C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1.

C. elegans develops through four larval stages that are rhythmically terminated by molts, that is, the synthesis and shedding of a cuticular exoskeleton. Each larval cycle involves rhythmic accumulation of thousands of transcripts, which we show here relies on rhythmic transcription. To uncover the responsible gene regulatory networks (GRNs), we screened for transcription factors that promote progression through the larval stages and identified GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1, and BED-3. We further characterize GRH-1, a Grainyhead/LSF transcription factor, whose orthologues in other animals are key epithelial cell-fate regulators. We find that GRH-1 depletion extends molt durations, impairs cuticle integrity and shedding, and causes larval death. GRH-1 is required for, and accumulates prior to, each molt, and preferentially binds to the promoters of genes expressed during this time window. Binding to the promoters of additional genes identified in our screen furthermore suggests that we have identified components of a core molting-clock GRN. Since the mammalian orthologues of GRH-1, BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.

NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Identification of genes regulated by 20-Hydroxyecdysone in Macrobrachium nipponense using comparative transcriptomic analysis.

BACKGROUND: Macrobrachium nipponense is a freshwater prawn of economic importance in China. Its reproductive molt is crucial for seedling rearing and directly impacts the industry's economic efficiency. 20-hydroxyecdysone (20E) controls various physiological behaviors in crustaceans, among which is the initiation of molt. Previous studies have shown that 20E plays a vital role in regulating molt and oviposition in M. nipponense. However, research on the molecular mechanisms underlying the reproductive molt and role of 20E in M. nipponense is still limited. RESULTS: A total of 240.24 Gb of data was obtained from 18 tissue samples by transcriptome sequencing, with > 6 Gb of clean reads per sample. The efficiency of comparison with the reference transcriptome ranged from 87.05 to 92.48%. A total of 2532 differentially expressed genes (DEGs) were identified. Eighty-seven DEGs associated with molt or 20E were screened in the transcriptomes of the different tissues sampled in both the experimental and control groups. The reliability of the RNA sequencing data was confirmed using Quantitative Real-Time PCR. The expression levels of the eight strong candidate genes showed significant variation at the different stages of molt. CONCLUSION: This study established the first transcriptome library for the different tissues of M. nipponense in response to 20E and demonstrated the dominant role of 20E in the molting process of this species. The discovery of a large number of 20E-regulated strong candidate DEGs further confirms the extensive regulatory role of 20E and provides a foundation for the deeper understanding of its molecular regulatory mechanisms.

Dietary restriction promote sperm remodeling in aged roosters based on transcriptome analysis.

BACKGROUND: The breeder rooster has played a pivotal role in poultry production by providing high-quality semen. Typically, fertility peaks between 30 and 40 weeks of age and then declines rapidly from 45 to 55 weeks of age. Research into improving fertility in aging roosters is essential to extend their productive life. While progress has been made, enhancing fertility in aging roosters remains a significant challenge. METHODS: To identify the genes related to promoting sperm remodeling in aged Houdan roosters, we combined changes in testis and semen quality with transcriptome sequencing (RNA-seq) to analyze the synchrony of semen quality and testis development. In this study, 350-day-old Houdan breeder roosters were selected for RNA-seq analysis in testis tissues from induced molting roosters (D group) and non-induced molting roosters (47DG group). All analyses of differentially expressed genes (DEGs) and functional enrichment were performed. Finally, we selected six DEGs to verify the accuracy of the sequencing by qPCR. RESULTS: Compared with the 47DG group, sperm motility (P < 0.05), sperm density (P < 0.01), and testis weight (P < 0.05) were significantly increased in roosters in the D group. Further RNA-seq analysis of the testis between the D group and 47DG group identified 61 DEGs, with 21 up-regulated and 40 down-regulated. Functional enrichment analysis showed that the DEGs were primarily enriched in the cytokine-cytokine receptor interaction, Wnt signaling pathway, MAPK signaling pathway, TGF-ß signaling pathway, and focal adhesion pathway. The qRT-PCR results showed that the expression trend of these genes was consistent with the sequencing results. WNT5A, FGFR3, AGTR2, TGFß2, ROMO1, and SLC26A7 may play a role in testis development and spermatogenesis. This study provides fundamental data to enhance the reproductive value of aging roosters.

Evidence for cryptic molting behavior in the trilobite Toxochasmops vormsiensis from the Upper Ordovician Katian Kõrgessaare Formation, Estonia.

Documentation of cryptic trilobite behavior has presented important insights into the paleoecology of this fully extinct arthropod group. One such example is the preservation of trilobites inside the remains of larger animals. To date, evidence for trilobites within cephalopods, gastropods, hyoliths, and other trilobites has been presented. Importantly, most of these interactions show trilobite molts, suggesting that trilobites used larger animals for protection during molting. To expand the record of molted trilobites within cephalopods, we present a unique case of a Toxochasmops vormsiensis trilobite within the body chamber of a Gorbyoceras textumaraneum nautiloid from the Upper Ordovician Kõrgessaare Formation of Estonia. By examining this material, we present new insights into the ecology of pterygometopid trilobites, highlighting how these forms used large cephalopods as areas to successfully molt.

Biological and physiological responses of marine crabs to ocean acidification: A review.

