E93 is indispensable for reproduction in ametabolous and hemimetabolous insects.

Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. Although E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discover that, in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNA is broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we reveal that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis that is necessary to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects, and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.

Vitellogenin and its upstream gene TOR play essential roles in the reproduction of Dermanyssus gallinae.

In Dermanyssus gallinae, a hematophagous mite, the initiation of vitellogenesis induced by blood feeding is essential for its reproduction. However, the precise gene structures and physiological functions of Vg in D. gallinae and its upstream gene, Target of Rapamycin (TOR), have not been fully understood. This study revealed the presence of four homologous genes within D. gallinae, named Dg-Vg1, Dg-Vg1-like, Dg-Vg2, and Dg-Vg2-like, especially, Dg-Vg2-like was firstly identified in the mites. The expression levels of all these Vg genes were significantly higher in adult females than other stages. Following blood feeding, the expression levels of these genes increased significantly, followed by a subsequent decrease, aligning with egg production. Silencing Dg-Vgs by RNA interference (RNAi) led to decreased fecundity and egg hatching rates, as well as abnormal embryonic development, suggesting a vital role for Dg-Vgs in both egg formation and embryonic development. Furthermore, the knockdown of Dg-TOR significantly reduced the expression of Dg-Vgs and negatively impacted the reproductive capabilities of PRMs, indicating that TOR influences PRM reproduction by regulating the expression of Dg-Vgs. In summary, these findings demonstrated the crucial roles of Dg-Vgs and Dg-TOR in PRM reproduction, highlighting their potential as targets for pest control.

The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis.

The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated "posterior dorsal neuron 1" (DN1p), which are among the ∼150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). Here, we report that six pairs of AstC-DN1p send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. We show evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms.

Genome-Wide Identification, Expression and Response to Estrogen of Vitellogenin Gene Family in Sichuan Bream (Sinibrama taeniatus).

To enhance our understanding of teleost reproductive physiology, we identified six Sichuan bream (Sinibrama taeniatus) vitellogenin genes (vtg1-6) and characterized their sequence structures. We categorized them into type Ⅰ (vtg1,4,5 and 6), type Ⅱ (vtg2) and type Ⅲ (vtg3) based on differences in their subdomain structure. The promoter sequence of vtgs has multiple estrogen response elements, and their abundance appears to correlate with the responsiveness of vtg gene expression to estrogen. Gene expression analyses revealed that the vitellogenesis of Sichuan bream involves both heterosynthesis and autosynthesis pathways, with the dominant pathway originating from the liver. The drug treatment experiments revealed that 17ß-estradiol (E2) tightly regulated the level of vtg mRNA in the liver. Feeding fish with a diet containing 100 µg/g E2 for three weeks significantly induced vtg gene expression and ovarian development, leading to an earlier onset of vitellogenesis. Additionally, it was observed that the initiation of vtg transcription required E2 binding to its receptor, a process primarily mediated by estrogen receptor alpha in Sichuan bream. The findings of this study provide novel insights into the molecular information of the vitellogenin gene family in teleosts, thereby contributing to the regulation of gonadal development in farmed fish.

Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis.

Vitellogenin (Vg) is a prerequisite for egg production and embryonic development after ovipositioning in oviparous animals. In many insects, juvenile hormone (JH) promotes fat body cell polyploidization for the massive Vg synthesis required for the maturation of multiple oocytes, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH induces the dephosphorylation of Forkhead box O transcription factor (FoxO) through a signaling cascade including leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2A (PP2A). JH promotes PP2A activity via LCMT1-mediated methylation, consequently triggering FoxO dephosphorylation. Dephosphorylated FoxO binds to the upstream region of two endocycle-related genes, cell-division-cycle 2 (Cdc2) and origin-recognition-complex subunit 5 (Orc5), and activates their transcription. Depletion of FoxO, Cdc2 or Orc5 results in blocked polyploidization of fat body cells, accompanied by markedly reduced Vg expression, impaired oocyte maturation and arrested ovarian development. The results suggest that JH acts via LCMT1-PP2A-FoxO to regulate Cdc2 and Orc5 expression, and to enhance ploidy of fat body cells in preparation for the large-scale Vg synthesis required for synchronous maturation of multiple eggs.

