by simultaneous transcription with T7 RNA polymerase (New England Biolabs) on templates containing T7 promoter sequences at both ends. dsRNA BicD was quantitated and injected into virgin females as described in Lavore, et al. 69 . Two days after injection, the females were fed to induce oogenesis and mated. After mating, eggs were collected and ovaries fixed as described 50 . A negative control was performed injecting virgin females with dsRNA corresponding to the β- lactamase gene (dsRNA β- lac ) of E. coli 69 . Amplicon was sequenced to confirm identity (Macrogen Inc.). Oligonucleotides used in this study are listed in the Additional file 2. Results and discussion 1. Assembling the ovarian and early embryonic transcriptomes of R. prolixus : characterization and completeness analysis The RNA-seq output comprised six R. prolixus samples that cover late oogenesis to the beginning of germ band extension (48 hPL ). Statistics on the sequencing and mapping are reported in Table 1A . According to completeness analysis, the coverage metrics obtained indicated that the assembled transcriptomes are sufficient for a meaningful analysis ( Table 1B ). As the genomic reference has a reasonable amount of positions that are not called, transcriptomes assembled by mapping genomic predictions ( ab initio ) were used to subsequent analyzes. In this respect, a review of zygotic genes 14 and of the regulatory pathways involved in egg production 52 has been reported based on these genomic annotations with a robust gene identification. View this table: View inline View popup Table 1. Summary of the RNA-seq metrics from R. prolixus transcriptomes from vitellogenic ovaries, unfertilized and 0 to 48 hPL eggs. A : Raw Reads: the original sequencing reads counts; Clean Reads: number of reads after filtering; % read mapping rate: percentages of the overall mapping rate using the annotated genome as reference, % GC: percentages of G and C in total bases. B : Transcriptome assembly’s statistics, Number, number of the total of reconstructed transcripts; Mean length: the mean length in base pairs; N50: is the size of the transcript which, along with the larger transcripts, contain half of sequence of the reference; GC: percentages of G and C in total bases; BUSCO: percentages of the transcriptome completeness by BUSCO analysis results. In order to conduct a transcriptome-composition representation analysis, eggNOG analysis was performed. A total of 25 eggNOG categories were detected ( Figure 1 and Additional file 3), in which the category “function unknown” was dominant followed by “Signal transduction mechanisms” and “Post-translational modification, protein turnover, and chaperones” in all the analyzed developmental times. To obtain information of the predicted proteins, InterproScan searched were performed to identified functional domains, repeats, sites and protein families conserved in the protein-coding transcripts ( Table 2 and Additional file 4). For all of the characterized transcripts, statistically over-represented GO terms were identified using the FDR adjusted relative to a reference set of 11.947 genes. These statistically highlighted GO terms were summarized to generic GO categories for each developmental time studied ( Table 2 and Additional file 5). GO analysis showed that mainly metabolic processes were enriched during embryo development, such as cellular macromolecule metabolic process (GO: 0044260), nucleobase-containing compound metabolic process (GO:0006139), organonitrogen compound biosynthetic process (GO:1901566), gene expression (GO: 0010467). This enrichment is in agreement with the requirements of the embryo during the transitions between the different embryonic stages with rapidly changing of the anabolic and catabolic demands 70 , 71 . Download figure Open in new tab Figure 1. Classification of eggNOG annotations in the R. prolixus transcriptomes. The capital letters on the X-axis represent different eggNOG categories. Y-axis shows the number of transcripts in each eggNOG category. A: “RNA processing and modification”, B: “Chromatin structure and dynamics”, C: “Energy production and conversion”, D: “Cell cycle control, cell division, chromosome partitioning”, E: “Amino acid transport and metabolism”, F: “Nucleotide transport and metabolism”, G: “Carbohydrate transport and metabolism”, H: “Coenzyme transport and metabolism”, I: “Lipid transport and metabolism”, J: “Translation, ribosomal structure and biogenesis”, K: “Transcription”, L: “Replication, recombination and repair”, O: “Post-translational modification, protein turnover, and chaperones”, P: “Inorganic ion transport and metabolism”, Q: “Secondary metabolites biosynthesis, transport, and catabolism”, R: “General function