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Dynamics of maternal gene expression in Rhodnius prolixus

The study of developmental processes in Rhodnius prolixus has recently advanced with the sequencing of the genome. In this work, we analyze the maternal gene expression driving oogenesis and early embryogenesis in R. prolixus. We examined 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 h post egg laying). We identified 81 putative maternal and ovary-related genes and validated their expression by qRT-PCR. We validate the function of the ortholog gene Bicaudal-D (Rp-BicD) by in situ hybridization and parental RNAi. Consistent with a role in oogenesis and early development of R. prolixus, we show that lack of Rp-BicD does not significantly affect oogenesis but impairs the formation of the blastoderm. Based on our findings, we propose three times of action for maternal genes during oogenesis and embryogenesis in R. prolixus.


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][11][12][13][14][15][16][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  However, the complete repertory of genes involved in oogenesis and early embryogenesis has been reported in detail only in D. melanogaster [25][26][27][28][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][35][36][37] . The embryonic development of R.
prolixus has been described from fertilization to hatching 7 , and the process of oogenesis studied in detail [38][39][40][41][42][43][44] . The genome was recently sequenced 45 and since then, R. prolixus is an emerging model for developmental biology [46][47][48][49][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.

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

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.

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

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.
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  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][74][75] ; and in fed females in R.

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).
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  (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 .
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 .

Maternal expression validation
To 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 .
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. melanogaster, but less so in other insects, include R. prolixus, 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.