The miRNAs let-7b and miR-141 Coordinately Regulate Vitellogenesis by Modulating Methyl Farnesoate Degradation in the Swimming Crab Portunus trituberculatus.

Methyl farnesoate (MF), a crucial sesquiterpenoid hormone, plays a pivotal role in the reproduction of female crustaceans, particularly in the vitellogenesis process. Despite extensive research on its functions, the molecular mechanisms that regulate MF levels during the vitellogenic phase remain largely elusive. This study investigates the roles of microRNAs (miRNAs), significant post-transcriptional regulators of gene expression, in controlling MF levels in the swimming crab Portunus trituberculatus. Through bioinformatic analysis, four miRNAs were identified as potential regulators targeting two genes encoding Carboxylesterases (CXEs), which are key enzymes in MF degradation. Dual luciferase reporter assays revealed that let-7b and miR-141 suppress CXE1 and CXE2 expression by directly binding to their 3' UTRs. In vivo overexpression of let-7b and miR-141 significantly diminished CXE1 and CXE2 levels, consequently elevating hemolymph MF and enhancing vitellogenin expression. Spatiotemporal expression profile analysis showed that these two miRNAs and their targets exhibited generally opposite patterns during ovarian development. These findings demonstrate that let-7b and miR-141 collaboratively modulate MF levels by targeting CXEs, thus influencing vitellogenesis in P. trituberculatus. Additionally, we found that the expression of let-7b and miR-141 were suppressed by MF, constituting a regulatory loop for the regulation of MF levels. The findings contribute novel insights into miRNA-mediated ovarian development regulation in crustaceans and offer valuable information for developing innovative reproduction manipulation techniques for P. trituberculatus.

The transition from vitellogenesis to choriogenesis triggers the downregulation of the UPR sensors IRE1 and PERK and alterations in the ER architecture in the follicle cells of the vector Rhodnius prolixus.

In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development.

CEH-60/PBX regulates vitellogenesis and cuticle permeability through intestinal interaction with UNC-62/MEIS in Caenorhabditis elegans.

The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis.

Potential receptors in Fenneropenaeus merguiensis ovary and role of saxophone, the bone morphogenetic protein receptor, in ovarian development.

Receptors, which play an initial role in signaling pathways in several physiological processes, including reproduction, are among the several molecular factors that control ovarian development in organisms. This study aimed to identify and study receptors potentially involved in controlling the reproductive process of female banana shrimp, Fenneropenaeus merguiensis. Ovarian transcriptomes derived from 4 developmental stages were generated by RNA sequencing. A total of 53,763 transcripts were obtained from the de novo assembled transcriptome, and 663 genes were identified as receptors. Among them, 185 receptors were differentially expressed during ovarian development. Fifteen of these differentially expressed receptors showed distinct expression patterns that were validated by RT-qPCR. Bone morphogenetic protein receptors (BMPR) and their signaling genes were investigated for their roles in shrimp vitellogenesis. The expressions of F. merguiensis saxophone (FmSax), a BMP type I receptor, and BMP type II receptor (FmBMPRII) as well as FmMad, FmMed, and FmSMAD3 were significantly altered during ovarian development. RNA interference was used to investigate the role of FmSax in vitellogenesis. The result indicated that the expression of vitellogenin (Vg) was significantly reduced in both ovary and hepatopancreas of FmSax-knockdown shrimp compared to control shrimp. Furthermore, in FmSax-silencing shrimp, FmBMPRII, FmMad, and FmMed expressions were decreased as well as Vg expression. These findings suggest that FmSax positively regulates Vg synthesis via the BMP signaling pathway.

