Ecdysteroid Titer Research Articles

Larval development of a parasitoid depends on host ecdysteroids

Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20E signaling and the subsequent 2nd- instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ)

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

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.

Effects of mating on female reproductive physiology in the insect model, Rhodnius prolixus, a vector of the causative parasite of Chagas disease.

The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

Effects of neuropeptide F2 on female reproduction in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

AbstractRecently, neuropeptide F2 (NPF2) has been demonstrated to increase food consumption and body weight in the cotton bollworm, Helicoverpa armigera. This study evaluated the effect of NPF2 on the adult female reproductive system of H. armigera. Daily NPF2 injections increased oocyte size, ecdysteroid titre in ovaries and haemolymph and the expression level of ecdysone receptor (EcR), ultraspiracle (USP) and vitellogenin (Vg) genes. However, NPF2 injection had no effect on the expression level of hormone receptor 3 (HR3). Fecundity and fertility were increased after the injection of NPF2. The knockdown of the NPF2 gene using RNA interference (RNAi) had opposite effects compared to NPF2 injection. Overall, these results revealed that NPF2 has a regulatory role in the female reproduction of the cotton bollworm.

20-hydroxyecdysone Upregulates Ecdysone Receptor (ECR) Gene to Promote Pupation in the Honeybee, Apis mellifera Ligustica.

A heterodimeric complex of two nuclear receptors, the ecdysone receptor (ECR) and ultraspiracle (USP), transduces 20-hydroxyecdysone (20E) signaling to modulate insect growth and development. Here, we aimed to determine the relationship between ECR and 20E during larval metamorphosis and also the specific roles of ECR during larval-adult transition in Apis mellifera. We found that ECR gene expression peaked in the 7-day-old larvae, then decreased gradually from the pupae stage. 20E slowly reduced food consumption and then induced starvation, resulting in small-sized adults. In addition, 20E induced ECR expression to regulate larval development time. Double-stranded RNAs (dsRNAs) were prepared using common dsECR as templates. After dsECR injection, larval transition to the pupal stage was delayed, and 80% of the larvae showed prolonged pupation beyond 18 h. Moreover, the mRNA levels of shd, sro, nvd, and spo, and ecdysteroid titers were significantly decreased in ECR RNAi larvae compared with those in GFP RNAi control larvae. ECR RNAi disrupted 20E signaling during larval metamorphosis. We performed rescuing experiments by injecting 20E in ECR RNAi larvae and found that the mRNA levels of ECR, USP, E75, E93, and Br-c were not restored. 20E induced apoptosis in the fat body during larval pupation, while RNAi knockdown of ECR genes reduced apoptosis. We concluded that 20E induced ECR to modulate 20E signaling to promote honeybee pupation. These results assist our understanding of the complicated molecular mechanisms of insect metamorphosis.

Drosophila postembryonic nervous system development: a model for the endocrine control of development.

During postembryonic life, hormones, including ecdysteroids, juvenile hormones, insulin-like peptides, and activin/TGFβ ligands act to transform the larval nervous system into an adult version, which is a fine-grained mosaic of recycled larval neurons and adult-specific neurons. Hormones provide both instructional signals that make cells competent to undergo developmental change and timing cues to evoke these changes across the nervous system. While touching on all the above hormones, our emphasis is on the ecdysteroids, ecdysone and 20-hydroxyecdysone (20E). These are the prime movers of insect molting and metamorphosis and are involved in all phases of nervous system development, including neurogenesis, pruning, arbor outgrowth, and cell death. Ecdysteroids appear as a series of steroid peaks that coordinate the larval molts and the different phases of metamorphosis. Each peak directs a stereotyped cascade of transcription factor expression. The cascade components then direct temporal programs of effector gene expression, but the latter vary markedly according to tissue and life stage. The neurons read the ecdysteroid titer through various isoforms of the ecdysone receptor, a nuclear hormone receptor. For example, at metamorphosis the pruning of larval neurons is mediated through the B isoforms, which have strong activation functions, whereas subsequent outgrowth is mediated through the A isoform through which ecdysteroids play a permissive role to allow local tissue interactions to direct outgrowth. The major circulating ecdysteroid can also change through development. During adult development ecdysone promotes early adult patterning and differentiation while its metabolite, 20E, later evokes terminal adult differentiation.

