Phylogenomics of the Ecdysteroid Kinase-like (EcKL) Gene Family in Insects Highlights Roles in Both Steroid Hormone Metabolism and Detoxification.

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid molting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown, and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and we also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. Our evolutionary analyses provide hypotheses of function and a robust framework for future experimental studies of the EcKL gene family. They also open promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied coevolution of butterflies with their host plants.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Corticotropin-releasing factor-like diuretic hormone acts as a gonad-inhibiting hormone in adult female, Rhodnius prolixus.

Within insects, corticotropin-releasing factor/diuretic hormones (CRF/DHs) are responsible for the modulation of a range of physiological and behavioural processes such as feeding, diuresis, and reproduction. Rhopr-CRF/DH plays a key role in feeding and diuresis in Rhodnius prolixus, a blood-gorging insect and a vector for human Chagas disease. Here, we extend our understanding on the role of this neurohormone in reproduction in adult female R. prolixus. Double-label immunohistochemistry displays co-localized staining of CRF-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in the same neurosecretory cells (NSCs) in the mesothoracic ganglionic mass (MTGM) and in their neurohemal sites in adult female R. prolixus, suggesting these peptides could work together to regulate physiological processes. qPCR analysis reveals that the transcript for Rhopr-CRF/DH receptor 2 (Rhopr-CRF/DH-R2) is expressed in reproductive tissues and fat body (FB) in adult female R. prolixus, and its expression increases post blood meal (PBM), a stimulus that triggers diuresis and reproduction. Using RNA interference, transcript expression of Rhopr-CRF/DH-R2 was knocked down, and egg production monitored by examining the major yolk protein, vitellogenin (Vg), the number and quality of eggs laid, and their hatching ratio. Injection of dsCRFR2 into adult females reduces Rhopr-CRF/DH-R2 transcript expression, accelerates oogenesis, increases the number of eggs produced, and reduces hatching rate in female R. prolixus. Downregulation of Rhopr-CRF/DH-R2 leads to an increase in the transcript expression of RhoprVg1 in the fat body and ovaries, and increases the transcript level for the Vg receptor, RhoprVgR, in the ovaries. A significant increase in Vg content in the fat body and in the hemolymph is also observed. Incubation of isolated tissues with Rhopr-CRF/DH leads to a significant decrease in transcript expression of RhoprVg1 in the fat body and RhoprVg1 in the ovaries. In addition, Rhopr-CRF/DH reduces transcript expression of the ecdysteroid biosynthetic enzymes and reduces ecdysteroid titer in the culture medium containing isolated ovaries. These results suggest the involvement of the CRF-signaling pathway in reproduction, and that Rhopr-CRF/DH acts as a gonad-inhibiting hormone in the adult female R. prolixus, as previously shown for the colocalized glycoprotein, GPA2/GPB5.

Expressions of sugar transporter genes during Bombyx mori embryonic development.

Sugar transporters (Sts) play important roles in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. Few studies have been conducted on expressions of Sts during insect embryonic development. In the present study, we investigated temporal expressions of St genes during the embryonic diapause process in Bombyx mori. We found that in HCl-treated developing eggs, high gene expressions of trehalose transporter 1 (Tret1) were detected during middle and later embryonic development. St4 and St3 gene expressions gradually increased during the early stages, reached a small peak on Day 3, and large peaks were again detected on Day 7. However, in diapause eggs, expression levels of the Tret1, St4, and St3 genes all remained at low levels. Differential temporal changes in expressions of the Tret1, St4, and St3 genes found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited similar changing patterns as those of HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development. In addition, high gene expressions of Tret1 were also detected when dechorionated eggs were incubated in the medium. The addition of LY294002 (a specific phosphatidylinositol 3-kinase [PI3K] inhibitor) and U0126 (a mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase [MEK] inhibitor) partially inhibited Tret1 gene expression in dechorionated eggs, but did not affect either ecdysteroid-phosphate phosphatase gene expression or ecdysteroid biosynthesis, clearly indicating that both PI3K and ERK are involved in increased gene expression of Tret1 that was independent of ecdysteroid levels. To our knowledge, this is the first comprehensive report to demonstrate the transcriptional regulation of St genes during embryonic development, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster.

Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C27 and C28 ecdysteroids have been identified. While the biosynthetic pathway of the C27 ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C28 ecdysteroids from C28 or C29 dietary sterols remains unknown. In this study, we found that different dietary sterols (including the C27 sterols cholesterol and 7-dehydrocholesterol, the C28 sterols brassicasterol, campesterol, and ergosterol, and the C29 sterols ß-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. We also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, our study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C27 ecdysteroid 20E biosynthetic pathway also participated in the conversion of C28 and C29 dietary sterols into C28 ecdysteroids.