prediction only”, S: “Function unknown”, T: “Signal transduction mechanisms”, U: “Intracellular trafficking, secretion, and vesicular transport”, V: “Defense mechanisms”, W: “Extracellular structures”, Y: “Nuclear structure”, Z: “Cytoskeleton”. View this table: View inline View popup Download powerpoint Table 2. Summary of the functional annotation. Top 10 Interpro domains and GO terms enriched in each transcriptome analyzed. TopGO results were shown in different colors according to the three sub-ontologies: green “Biological Process”, blue “Molecular functions” and orange “Cellular component”. A total of 1.192 annotated transcripts common to all developmental stages studied were further examined to determine GO enrichment. The represented GO terms ( Figure 2 and Additional file 6) were categorized in two main groups: cellular components and biological processes. The main terms of cellular components are cell structures involved in protein synthesis, and the biological processes significantly over-represented terms are related to lipid, carbohydrate, nucleic acid and protein metabolism. As expected in the enrichment observed for each developmental time, these biological processes play key roles in the embryonic development of R. prolixus . The energy is supplied to the embryogenesis to proceed through the breakdown of biomolecules stored in the yolk 72 . These, in turn, drive converging biosynthetic pathways such as protein and nucleotide biosynthesis required to meet the needs of the developing embryo 70 . These results agree with the increment of lipid, protein and carbohydrate reported during embryonic stages of T. castaneum, Boophilus microplus and Aedes aegypti 73 - 75 ; and in fed females in R. prolixus and also other triatomines 76 - 79 . Download figure Open in new tab Figure 2. Bar graph of Gene Ontology (GO) enrichment analysis of the common transcripts across the six developmental stages. Upper: GO category “Biological process”, lower: “Cellular component”. X-axis: number of transcripts involved in the distinct GO terms. Y-axis: description of GO terms with the GO ID. 2. Gene identification for developmental processes In order to study genes involved in early embryogenesis and oogenesis, a list of 84 developmental genes (37 segmentation-related genes, 16 of the dorsal patterning pathway and 31 linked to oogenesis) related to these processes in D. melanogaster was used to identified orthologues sequences in the R. prolixus transcriptomes. ( Table 3A-C ). View this table: View inline View popup Table 3 Developmental genes identified in the R. prolixus transcriptomes. Gene ID: VectorBase code (the official gene number in the RproC3 genome assembly); Annotations: protein name we are assigning. For each annotation, presence/absence was assessed by sequence similarity search. Green boxes indicate presence of expression; white boxes indicate absence of expression. A: Segmentation genes, B: Dorso-ventral genes, C: oogenesis, orthologues piwi genes were annotated as Brito, et al. 48 . The approach identified 81 expressed genes conserved in R. prolixus . We cannot rule out that the absence of a transcript is due to an incomplete transcriptome coverage rather than evolutionary divergence. It is plausible to consider that some genes are expressed at very low levels or in a small subset of cells in the developmental stages analyzed and that enrichment analysis may be necessary for their identification. The genes gurken ( grk ), bicoid ( bcd ), oskar ( osk ), were not included in this searched because they have been reported absent in triatomines 14 . The annotation in our work showed limitations in the current version of the R. prolixus genome as more than one gene ID was identified to correspond to the same assembled transcription unit, which was verified by manual assembly. This implies that the genome needs extensive resequencing although a remarkable progress in the annotation of early genes was put forward by Coelho, et al. 54 correctly mapping thousands of transcripts even in non-annotated genomic regions. It would be expected that genes related to oogenesis are not expressed in eggs at times related to gastrulation and germ band formation (48 hPL ), as well as zygotic expression genes were not expressed in unfertilized eggs. However, a comparison of the different times of development revealed the identification of a stable number of genes of interest in all the times, not allowing to discriminate between presence or absence of gene expression according to a specific time of development. However, all genes showed a maternal expression. These results agree with the assumed role of the maternal genome as director of virtually all aspects of early animal development 15 , 80 - 