RNAi silencing of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene inhibits vitellogenesis in Chinese mitten crab Eriocheir sinensis.

The sesquiterpenoid methyl farnesoate (MF), a de-epoxide form of insect juvenile hormone III (JH III), plays an essential role in regulating many crucial physiological processes in crustaceans including vitellogenesis and reproduction. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of JH III and MF. In the present study, a full-length cDNA encoding HMGR (EsHMGR) in Eriocheir sinensis was isolated and characterised. Sequence analysis of EsHMGR revealed that it belongs to Class I HMGR family proteins with HMG-CoA-binding and NADPH-binding domains, both important for HMGR activity. In addition to its ubiquitous tissue expression, expression of EsHMGR was highly specific to the ovary, the main site of Vg synthesis. During ovarian development, EsHMGR expression in ovary displayed a stage-specific pattern, and was correlated with expression of vitellogenin (EsVg) in hepatopancreas, which suggests that EsHMGR possibly involved in vitellogenesis. To further investigate the functional role of EsHMGR in vitellogenin biosynthesis in E. sinensis, RNA interference-mediated gene silencing was carried out both in vitro and in vivo. Quantitative PCR results showed that injection of EsHMGR double-stranded RNA (dsRNA) led to a significant decrease in EsVg expression levels in ovary and hepatopancreas both in vitro and in vivo. Taken together, the results suggest that EsHMGR is involved in vitellogenin biosynthesis in female E. sinensis, which may provide a new resource for HMGR enzymes participating in reproduction in crustaceans.

Molecular Characterization of Vitellogenin and Its Receptor in Spodoptera frugiperda (J. E. Smith, 1797), and Their Function in Reproduction of Female.

The fall armyworm Spodoptera frugiperda is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of S. frugiperda. Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in insect reproduction. As the precursor of vitellin (Vn), Vg provides essential nutrition for embryonic development, and VgR mediates the uptake of Vg by oocytes. In this context, we cloned and characterized these two genes of S. frugiperda (SfVg and SfVgR) and evaluated their expression profiles in different developmental stages and tissues. The RNA interference experiment was used to investigate their function in vitellogenesis. The ORF values of SfVg and SfVgR were 5250 and 5445 bp, encoding 1749 and 1815 amino acid residues, respectively. The qRT-PCR results revealed that both SfVg and SfVgR were highly expressed in female adults; SfVg was specifically expressed in the fat body, whereas SfVgR was highly expressed in the ovary. In addition, the depletion of either SfVg or SfVgR hindered oocyte maturation and ovarian development, leading to a significant decrease in fecundity. The present study reveals the importance of SfVg and SfVgR in the vitellogenesis of S. frugiperda, laying a theoretical foundation for the development of pollution-free pest control strategies with SfVg and SfVgR as new targets.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Molecular cloning, characterisation and expression analysis of the vitellogenin genes vtgAo1 and vtgC during ovarian development in Chinese hook snout carp Opsariichthys bidens.

Vitellogenesis is essential for oocyte maturation. Vitellogenin (Vtg), a yolk precursor protein, plays an important role in oogenesis and vitellogenesis. Chinese hook snout carp Opsariichthys bidens is an economically important freshwater fish in China whose reproductive and developmental biology are not well understood. In this study, we undertook histological analysis to examine ovary development and oogenesis in O. bidens. The ovaries were divided into Stages II-V and oocytes were divided into perinuclear oocytes, cortical alveoli oocytes, vitellogenic oocytes and mature oocytes. Full-length cDNA sequences were cloned of two vtg genes from the liver of O. bidens, namely Ob-vtgAo1 and Ob-vtgC. Ob-vtgAo1 and Ob-vtgC cDNA are made up of 4136 and 4392 bases respectively and encode proteins containing 1335 and 1250 amino acids respectively. Ob-vtgAo1 contains three yolk protein domains: lipovitellin heavy chain (LvH), phosvitin (Pv) and lipovitellin light chain (LvL), whereas Ob-VtgC contains LvH and LvL, which are incomplete Vtgs. Ob-vtgAo1 and Ob-vtgC mRNA expression was significantly higher in the liver of O. bidens than in all other tissues. In oocytes of Stage II-III ovaries, yolk granules are almost absent and ovarian and hepatic Ob-vtgAo1 and Ob-vtgC expression is low. At Stage IV, the oocyte is filled with yolk granules and ovarian and hepatic Ob-vtgAo1 and Ob-vtgC expression is significantly increased. Collectively, these findings help us better understand vitellogenesis in O. bidens.

