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Abstract Background The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1) and USP (NR2B4), was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD) of USP during evolution of Mecopterida. Results We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP) underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera) diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation. Conclusions In order to explain the episodic mode of evolution of USP, we propose a model in which the molecular adaptation of this protein is seen as a process of resilience for the maintenance of the ecdysone receptor functionality.

Abstract Background Previous work showed that miRNAs play key roles in the regulation of metamorphosis in the hemimetabolan species Blattella germanica. To gain insight about which miRNAs might be important, we have constructed two miRNA libraries, one of the penultimate, pre-metamorphic nymphal instar (N5) and the other of the last, metamorphic nymphal instar (N6). Results High throughput sequencing gave 61 canonical miRNAs present in the N5 and N6 libraries, although at different proportions in each. Comparison of both libraries led to the identification of three and 37 miRNAs significantly more expressed in N5 and N6 respectively. Twelve of these 40 miRNAs were then investigated further by qRT-PCR and results indicated that miR-252-3p was well expressed in N5 but not in N6, whereas let-7-5p, miR-100-5p and miR-125-5p showed the reverse pattern. 20-Hydroxyecdysone (20E) tended to stimulate miRNA expression, whereas juvenile hormone (JH) inhibited the 20E stimulatory effect. Expression of let-7, miR-100 and miR-125 was increased by 20E, which has also been observed in D. melanogaster. The only miRNA that was inhibited by 20E was miR-252-3p. The involvement of let-7, miR-100 and miR-125 in metamorphosis has been demonstrated in other insects. Depletion of miR-252-3p caused growth and developmental delays, which suggests that this miRNA is involved in regulating these processes prior to metamorphosis. Conclusions The comparative analysis of miRNA libraries from pre-metamorphic (N5) and metamorphic stages (N6) of B. germanica proved to be a useful tool to identify miRNAs with roles in hemimetabolan metamorphosis. Three miRNAs emerged as important factors in the metamorphic stage (N6): let-7-5p, miR-100-5p and miR-125-5p, whereas miR-252-3p appears to be important in the pre-metamorphic stage (N5).

Abstract Background Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model. Results We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis. Conclusion The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

Hypertrehalosemic hormone (HTH) is a peptide hormone that belongs to the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family, which exerts pleiotropic actions related to catabolic reaction and stress response. AKH peptides have been demonstrated to participate in stress response including oxidative stress in several insects. In order to study the signaling pathway of HTH involved in anti-oxidative stress, we have characterized a HIH receptor cDNA in Blattella germanica (Blage-HTHR) in structural and in functional terms using RNA interference (RNAi). Blage-HTHR is expressed in various female adult tissues (brain-CC-CA, ventral nerve cord, midgut, fat body, oviduct), but maximal expression is observed in the fat body. RNAi-mediated knockdown of Blage-HTHR expression results in a significantly lower level of hemolymph trehalose, even though HTH is exogenously administered. Paraquat elicits lethal oxidative stress in B. germanica, and co-injection of paraquat and HTH reduces this detrimental effect and extends the median survival time. Interestingly, the "rescue" effect of HTH on mortality caused by paraquat is diminished in specimens with depleted expression of Blage-HTH and Blage-HTHR. Finally, lipid peroxidation in the hemolymph increases 4 h after paraquat treatment, in comparison with control specimens or with HTH-treated specimens. However, lipid peroxidation induced by paraquat was not "rescued" by HTH in Blage-HTH and Blage-HTHR knockdown specimens. Our results demonstrate that HTH acts as a stress hormone mediating anti-oxidative protection in B. germanica, and that its receptor, Blage-HTHR, is essential for this action.

Aquaporins (AQPs) are membrane proteins that form water channels, allowing rapid movement of water across cell membranes. AQPs have been reported in species of all life kingdoms and in almost all tissues, but little is known about them in insects. Our purpose was to explore the occurrence of AQPs in the ovary of the phylogenetically basal insect Blattella germanica (L.) and to study their possible role in fluid homeostasis during oogenesis. We isolated an ovarian AQP from B. germanica (BgAQP) that has a deduced amino acid sequence showing six potential transmembrane domains, two NPA motifs and an ar/R constriction region, which are typical features of the AQP family. Phylogenetic analyses indicated that BgAQP belongs to the PRIP group of insect AQPs, previously suggested to be water specific. However, ectopic expression of BgAQP in Xenopus laevis oocytes demonstrated that this AQP transports water and modest amounts of urea, but not glycerol, which suggests that the PRIP group of insect AQPs may have heterogeneous solute preferences. BgAQP was shown to be highly expressed in the ovary, followed by the fat body and muscle tissues, but water stress did not significantly modify the ovarian expression levels. RNA interference (RNAi) reduced BgAQP mRNA levels in the ovary but the oocytes developed normally. The absence of an apparent ovarian phenotype after BgAQP RNAi suggests that other functionally redundant AQPs that were not silenced in our experiments might exist in the ovary of B. germanica.

In Drosophila melanogaster, male courtship behaviour is regulated by the fruitless gene. In D. melanogaster, fruitless encodes a set of putative transcription factors that are sex-specifically spliced. Male-specific variants are necessary and sufficient to elicit male courtship behaviour. Fruitless sequences have been reported in other insect species, but there are no data available on their functional role. In the present work, we cloned and sequenced fruitless in males of the German cockroach, Blattella germanica, and we studied its expression in male brain and testes. B. germanica fruitless encodes a 350-amino acid protein with BTB and Zinc finger domains typical of fruitless sequences. Upon RNAi-mediated knockdown of fruitless in B. germanica, males no longer exhibit courtship behaviour, thus implying that fruitless is necessary for male sexual behaviour in our cockroach model. This suggests that the role of fruitless as master regulator of male sexual behaviour has been conserved along insect evolution, at least from cockroaches to flies.