83 . Specifically, in D. melanogaster and T. castaneum a high percentage of maternal transcripts were deposited during oogenesis, and so were detected (approximately 58 – 65 %) in the course of the first hours of embryo development, while zygotic transcription was not detected 15 , 25 , 27 , 83 . These maternal mRNAs have been reported participating in several biological functions, including the establishment of morphogen gradients 84 - 86 , segregation of cell-fate determinants 87 - 95 , and targeting of protein synthesis to specialized organelles or cellular domains 96 - 100 . To gain insight into this maternal contribution, ovary, unfertilized and 0 hPL transcriptomes were used to examine the expression of R. prolixus orthologues of reported maternal genes in D. melanogaster . The dataset was comprised of 10.277 sequences, of which the 54 % had a R. prolixus ortholog to the D. melanogaster genes ( Figure 3 and Additional file 7). 40,7 % were expressed during the three stages, oogenesis, unfertilized and 0 hPL eggs. 1,46 % of the maternal genes had R. prolixus orthologs which were expressed only during oogenesis but not in deposited eggs, 1,72 % had R. prolixus orthologs that were only maternally loaded into unfertilized eggs, and 2,03 % showed only expression in the first hours of fertilized eggs. At difference to the maternal genes reported in T. castaneum , here we identified 306 maternal orthologs more to D. melanogaster 15 . Out of 81 developmental genes investigated, eight were not reported to be maternal in both D. melanogaster and T. castaneum ( Table 4 ). These eight genes were found in unfertilized eggs of R. prolixus ( gooseberry: RPRC008887, odd paired : RPRC013047, odd skipped : RPRC011812, rhomboid: RPRC008474, sloppy paired: RPRC000987) or in both, vitellogenic ovary and unfertilized eggs transcriptomes ( windbeutel : RPRC007848 and proboscipedia: RPRC002128). We also identified a subset of transcript derived from the Hox cluster abdominal-A (RPRC000598), Abdominal-B (RPRC012974), Deformed (RPRC000437), proboscipedia and Ultrabithorax (RPRC000565) ( Table 3A , Table 4 ), suggesting differential maternal/zygotic regulation of the HOX genes in R. prolixus that deserves further investigation and that are out of the scope of this work 101 . Download figure Open in new tab Figure 3. Venn diagram depicting the maternal transcripts across developmental stages. Comparison among the expressed transcripts of the Vitellogenic ovary, Unfertilized and 0 hPL , eggs and reference maternal genes. View this table: View inline View popup Download powerpoint Table 4. Annotated developmental genes identified as maternal gene in R. prolixus . +: detected with maternal expression; -: not detected. Taken together, our data support the notion that maternal expression of developmental genes that are mostly zygotic in other species might be widespread in R. prolixus and the idea that the expression of maternal genes is maintained (either by stability or zygotic expression) during early developmental stages. The results agree with other gene specific studies that have reported maternal expression in the triatomine, during oogenesis and embryo development 46 , 48 , 50 , 69 . 3. Maternal expression validation To confirm the gene expression patterns revealed by the transcriptomic analyses, 11 genes (from the 81 previously annotated) involved in oogenesis and early embryo development were chosen for qRT-PCR. This analysis was performed with three independent biological replicates different from those used for RNA-seq analysis. The relative expression levels of these genes over time are shown in Figure 3 . All of the selected genes showed expression patterns consistent with the results derived from the transcriptome analysis, indicating that our approach, although not intended to be quantitative, was valid for the identification of expressed genes. All genes analyzed showed expression in unfertilized eggs, which confirm that the mRNAs were maternally provided. The different expression level between the vitellogenic ovary and the unfertilized egg could be explained by the nature of the samples, which implied ovarioles depleted of choriogenic egg chambers. Rp-sqd showed greater relative expression level than the other genes of interest at the different analyzed times. 