Sublethal effects of fenoxycarb on female reproduction in Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract The cotton bollworm, Helicoverpa armigera (Hünber) (Lepidoptera: Noctuidae), is a polyphagous pest that causes severe damage to economical crops in most parts of the world. The juvenile hormone analogues are a group of insect growth regulators that increase larval ages and disrupt larval development. Moreover, they affect reproductive system in adult insects. In this study, the effects of sublethal doses of fenoxycarb on fecundity, fertility, and longevity parameters, as well as on oocyte size, ecdysteroid titer in ovaries, and expression level of the ultraspiracle (USP), ecdysone receptor (EcR), hormone receptor 3 (HR3), and vitellogenin (Vg) genes in adult female H. armigera, were examined. Feeding on artificial diet containing fenoxycarb (LC10 and LC25) significantly decreased the percentage of larval survival rate, pupation rate, pupal weight, fecundity, fertility, and oviposition period. It also clearly shortened female longevity. Fenoxycarb treatment suppressed the expression of the USP, EcR, and Vg genes in H. armigera but had no effect on the HR3 gene expression level in either pupae or adults. Overall, our results show that using fenoxycarb as a juvenile hormone analogue can reduce reproduction in H. armigera and may work as an alternative to the use of insecticides for controlling this important agricultural pest.

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.

Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

RNAi-Mediated Silencing of Putative Halloween Gene Phantom Affects the Performance of Rice Striped Stem Borer, Chilo suppressalis.

Simple SummaryEcdysteroids have a significant role in insect development and reproduction. Ecdysteroidogensis is regulated by a group of genes known as Halloween genes. We provide experimental evidence that Chilo suppressalis-phantom (Cs-Phm) in rice striped stem borer is a Halloween gene phantom and has important role in specific steps in ecdysteroidogensis. Three lines of experiments were done on typical protein domains of Cs-Phm, RNAi-mediated knockdown of Cs-Phm, and the rescue of larval development in dsRNA-treated groups. The results strongly suggest that Cs-Phm plays a critical role in ecdysteroidogensis in rice striped stem borer.The physiological and biochemical characterization of the “Halloween” genes has fundamental importance in the biosynthesis pathway of ecdysteroids. These genes were found to catalyze the final phases of ecdysteroid biosynthesis from dietary cholesterol to the molting hormone 20-hydroxyecdysone. We report the characterization of the Cs-Phm in a major insect pest in agriculture, the rice striped stem borer, Chilo suppressalis (C. suppressalis). A full-length transcript of Cs-Phm was amplified with an open reading frame (ORF) of 478 amino acids through 5′ and 3′ RACE. Cs-Phm shows five insect-conserved P450 motifs: Helix-C, Helix-I, Helix-K, PERF, and heme-binding motifs. Phylogenetic analysis clearly shows high similarity to Lepidoptera and evolutionary conservation in insects. The relative spatial and temporal transcript profile shows that Cs-Phm is highly expressed in the prothoracic glands and appears throughout the larval development, but with low expression at the start of the larval instar. It seems to peak in 3–4 days and decreases again before the larvae molt. Double-stranded RNA (dsRNA) injection of Cs-Phm at the larval stage efficiently knocked down the target gene and decreased its expression level. The dsRNA-treated group showed significantly decreased ecdysteroid titers, which leads to delayed larval development and higher larval mortality. Negative effects of larval development were rescued by treating 20E in the dsRNA-treated group. Thus, in conclusion, our results suggest that Cs-Phm is functionally conserved in C. suppressalis and encodes functional CYP that contributes to the biogenesis of 20E.