Signaling in cAMP-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

In the present study, we investigated downstream pathways of cyclic adenosine monophosphate (cAMP) signaling (which is related to prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis) in Bombyx mori prothoracic glands (PGs). Results showed that treatment with either dibutyryl cAMP (dbcAMP) or 1-methyl-3-isobutylxanthine (MIX) inhibited phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and activated phosphorylation of the translational repressor, 4E-binding protein (4E-BP), a marker of target of rapamycin (TOR) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, AICAR) increased dbcAMP-inhibited AMPK phosphorylation and blocked dbcAMP-stimulated phosphorylation of 4E-BP, indicating that inhibition of AMPK phosphorylation lies upstream of dbcAMP-stimulated TOR signaling. Treatment of PGs with dbcAMP and MIX also stimulated phosphorylation of a 37-kDa protein, as recognized by a protein kinase C (PKC) substrate antibody, indicating that cAMP activates PKC signaling. Treatment with either LY294002 or AICAR did not affect dbcAMP-stimulated phosphorylation of the PKC-dependent 37-kDa protein, indicating that cAMP-stimulated PKC signaling is not related to phosphoinositide 3-kinase (PI3K) or AMPK. In addition, dbcAMP-stimulated ecdysteroidogenesis in PGs was partially inhibited by pretreatment with either LY294002, AICAR, or calphostin C. From these results, we concluded that AMPK/TOR/4E-BP and PKC pathways are involved in ecdysteroidogenesis of PGs stimulated by cAMP signaling in B. mori.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis.

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.

An ecdysteroid-regulated 16-kDa protein homolog participates in the immune response of the crayfish Procambarus clarkii.

An ecdysteroid-regulated 16-kDa protein homolog (named Pc-E16), encoding 150 amino acid residues with a conserved MD-2-related lipid-recognition domain, was first identified in Procambarus clarkii. Phylogenetic analyses indicated similarity between Pc-E16 and 16-kDa proteins from Aplysia californica and insects. Recombinant Pc-E16 protein was successfully expressed in BL21 (DE3) Escherichia coli cells, and polyclonal antibodies against purified Pc-E16 proteins were prepared. In comparison with other tissues, Pc-E16 was highly expressed in the intestine; real-time PCR and Western blotting results indicated that Pc-E16 expression was significantly induced by lipopolysaccharides in hepatopancreas and hemocytes. Pc-E16-mediated signaling pathways were investigated by digital gene expression analysis following RNA interference targeting Pc-E16. A total of 6103 differentially expressed genes (DEGs) were identified, of which 3318 were up- and 2785 were downregulated. Many DEGs were involved in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that DEGs were clustered into 225 pathways, and 15 significantly enriched pathways were identified at the immune system level. In addition, the expression level of Pc-E16 in hemocytes and hepatopancreas was obviously downregulated at 48 h after dsRNA injection, and Pc-E16-RNAi treatment affected the expression levels of immune-related genes. Altogether, our results suggest that Pc-E16 is involved in the innate immune response of P. clarkii.

The MicroRNA Ame-Bantam-3p Controls Larval Pupal Development by Targeting the Multiple Epidermal Growth Factor-like Domains 8 Gene (megf8) in the Honeybee, Apis mellifera.

20-Hydroxyecdysone (20E) plays an essential role in coordinating developmental transitions in insects through responsive protein-coding genes and microRNAs (miRNAs). However, the interplay between 20E and miRNAs during insect metamorphosis is unknown. In this study, using small RNA sequencing, a comparative miRNA transcriptomic analysis in different development stages, and 20E treatment, we identified ame-bantam-3p as a key candidate miRNA involved in honeybee metamorphosis. Target prediction and in vitro dual-luciferase assays confirmed that ame-bantam-3p interacts with the coding region of the megf8 gene and promotes its expression. Meanwhile, temporal expression analysis revealed that the expression of ame-bantam-3p is higher in the larval stage than in prepupal and pupal stages, and that this expression pattern is similar to that of megf8. In vivo, we found that the mRNA level of megf8 was significantly increased after the injection of ame-bantam-3p agomir. A 20E feeding assay showed that 20E downregulated the expression of both ame-bantam-3p and its target gene megf8 on larval days five, six, and seven. Meanwhile, the injection of ame-bantam-3p agomir also reduced the 20E titer, as well as the transcript levels of essential ecdysteroid synthesis genes, including Dib, Phm, Sad, and Nvd. The transcript levels of 20E cascade genes, including EcRA, ECRB1, USP, E75, E93, and Br-c, were also significantly decreased after ame-bantam-3p agomir injection. However, ame-bantam-3p antagomir injection and dsmegf8 injection showed the opposite effect to ame-bantam-3p agomir injection. Ame-bantam-3p agomir treatment ultimately led to mortality and the failure of larval pupation by inhibiting ecdysteroid synthesis and the 20E signaling pathway. However, the expression of 20E signaling-related genes was significantly increased after megf8 knockdown, and larvae injected with dsmegf8 showed early pupation. Combined, our results indicate that ame-bantam-3p is involved in the 20E signaling pathway through positively regulating its target gene megf8 and is indispensable for larval-pupal development in the honeybee. These findings may enhance our understanding of the relationship between 20E signaling and small RNAs during honeybee development.