Anti-masculinization induced by aromatase inhibitors in adult female zebrafish.

BACKGROUND: Early sex differentiation genes of zebrafish remain an unsolved mystery due to the difficulty to distinguish the sex of juvenile zebrafish. However, aromatase inhibitors (AIs) could direct juvenile zebrafish sex differentiation to male and even induce ovary-to-testis reversal in adult zebrafish. RESULTS: In order to determine the transcriptomic changes of sex differentiation in juvenile zebrafish and early sex-reversal in adult zebrafish, we sequenced the transcriptomes of juvenile and adult zebrafish treated with AI exemestane (EM) for 32 days, when juvenile zebrafish sex differentiation finished. EM treatment in females up-regulated the expression of genes involved in estrogen metabolic process, female gamete generation and oogenesis, including gsdf, macf1a and paqr5a, while down-regulated the expression of vitellogenin (vtg) genes, including vtg6, vtg2, vtg4, and vtg7 due to the lower level of Estradiol (E2). Furthermore, EM-juveniles showed up-regulation in genes related to cell death and apoptosis, such as bcl2l16 and anax1c, while the control-juveniles exhibited up-regulation of genes involved in positive regulation of reproductive process and oocyte differentiation such as zar1 and zpcx. Moreover, EM-females showed higher enrichment than control females in genes involved in VEGF signaling pathway, glycosaminoglycan degradation, hedgehog signaling pathway, GnRH signaling pathway and steroid hormone biosynthesis. CONCLUSIONS: Our study shows anti-masculinization in EM-treated adult females but not in EM-treated juveniles. This may be responsible for the lower sex plasticity in adults than juveniles.

The unfolded protein response modulates the autophagy-mediated egg production in the mosquito Aedes aegypti.

Mosquitoes must feed on vertebrate blood for egg development. As a consequence, some mosquito species are vectors for pathogens that cause devastating diseases in humans. Hence, understanding the mechanisms that control egg developmental cycles is important for developing novel approaches for the control of mosquito-borne diseases. The unfolded protein response (UPR) is a cellular stress response related to endoplasmic reticulum (ER) stress. The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the ER. Massive proteins have been shown to be produced during egg development, and it is obvious that unfolded or misfolded proteins may arise during vitellogenesis. It has been shown that autophagy in the mosquito fat body plays a central role in the progression of gonadotrophic cycles in the mosquito Aedes aegypti. However, the molecular mechanisms underlying the induction of UPR and the correlation between UPR and autophagy remain unclear. Here, we demonstrate that autophagy is activated during vitellogenesis and that the activation of autophagy is correlated with the UPR. We also show that the expressions of UPR and autophagy can be induced in an in vitro fat body culture system through an amino acid treatment. In addition, the expressions of UPR, autophagy-specific markers and vitellogenin were also induced during dithiothreitol treatment. Interestingly, the silencing of UPR-related genes significantly reduced the expression of autophagy-specific markers and inhibited mosquito fecundity. Taken together, we conclude that autophagy-mediated egg production in the mosquito A. aegypti is regulated by UPR.

RNA interference-mediated knockdown of the transcription factor Krüppel homologue 1 suppresses vitellogenesis in Chilo suppressalis.