Characterization of the moult cycle in Neocaridina denticulata sinensis Kemp, 1918: the moulting frequency, moulting stages, and haemolymph ecdysteroid levels

Abstract This study aims to elucidate the physical evidence of the moulting stages and the characteristics of ecdysteroid levels in the moulting cycle of Neocaridina denticulata sinensis. From hatching to the first complete pleopod development, N. denticulata sinensis moults 14 times at 25°C in the laboratory. The whole process was divided into four stages based on the moulting characteristics during the various intervals. Using the structural changes of the telson and Drach’s classification system, four moulting periods (postmoult, intermoult, premoult, and ecdysis) were recognized. In addition, based on the changes of the setal lumen, internal cone, and the epidermis, the premoult period was further divided into five substages (D0, D1, D2, D3, and D4). Despite similar fluctuation patterns in mature males and females, significant differences of haemolymph ecdysteroid titers were revealed in postmoult, intermoult, and premoult. These works will provide an important additional reference for the exploration of the moulting mechanism in crustaceans.

Growth Regulating Effects of Natural Pesticide, Azadirachtin from Neem Tree (Azadirachta indica A. Juss) on Insects: A Review

Azadirachtin is a tetranortriterpenoid derived from the neem tree Azadirachta indica A. juss. Azadirachtin has attracted worldwide attention as promising feeding deterrent, insect growth disruptor and sterilent. Azadirachtin based pesticides become very much popular because of its biodegradability and least toxicity to non- target organisms. It has now been promoted as an alternative insecticide in integrated pest management programmes. Azadirachtin has an ability to modify or suppress haemolymph ecdysteroid and juvenile hormone titres through inhibition of the secretion of brain peptides prothoracicotropic hormone and allatotropic hormone, which are responsible for inducing the synthesis and release of ecdysone from the prothoracic gland and juvenile hormone from corpora allata, respectively. Blocking the synthesis and release of ecdysone and juvenile hormone leading to disruption of coordination of moulting process. Furthermore, this compound is known to cause degenerative changes in the cells of insect endocrine glands. This review focuses on growth regulating effects of natural pesticide, azadirachtin from neem tree (Azadirachta indica A. Juss) on insects.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata

Fushi-tarazu factor 1 (FTZF1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn.G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20E-related genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZ-F1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

Hemolymph Ecdysteroid Titer Affects Maternal mRNAs during Bombyx mori Oogenesis.

Simple SummaryBoth maternal genes and ecdysteroids play important roles during embryonic development. In this study, we aimed to characterize the dynamic landscape of maternal mRNAs and the relationship between maternal genes and ecdysteroids during silkworm oogenesis. For the first time, we determined the start of the accumulation of maternal mRNAs in the ovary at the wandering stage during the larval period. We detected the developmental expression profiles of each gene in the ovary or ovariole. We finally confirmed the role of 20-hydroxyecdysone in regulating maternal gene expression. Taken together, our findings expand the understanding of insect oogenesis and provide a perspective on the embryonic development of the silkworm.Silkworm larval–pupal metamorphosis and the first half of pupal–adult development occur during oogenesis from previtellogenesis to vitellogenesis and include two peaks of the hemolymph ecdysteroid titer. Moreover, a rise in 20-hydroxyecdysone titer in early pupae can trigger the first major transition from previtellogenesis to vitellogenesis in silkworm oogenesis. In this study, we first investigated the expression patterns of 66 maternal genes in the ovary at the wandering stage. We then examined the developmental expression profiles in six time-series samples of ovaries or ovarioles by reverse transcription–quantitative PCR. We found that the transcripts of 22 maternal genes were regulated by 20-hydroxyecdysone in the isolated abdomens of the pupae following a single injection of 20-hydroxyecdysone. This study is the first to determine the relationship between 20-hydroxyecdysone and maternal genes during silkworm oogenesis. These findings provide a basis for further research into the embryonic development of Bombyx mori.

Induction of diapause by clock proteins period and timeless via changes in PTTH and ecdysteroid titer in the sugar beet moth, Scrobipalpa ocellatella (Lepidoptera: Gelechiidae).