Compounds Inhibiting Noppera-bo, a Glutathione S-transferase Involved in Insect Ecdysteroid Biosynthesis: Novel Insect Growth Regulators.

Glutathione S-transferases (GSTs) are conserved in a wide range of organisms, including insects. In 2014, an epsilon GST, known as Noppera-bo (Nobo), was shown to regulate the biosynthesis of ecdysteroid, the principal steroid hormone in insects. Studies on fruit flies, Drosophila melanogaster, and silkworms, Bombyx mori, demonstrated that loss-of-function mutants of nobo fail to synthesize ecdysteroid and die during development, consistent with the essential function of ecdysteroids in insect molting and metamorphosis. This genetic evidence suggests that chemical compounds that inhibit activity of Nobo could be insect growth regulators (IGRs) that kill insects by disrupting their molting and metamorphosis. In addition, because nobo is conserved only in Diptera and Lepidoptera, a Nobo inhibitor could be used to target IGRs in a narrow spectrum of insect taxa. Dipterans include mosquitoes, some of which are vectors of diseases such as malaria and dengue fever. Given that mosquito control is essential to reduce mosquito-borne diseases, new IGRs that specifically kill mosquito vectors are always in demand. We have addressed this issue by identifying and characterizing several chemical compounds that inhibit Nobo protein in both D. melanogaster and the yellow fever mosquito, Aedes aegypti. In this review, we summarize our findings from the search for Nobo inhibitors.

A critical role for ecdysone response genes in regulating egg production in adult female Rhodnius prolixus.

Ecdysteroids control ovary growth and egg production through a complex gene hierarchy. In the female Rhodnius prolixus, a blood-gorging triatomine and the vector of Chagas disease, we have identified the ecdysone response genes in the ovary using transcriptomic data. We then quantified the expression of the ecdysone response gene transcripts (E75, E74, BR-C, HR3, HR4, and FTZ-F1) in several tissues, including the ovary, following a blood meal. These results confirm the presence of these transcripts in several tissues in R. prolixus and show that the ecdysone response genes in the ovary are mostly upregulated during the first three days post blood meal (PBM). Knockdown of E75, E74, or FTZ-F1 transcripts using RNA interference (RNAi) was used to understand the role of the ecdysone response genes in vitellogenesis and egg production. Knockdown significantly decreases the expression of the transcripts for the ecdysone receptor and Halloween genes in the fat body and the ovaries and reduces the titer of ecdysteroid in the hemolymph. Knockdown of each of these transcription factors typically alters the expression of the other transcription factors. Knockdown also significantly decreases the expression of vitellogenin transcripts, Vg1 and Vg2, in the fat body and ovaries and reduces the number of eggs produced and laid. Some of the laid eggs have an irregular shape and smaller volume, and their hatching rate is decreased. Knockdown also influences the expression of the chorion gene transcripts Rp30 and Rp45. The overall effect of knockdown is a decrease in number of eggs produced and a severe reduction in number of eggs laid and their hatching rate. Clearly, ecdysteroids and ecdysone response genes play a significant role in reproduction in R. prolixus.

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Transcriptome-wide identification of development related genes and pathways in Tribolium castaneum.

The growth and development in Tribolium castaneum were poorly understood at the transcriptome level. Currently, we identified 15,756, 9941 and 10,080 differentially expressed transcripts between late eggs VS early larvae, late larvae VS early pupae, and late pupae VS early adults of T. castaneum by RNA-seq, which was confirmed by qRT-PCR analysis on nine genes expression. Functional enrichment analysis indicated that DNA replication, cell cycle and insect hormone biosynthesis significantly enriched differentially expressed genes. The transcription of DNA replication and cell cycle genes decreased after hatching but increased after pupation. The juvenile hormone (JH) and ecdysteroid biosynthesis genes decreased after hatching, and the JH degradation genes were stimulated after pupation and eclosion while the ecdysteroid degradation gene CYP18A1 decreased after pupation. Silencing CYP18A1 elevated the titer of ecdysteroids and caused developmental arrest at the late larval stage. This study promotes the understanding of insect growth and development.

The prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Unusual functions of insect vitellogenins: minireview.

Insect vitellogenins are an intriguing class of complex proteins. They primarily serve as a source of energy for the developing embryo in insect eggs. Vitellogenesis is a complex hormonally and neurally controlled process that command synthesis of vitellogenin molecules and ensures their transport from the female fat bodies or ovarial cells into eggs. The representatives of all insect hormones such as juvenile hormones, ecdysteroids, and neurohormones participate in vitellogenesis, but juvenile hormones (most insect species) and ecdysteroids (mostly Diptera) play the most important roles in the process. Strikingly, not only insect females, but also males have been reported to synthesize vitellogenins indicating their further utility in the insect body. Indeed, it has recently been found that vitellogenins perform a variety of biological functions in the insect body. They participate in defense reactions against entomopathogens such as nematodes, fungi, and bacteria, as well as against venoms such as the honeybee Apis mellifera venom. Interestingly, vitellogenins are also present in the venom of the honeybee itself, albeit their exact role is unknown; they most likely increase the efficacy of the venom in the victim's body. Within the bee's body vitellogenins contribute to the lifespan regulation as anti-aging factor acting under tight social interactions and hormonal control. The current minireview covers all of these functions of vitellogenins and portrays them as biologically active substances that play a variety of significant roles in both insect females and males, and not only acting as passive energy sources for developing embryo.

Effects of triazole plant growth regulators on molting mechanism in Chinese mitten crab (Eriocheir sinensis).

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-ß-d-aminoglucosidase (ß-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis.

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

Lowfat functions downstream of Myo20 to regulate wing and leg morphogenesis in Tribolium castaneum.

Myosin Myo20 plays vital roles in the morphogenesis of wings and legs among insects, but the function and signalling of Myo20 remain unclear. We show that Myo20 regulates wing cell division, ecdysteroid and amino acid metabolism, and gene expression in Tribolium castaneum. By RNA-seq, we identified 582 differentially expressed genes (DEGs) between control and ds-Myo20 larvae of T. castaneum. Of these DEGs, silencing Myo20 significantly decreased the mRNA and protein levels of lowfat. During development, lowfat has the highest expression in early pupae and the lowest level in 1-day embryos. Tissue-specific analysis indicated that lowfat was abundantly expressed in the head, fat body and epidermis of late-stage larvae and in wings and legs of 1, 2 and 5-day pupae. Likewise, knockdown of lowfat affected wing and leg morphogenesis, ecdysteroid and amino acid metabolism, and gene expression in T. castaneum. Silencing Myo20 or lowfat activated CYP18A1 to degrade ecdysteroids, stimulated amino acids catabolism to increase the transcription of 4E-BP but reduce S6K and cycE expression. These results suggest that Lowfat works downstream of Myo20 to employ target of rapamycin (TOR) signalling for wing and leg morphogenesis in insects.

Knockdown of the Halloween Genes spook, shadow and shade Influences Oocyte Development, Egg Shape, Oviposition and Hatching in the Desert Locust.

Ecdysteroids are widely investigated for their role during the molting cascade in insects; however, they are also involved in the development of the female reproductive system. Ecdysteroids are synthesized from cholesterol, which is further converted via a series of enzymatic steps into the main molting hormone, 20-hydoxyecdysone. Most of these biosynthetic conversion steps involve the activity of cytochrome P450 (CYP) hydroxylases, which are encoded by the Halloween genes. Three of these genes, spook (spo), phantom (phm) and shade (shd), were previously characterized in the desert locust, Schistocerca gregaria. Based on recent sequencing data, we have now identified the sequences of disembodied (dib) and shadow (sad), for which we also analyzed spatiotemporal expression profiles using qRT-PCR. Furthermore, we investigated the possible role(s) of five different Halloween genes in the oogenesis process by means of RNA interference mediated knockdown experiments. Our results showed that depleting the expression of SchgrSpo, SchgrSad and SchgrShd had a significant impact on oocyte development, oviposition and hatching of the eggs. Moreover, the shape of the growing oocytes, as well as the deposited eggs, was very drastically altered by the experimental treatments. Consequently, it can be proposed that these three enzymes play an important role in oogenesis.