4. Expression and functional analysis of Bicaudal D ( Rp-BicD ) during oogenesis and embryonic development To further extend the analysis, in situ hybridization was performed to evaluated if Rp-BicD transcript (Additional file 8) display stage- or tissue-specific expression patterns during oogenesis and/or early development of R. prolixus . The structure of an ovariole is shown in Figure 4A-C . The expression of Rp-BicD is cytoplasmic and the mRNA is detected in both, the germarium and follicular epithelium of previtellogenic and vitellogenic oocytes. In the germarium, Rp-BicD expression is enriched in the region close to the vitellarium (Z3; Figure 4D-E ). The expression of Rp-BicD is also detected in unfertilized eggs confined in the central region of the egg, with a diffusion of the signal towards the embryo surface ( Figure 4F ). In embryos up to the onset of blastoderm stage, Rp-BicD mRNA could not be detected (data not shown), in agreement with our qRT-PCR data, although we cannot rule out transcripts below the detection limit of the in situ hybridization technique. It also shows similarity with D. melanogaster , the only insect species in which BicD was studied - BicD expression drops just prior to cellular blastoderm formation 102 . This dynamic of expression is similar to that be observed in other maternally supplied RNAs, in which the maternal transcripts persist during the very early embryonic cleavage stages but are degraded just prior to the reach the stage of blastoderm cellularization 9 , 103 . Download figure Open in new tab Figure 4. Real-time quantitative PCR of expression of candidate genes in the different stages. X axis: developmental times analyzed. Y axis: expression relative to the reference gene. Values are expressed as mean ± SEM of 3 independent experiments. Rp-arm : Armadillo; Rp-BicD: Bicaudal D; Rp-cact: Cactus; Rp-capu: Cappuccino; Rp-dl: Dorsal; Rp-egh: Egghead; Rp-egl: Egalitarian; Rp-exu: Exuperantia; Rp-pum: Pumilio; Rp-sqd: Squid; Rp-stau: Staufen . Graphs were performed using GraphPad Prism 7. *<0,1; **<0,05. Download figure Open in new tab Figure 5. Silencing of Rp-BicD produces anembryonic eggs. A Schematic of the ovariole showing the germarium host mitotically active cells (i.e. nurse cells) in zone 1 (Z1), zone 2 (Z2) and zone 3 (Z3), and previtellogenic (Pv), vitellogenic (V) and choriogenic (Ch) oocytes. Each oocyte becomes encapsulated by follicle cells (Fc), and remains connected to the germarium through the trophic cords (Tch). Germinal vesicle (Gv). B Structure of the ovariole showing the different stages that characterized oogenesis: previtellogenic, vitellogenic and choriogenic stage. C Ovariole of a control female showing the nuclei distribution by DAPI staining and the actin filaments by phalloidin staining 50 , 116 . D Detection of Rp-BicD transcript in the ovarioles by in situ hybridization. The arrowhead indicates the expression in Z3 of the germarium, the arrow indicates the expression in the vitellogenic oocyte. Scale Bar: 100 µm. E Different focal plane of the vitellogenic oocyte showed in D . Note the expression of Rp-BicD in the follicular cells. F Detection of Rp-BicD transcript in unfertilized eggs by in situ hybridization. The arrowhead indicated the pattern of expression. G Egg from control females showing nuclei distribution by DAPI staining. Scale bar: 200 µm. A: Anterior pole of the egg. P: Posterior pole of the egg. H Egg from silenced (RNAi BicD ) females showing nuclei distribution by DAPI staining. Scale bar: 100 µm. A: Anterior pole of the egg. P: Posterior pole of the egg. The development stages corresponding to both, control (G) and silenced (H), represent a germ band extension. I Ovariole of a control females showing the nuclei distribution by DAPI staining. Scale bar: 100 µm. J Ovariole of a silenced (RNAi BicD ) females showing the nuclei distribution by DAPI staining. Scale bar: 100 µm. In order to determine the function of Rp-BicD we performed parental RNAi. We injected non-fed virgin females (n=20) with different concentrations (0.5 to 2.5 µg/female) of dsRNA BicD . As control, we used dsRNA dsRNA β- lac (see methods). After feeding and mating, dsRNA BicD and dsRNA β- lac injected females were evaluated for survival, egg deposition, embryo lethality, and ovary and embryo phenotype (Additional file 9). In our hands, 45% females injected with dsRNA BicD survived until the 5th day after injection and did not reach the oviposition phase. From the survivors (n=9), we investigated the number of egg laid and incubated them to develop for the expected time of embryogenesis to finish (>14 days) to determine lethality. Compared to the control ones, the silenced females laid less eggs (n=43), and of these only 9%, corresponding to the cohort from females injected with the lowest concentration of dsRNA BicD , hatched to first-instar larvae. The dissection of the not hatching eggs did not show any distinguishable embryonic structure, suggesting that BicD might act at very early stages of embryogenesis ( Figure 4G-H ). The morphology of the ovary analyzed under the dissection microscope and the cellular pattern, as judged by nuclear