The sugar beet moth, Scrobipalpa ocellatella (Boyd), one of the most severe sugar beet pests, causes quantitative and qualitative yield losses late in the autumn. Previously, it was shown that low temperature and short-day photoperiod together cause diapause induction in pupae. Here, the interaction of the critical elements of the diapause induction, including the period (PER), timeless (TIM), prothoracicotropic hormone (PTTH), and ecdysteroid titer, were investigated. Immunohistochemistry results showed that the number of period immunoreactivity (PER-ir) and TIM-ir cells in nondiapause pupae (NDP) was lower than in the brain of the diapause pupae (DP). Moreover, the number of PER-ir and TIM-ir cells in the protocerebrum and optic lobe (OL) of NDP was lower than DP. Moreover, lower PTTH content in the brain and hemolymph of DP was confirmed by competitive enzyme-linked immunosorbent assay. Enzyme immunoassay showed a lower 20-hydroxyecdysone (20E) titer in the hemolymph of the DP compared with the NDP. Within a short-day condition, PER and TIM titers increased in the brain leading to decreasing PTTH titers in the brain and hemolymph that caused decreasing 20E titer in the hemolymph, leading to the induction of diapause. This study suggests that PER and TIM could be one of the brain factors that play an essential role in regulating diapause in S. ocellatella.

Effects of Pergularia tomentosa active fraction on the oogenesis of the desert locust, Schistocerca gregaria: ovarian biochemical composition, and effects on the ecdysteroid pathway

AbstractFractionation of the methanolic extract of Pergularia tomentosa, using column chromatography, resulted in the separation of 10 fractions and only the active one (that gave mortality >70%) was kept and denoted the active fraction (AFPt). Adult female Schistocerca gregaria were treated with two concentrations of AFPt, 0.24% and 0.96%, to investigate the effect on the female reproductive potential. Both concentrations of AFPt significantly increased the duration between adult emergence and first oviposition, as well as affected fecundity and fertility. Furthermore, the effects of AFPt on vitellogenesis and ecdysteroid titres were examined. Biochemical analyses revealed a significant decrease in protein, lipid and carbohydrate contents in ovaries. This study also showed that AFPt induced a significant decrease in the ovarian ecdysteroids levels, as well as the haemolymph ecdysteroid titres. The transcript levels of the ecdysteroid synthesis enzymes Spook and Shadow were significantly decreased when treated with the highest concentration of AFPt, while transcript levels of the ecdysteroid synthesis enzymes Phantom and Disembodied increased significantly in animals treated with AFPt. Treatment with AFPt also resulted in a significantly higher transcript level of both the ecdysone receptor (EcR) and its partner ultraspiracle/retinoid‐X‐receptor (USP/RXR).

Myosuppressin influences fecundity in the Colorado potato beetle, Leptinotarsa decemlineata

Insect neuropeptides regulate various physiological processes, such as reproduction, feeding, growth and development, and have been considered as viable targets in the development of alternative strategies for pest control. Amongst these neuropeptides is myosuppressin (MS), a very conserved neuropeptide that has been reported to regulate cardiac and skeletal muscle contractility, feeding and pupal diapause in insects. In this study, we investigated the involvement of MS in fecundity in a notorious defoliator of potato and other solanaceous plants, the Colorado potato beetle (CPB),Leptinotarsa decemlineata. We identified an MS-precursor-encoding transcript in the L. decemlineata transcriptomic database and then evaluated its transcript levels in various CPB tissues. MS transcript levels were found to be highest in the central nervous system, gut and muscle of CPB males and females. To investigate the role of MS in fecundity, MS was silenced in adult CPBs through RNA interference (RNAi). This resulted in a significant reduction in oviposition (over 80%) and oocyte size (69%) in the treated beetles compared to the controls. Also, the reduction in oviposition in treated females was confirmed to be dependent on MS knockdown and independent of male fertilization. Furthermore, MS-knockdown in females resulted in decreased levels of ecdysteroid hormone titers and the transcript levels of its receptor. Interestingly, the injection of 20-hydroxyecdysone into females following MS knockdown could rescue ovary development. Altogether, this study highlights the important role played by MS in regulating fecundity in CPB.