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Insect eggshells must combine protection for the yolk and embryo with provisions for respiration and for the entry of sperm, which are ensured by aeropyles and micropyles, respectively. Insects which oviposit the eggs in an egg-case have a double problem of respiration as gas exchange then involves two barriers. An example of this situation is found in the cockroach Blattella germanica, where the aeropyle and the micropyle are combined in a complex structure called the sponge-like body. The sponge-like body has been well described morphologically, but nothing is known about how it is built up., Methodology/principal Findings: In a library designed to find genes expressed during late chorion formation in B. germanica, we isolated the novel sequence Bg30009 (now called Brownie), which was outstanding due to its high copy number. In the present work, we show that Brownie is expressed in the follicle cells localized in the anterior pole of the oocyte in late choriogenesis. RNA interference (RNAi) of Brownie impaired correct formation of the sponge-like body and, as a result, the egg-case was also ill-formed and the eggs were not viable., Conclusions/significance: Results indicate that the novel gene Brownie plays a pivotal role in building up the sponge-like body. Brownie is the first reported gene involved in the construction of complex eggshell respiratory structures.

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Ecdysteroid hormones regulate key developmental processes throughout the life cycle of insects. 20-Hydroxyecdysone (20E) acts upon binding to a heterodimeric receptor formed by the nuclear receptors EcR and USP. The receptor, once 20E bounds to it, elicits cascades of gene expression that mediate and amplify the hormonal signal. The molecular characterization of the 20E-mediated hierarchy of transcription factors has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in more basal hemimetabolous species. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we have cloned and characterized five isoforms of B. germanica E75, a member of the nuclear receptor family participating in the 20E-triggered genetic hierarchy. The five isoforms present characteristic expression patterns during embryo and nymphal development, and experiments in vitro with fat body tissue have shown that the five isoforms display specific 20E responsiveness. RNAi experiments in vivo during the penultimate and last nymphal instars of B. germanica revealed that BgE75 is required for successfully complete nymphal-nymphal and nymphal-adult transitions. Detailed analysis of knockdown specimens during the last nymphal instar showed that BgE75 is required for the rise of circulating ecdysteroids that occurs towards the end of the instar. The main cause of ecdysteroid deficiency in BgE75 knockdowns is the premature stage-specific degeneration of the prothoracic gland. As a consequence, BgE75 knockdown nymphs do not molt, live for up to 90 days and start the adult developmental program properly, in spite of remaining as nymphs from a morphological point of view. Finally, RNAi of specific isoforms during the last nymphal instar of B. germanica has showed that they are functionally redundant. Furthermore, it also revealed the occurrence of a complex regulatory relationship among BgE75 isoforms, which is responsible of their sequential expression.

Background: Lipophorin receptors (LpRs) have been described in a number of insects, but functional studies have been reported only in locusts and mosquitoes. The aim of the present work was to characterize the LpR of the cockroach Blattella germanica, not only molecularly but also functionally using RNAi techniques, and to place LpRs in a phylogenetical context among lipoprotein receptors., Results: We cloned a putative LpR from B. germanica (BgLpR) using RT-PCR methods. Two isoforms of BgLpR that differ from each other by an insertion/deletion of 24 amino acids were obtained from the fat body and the ovary. A phylogenetical analysis of lipoprotein receptors showed that BgLpR grouped with other sequences annotated as LpR in a cluster placed as a sister group of vertebrate low density lipoprotein receptors (LDLR) + lipoprotein receptor-related proteins 8 (LPR8) + vitellogenin receptors (VgR) + very low density lipoprotein receptors (VLDLR). The two BgLpR isoforms are expressed in different adult female tissues (fat body, ovary, brain, midgut, muscle) and in embryos. In ovaries and fat body, the two isoforms are similarly expressed during the first gonadotrophic cycle. mRNA levels in the fat body increase in parallel to vitellogenesis, whereas they decrease in the ovaries. BgLpR protein levels increase in parallel to vitellogenesis in both organs. Treatment with juvenile hormone increases BgLpR protein. RNAi experiments show that females with lower BgLpR expression have less lipophorin in the growing oocytes with respect to controls., Conclusion: The two isoforms of BgLpR are structurally similar to other LpRs. Phylogenetical analyses show that LpRs and the group formed by vertebrate LDLR + LPR8 + VgR + VLDLR, diverged from a common ancestor and diversified in parallel. The different expression patterns in the fat body and the ovary, comparing mRNA and protein, indicate that the corresponding mechanisms regulating BgLpR expression are different. Experiments with JH III suggest that such a hormone regulates the expression of BgLpR posttranscriptionally. RNAi experiments indicate that BgLpR is a functional lipophorin receptor.

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we show that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, we reduced its expression using systemic RNAi in vivo, and we obtained knockdown specimens. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis.

During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)-mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre-adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Insects do not synthesize cholesterol; the 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) produced by HMG-CoA synthase is transformed to mevalonate by HMG-CoA reductase for the production of non-sterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG-CoA synthase, we have cloned a 1658 bp cDNA that encompasses the entire transcription unit of the HMG-CoA synthase gene from the cockroach Blattella germanica. This cDNA clone was isolated using as a probe a partial cDNA of B. germanica HMG-CoA synthase, amplified using the polymerase chain reaction. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 453 amino acids (M(r) 50338) that is similar to vertebrate HMG-CoA synthase (74-76% conserved residues). The B. germanica cDNA has been expressed as a fusion protein in Escherichia coli and exhibits HMG-CoA synthase activity. The HMG-CoA synthase transcript was differentially expressed throughout B. germanica development. Analysis of RNA samples from different adult female tissues shows high HMG-CoA synthase mRNA levels in the ovary and lower levels in brain and muscle.