staining, did not show conspicuous differences. Therefore, the silencing of Rp - BicD did not alter the normal morphology of the ovaries ( Figure 4I-J ). The results are similar to the BicD phenotypes of D. melanogaster . In heterozygous BicD females the progression into oogenesis is not affected, but causes sterility or inviable embryos that consist only of a mirror-image duplication of 2–4 posterior segments 104 , 105 . Homozygous BicD females show ovaries with no diploid germ cell nuclei visible in older egg chambers and no oocyte develops. Also the lack of BicD has effect in the zygotic viability, in which null flies die as pupae or young adults 106 . In D. melanogaster, BicD and Egalitarian ( Egl ) are part of a complex that transports and localize mRNAs in the early oocyte and in the early embryos, crucial for specifying their respective anteroposterior and dorsoventral axes 104 , 107 - 109 . Egl and BicD homologues have been identified in Caenorhabditis elegans and mammals, and proposed as a part of an evolutionarily conserved cytoskeletal system for transporting transcripts and others 110 , 111 . Our qRT-PCR results show similar expression dynamics of Rp-BicD and Rp-egl , suggesting that these co-expressed trajectories might be associated to establishment of BicD / Egl localization machinery in R. prolixus . Although genes involved in this polarization are well known in D. melanogaster , but less so in other insects, include R. prolixu s, our results provide evidence that Rp-BicD might be an upstream gene in a cascade that contributes to set the axis and the embryonic patterning. Conclusions Our data provide a framework for a better understanding of R. prolixus embryogenesis, and complement the remarkable recent work from Coelho, et al. 54 that contributed to a better annotation of the R. prolixus genome and provided a deep insight into the maternal genes complement of this hemimetabolous insect. In addition, several recent studies showed the expression and phenotype of different genes with maternal contribution. Based on the available data, we propose at least three hierarchies of maternal genes in R. prolixus : Genes affecting the early oogenesis , that results in germ cell survival and/or atresic ovaries: Rp-ATG-8 112 ; Rp-cactus 46 ; Rp-Piwi-2, Rp-Piwi-3, Rp-Argonauta-3 48 ; Rp-Me31B (Pascual and Rivera Pomar, unpublished data) Gene affecting late oogenesis , that results in eggs with less o incomplete yolk load or impaired choriogenesis: Rp-Bicaudal C 50 ; ULK/Rp-ATG-1 113 ; Rp-ATG-3 114 ; Rp-ATG-6 115 . Genes affecting embryonic patterning , that results in embryo lethality: Rp-giant 69 ; Rp-dorsal 46 ; Rp-Bicaudal D (this work), Genome analysis opened a broad and exciting path to study oogenesis and to decipher the genetic pathways of oogenesis in this emerging model organism, adding to the already well known cellular processes established by the pioneer work of Erwin Huebner. These studies will enrich the developmental evolutionary studies in the context of insect comparative genomics, and, in the case of R. prolixus , could contribute to devise new strategies to control Chagas disease insects. Supporting information (Excel files available under request) Additional file 1: Maternal D. melanogaster genes, reported as FlyBase symbol. Additional file 2: Primers used by RT-qPCR assays and Rp-BicD experiments. Additional file 3: COG functional category annotation using eggNOG-mapper. Additional file 4: InterproScan results. Additional file 5: GO-term enrichment. Additional file 6: Go-term enrichment form annotated transcripts common to all six developmental stages. Additional file 7: BLASTX from maternal genes. Additional file 8: The predicted sequences were aligned with orthologues from other species with Clustal Ω 117 . Alignment of the protein sequence of Rp-BicD with orthologues species, extracted from NCBI sequence database D. melanogaster (NP_001260531.1), Tribolium castaneum (EFA07458.1), Culex quinquefasciatus (EDS37197.1), Anopheles darlingi (ETN62092.1), Daphnia magna JAN71328.1, Homo sapiens (AAB94805.1), Mus musculus (NP_001034268.1), Caenorhabditis elegans (CDK13419.1). The amino acid conservation is visualized with black blocks, the amino acid group level conservation with gray blocks. Additional file 9: Summary of Parental RNAi experiment. Acknowledgements The authors thank all members of Rivera-Pomar lab, in particular to Elías Gazza and Agustín Rolandelli for fruitful discussions, and to Viviana Decker (Instituto Nacional de Tecnología Agropecuaria, Pergamino) for kindly share the qRT-PCR facility. R.R-P is professor at UNNOBA and investigator of CONICET. A.P is postdoctoral fellow of CONICET and UNNOBA. 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