Vitellogenesis in holometabolous insects involves the production and secretion of vitellogenin (Vg) and other yolk protein precursors in developing oocyte by the fat body, all of which is predominantly orchestrated by juvenile hormone (JH). Krüppel homologue 1 (Kr-h1) is a zinc finger transcription factor that has been demonstrated to be a JH-early inducible gene and to contribute to reproduction. However, the exact molecular function of Kr-h1 in insect reproduction is poorly understood. In the current study, we used the notorious pest Chilo suppressalis as a model system to investigate the role of Kr-h1 in female reproduction. Cloning and sequencing C. suppressalis Kr-h1 revealed that it shares high identity with its homologues from other lepidopteran insects. Moreover, RNA interference-mediated knockdown of CsKr-h1 substantially reduced the transcription of Vg in the fat body, dramatically decreased yolk protein deposition and also impaired oocyte maturation and ovarian development, indicating that Kr-h1 is indispensable for normal vitellogenesis in C. suppressalis. Based on these results, we conclude that Kr-h1 is crucial to reproduction in insects and that targeting this gene could potentially be a new way to suppress rice pests.

Role of neuroparsin 1 in vitellogenesis in the mud crab, Scylla paramamosain.

Neuroparsin (NP) is an important neuropeptide in invertebrates. It is well-known that NP displays multiple biological activities, including antidiuretic and inhibition of vitellogenesis in insects. However, the information about its effect in crustaceans is scarce. In this study, the sequence of Sp-NP1 was selected from the transcriptome database from the mud crab, Scylla paramamosain. Sequence analyses indicate that the Sp-NP1 amino acid (AA) sequences consist of a 27 AA signal peptide and a 74 AA mature peptide, which contains 12 cysteine residues. qRT-PCR analysis has revealed that the expressions of Sp-NP1 gene are high in the nervous tissues and extremely low in the ovary and hepatopancreas. In situ hybridization has shown that the positive signals are localized in cell cluster 6 of protocerebrum and cell clusters 10 and 11 of deutocerebrum. The presence of Sp-NP1 in the haemolymph has been detected in S. paramamosain through western blot, which indicates that Sp-NP1 serves as an endocrine factor in the regulation of physiological activities. In vitro experiments have further shown that the mRNA level of vitellogenin in the hepatopancreas notably decreases following administration of recombinant Sp-NP1, while the mRNA level of vitellogenin receptor and cyclin B in the ovary shows no significant differences. Collectively, Sp-NP1 possibly can inhibit the production of vitellogenin in the hepatopancreas and has no direct effect on the ovary in S. paramamosain.

Transcriptomic changes across vitellogenesis in the black tiger prawn (Penaeus monodon), neuropeptides and G protein-coupled receptors repertoire curation.

The black tiger prawn (Penaeus monodon) is one of the most commercially important prawn species world-wide, yet there are currently key issues that hinder aquaculture of this species, such as low spawning capacity of captive-reared broodstock females and lack of globally available fully domesticated strains. In this study, we analysed the molecular changes that occur from vitellogenesis to spawning of a fully domesticated population of P.monodon (Madagascar) using four tissues [brain and thoracic ganglia (central nervous system - CNS), eyestalks, antennal gland, and ovary] highlighting differentially expressed genes that could be involved in the sexual maturation. In addition, due to their key role in regulating multiple physiological processes including reproduction, transcripts encoding P.monodon neuropeptides and G protein-coupled receptors (GPCRs) were identified and their expression pattern was assessed. A few neuropeptides and their putative GPCRs which were previously implicated in reproduction are discussed. We identified 573 differentially expressed transcripts between previtellogenic and vitellogenic stages, across the four analysed tissues. Multiple transcripts that have been linked to ovarian maturation were highlighted throughout the study, these include vitellogenin, Wnt, heat shock protein 21, heat shock protein 90, teneurin, Fs(1)M3, hemolymph clottable proteins and some other candidates. Seventy neuropeptide transcripts were also characterized from our de novo assembly. In addition, a hybrid approach that involved clustering and phylogenetics analysis was used to annotate all P. monodon GPCRs, revealing 223 Rhodopsin, 100 Secretin and 27 Metabotropic glutamate GPCRs. Given the key commercial significance of P.monodon and the industry requirements for developing better genomic tools to control reproduction in this species, our findings provide a foundation for future gene-based studies, setting the scene for developing innovative tools for reproduction and/or sexual maturation control in P. monodon.

Expression profiles of types 2 and 3 iodothyronine deiodinase genes in relation to vitellogenesis in a tropical damselfish, Chrysiptera cyanea.

Thyroid hormone (TH) is involved in regulating the reproduction of vertebrates. Its physiological action in the target tissues is due to the conversion of TH by iodothyronine deiodinases. In this study, we aimed to clone and characterize type 2 (sdDio2) and type 3 (sdDio3) of the sapphire devil Chrysiptera cyanea, a tropical damselfish that undergoes active reproduction under long-day conditions, and to study the involvement of THs in the ovarian development of this species. When the cDNAs of sdDio2 and sdDio3 were partially cloned, they had deduced amino acid sequences of lengths 271 and 267, respectively, both of which were characterized by one selenocysteine residue. Real-time quantitative PCR (qPCR) revealed that both genes are highly expressed in the whole brain, and sdDio2 and sdDio3 are highly transcribed in the liver and ovary, respectively. In situ hybridization analyses showed positive signals of sdDio2 and sdDio3 transcripts in the hypothalamic area of the brain. Little change in mRNA abundance of sdDio2 and sdDio3 in the brain was observed during the vitellogenic phases. It is assumed that simultaneous activation and inactivation of THs occur in this area because oral administration of triiodothyronine (T3), but not of thyroxine (T4), upregulated mRNA abundance of both genes in the brain. The transcript levels of sdDio2 in the liver and sdDio3 in the ovary increased as vitellogenesis progressed, suggesting that, through the metabolism of THs, sdDio2 and sdDio3 play a role in vitellogenin synthesis in the liver and yolk accumulation/E2 synthesis in the ovary. Taken together, these results suggest that iodothyronine deiodinases act as a driver for vitellogenesis in tropical damselfish by conversion of THs in certain peripheral tissues.

Precocious Downregulation of Krüppel-Homolog 1 in the Migratory Locust, Locusta migratoria, Gives Rise to An Adultoid Phenotype with Accelerated Ovarian Development but Disturbed Mating and Oviposition.

Krüppel-homolog 1 (Kr-h1) is a zinc finger transcription factor maintaining the status quo in immature insect stages and promoting reproduction in adult insects through the transduction of the Juvenile Hormone (JH) signal. Knockdown studies have shown that precocious silencing of Kr-h1 in the immature stages results in the premature development of adult features. However, the molecular characteristics and reproductive potential of these premature adult insect stages are still poorly understood. Here we report on an adult-like or 'adultoid' phenotype of the migratory locust, Locusta migratoria, obtained after a premature metamorphosis induced by the silencing of LmKr-h1 in the penultimate instar. The freshly molted adultoid shows precocious development of adult features, corresponding with increased transcript levels of the adult specifier gene LmE93. Furthermore, accelerated ovarian maturation and vitellogenesis were observed in female adultoids, coinciding with elevated expression of LmCYP15A1 in corpora allata (CA) and LmKr-h1 and vitellogenin genes (LmVg) in fat body, whereas LmE93 and Methoprene-tolerant (LmMet) transcript levels decreased in fat body. In adultoid ovaries, expression of the Halloween genes, Spook (LmSpo) and Phantom (LmPhm), was elevated as well. In addition, the processes of mating and oviposition were severely disturbed in these females. L. migratoria is a well-known, swarm-forming pest insect that can destroy crops and harvests in some of the world's poorest countries. As such, a better understanding of factors that are capable of significantly reducing the reproductive potential of this pest may be of crucial importance for the development of novel locust control strategies.