Marine crabs play an integral role in the food chain and scavenge the debris in the ecosystem. Gradual increases in global atmospheric carbon dioxide cause ocean acidification (OA) and global warming that leads to severe consequences for marine organisms including crabs. Also, OA combined with other stressors like temperature, hypoxia, and heavy metals causes more severe adverse effects in marine crabs. The present review was made holistic discussion of information from 111 articles, of which 37 peer-reviewed original research papers reported on the effect of OA experiments and its combination with other stressors like heavy metals, temperature, and hypoxia on growth, survival, molting, chitin quality, food indices, tissue biochemical constituents, hemocytes population, and biomarker enzymes of marine crabs. Nevertheless, the available reports are still in the infancy of marine crabs, hence, this review depicts the possible gaps and future research needs on the impact of OA on marine crabs.

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.

Neochloris aquatica induces larval mortality, molting defects, and unstable flightless adults in the Asian tiger mosquito.

The Asian tiger mosquito, Aedes albopictus, is a highly invasive and aggressive species capable of transmitting a large number of etiological agents of medical and veterinary importance, posing a high risk for the transmission of emerging viruses between animals and humans. In this work, we evaluated the mosquitocidal activity of Neochloris aquatica against A. albopictus throughout its development and analyzed whether this effect was potentiated when the microalga was cultivated under stress conditions due to nutrient deprivation. Our results suggest that N. aquatica produces metabolites that have negative effects on these insects, including larval mortality, interruption of pupal development, and incomplete emergence of adults when fed on microalgae in the larval stages. When microalgae were cultured under stress conditions, an increase in molting defects was recorded, and the number of healthy adults emerged drastically decreased. Histological studies revealed severe signs of total disintegration of different tissues and organs in the thorax and abdomen regions. The muscles and fat bodies in the midgut and foregut were severely distorted. In particular, larval intestinal tissue damage included vacuolization of the cytoplasm, destruction of brush border microvilli, and dilation of the intercellular space, which are distinctive morphological characteristics of apoptotic cells. Evidence suggests that N. aquatica produces metabolites with mosquitocidal effects that affect development and, therefore, the ability to vector etiological agents of medical and veterinary importance.

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development.

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

Buprofezin delayed the molting of Pardosa pseudoannulata, a predatory enemy for insect pests, by suppressing chitin synthase 1 expression.

Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species.

Four New Furofuran Lignans from Phryma leptostachya Inhibit the Accumulation of Molting Hormones in Armyworm.

Furofuran lignans have been identified as the main substances responsible for the biological activities of the plant genus Phryma. Here, four new phrymarolin-type leptolignans A-D (7-10) and eight previously known lignans were isolated from P. leptostachya. Of these, nine exhibited significant antifeedant activity against armyworm (Mythimna separata) through a dual-choice bioassay, with the EC50 values ranging from 0.58 to 10.08 µg/cm2. In particular, the newly identified lignan leptolignan A (7) showed strong antifeedant activity, with an EC50 value of 0.58 ± 0.34 µg/cm2. Further investigation found that leptolignan A can inhibit the growth and nutritional indicators in the armyworm M. separata. The concentrations of two molting hormones, 20-hydroxyecdysone and ecdysone, were also found to decrease significantly following the treatment of the armyworms with the lignan, implying that the target of the P. leptostachya lignan may be involved in 20-hydroxyecdysone and ecdysone synthesis. These results enrich our knowledge of P. leptostachya metabolite structural diversity, and provide a theoretical basis for the control of armyworm using lignans.

The nuclear receptor gene E75 plays a key role in regulating the molting process of the spider mite, Tetranychus urticae.

The nuclear receptor gene Ecdysone-induced protein 75 (E75), as the component of ecdysone response genes in the ecdysone signaling pathway, has important regulatory function for insect molting. However, the regulatory function of E75 during the molting process of spider mites is not yet clear. In this study, the expression pattern of E75 in the molting process of the spider mite Tetranychus urticae was analyzed. The results showed that there was a peak at 8 h post-molting, followed by a decline 8 h after entering each respective quiescent stage across various developmental stages. During the deutonymph stage, the expression dynamics of E75, observed at 4-h intervals, indicated that the transcript levels of TuE75 peaked at 24 h, coinciding with the onset of molting in the mites. To investigate the function of TuE75 during the molting process, silencing TuE75 through dsRNA injection into deutonymph mites at the age of 8 h yielded a notable outcome: 78% of the deutonymph mites were unable to progress to the adult stage. Among these phenotypic mites, 37% were incapable of transitioning into the quiescent state and eventually succumbed after a certain period. An additional 41% of the mites successfully entered the quiescent state but encountered difficulties in shedding the old epidermis, leading to eventual mortality. In summary, these results suggested that TuE75 plays a key role in the molting process of T. urticae.

A life cycle alteration can correct molting defects in Caenorhabditis elegans.

Molting is a widespread feature in the development of many invertebrates, including nematodes and arthropods. In Caenorhabditis elegans, the highly conserved protein kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (NEKLs) promote molting through their involvement in the uptake and intracellular trafficking of epidermal cargos. We found that the relative requirements for NEKL-2 and NEKL-3 differed at different life-cycle stages and under different environmental conditions. Most notably, the transition from the second to the third larval stage (L2→L3 molt) required a higher level of NEKL function than during several other life stages or when animals had experienced starvation at the L1 stage. Specifically, larvae that entered the pre-dauer L2d stage could escape molting defects when transiting to the (non-dauer) L3 stage. Consistent with this, mutations that promote entry into L2d suppressed nekl-associated molting defects, whereas mutations that inhibit L2d entry reduced starvation-mediated suppression. We further showed that loss or reduction of NEKL functions led to defects in the transcription of cyclically expressed molting genes, many of which are under the control of systemic steroid hormone regulation. Moreover, the timing and severity of these transcriptional defects correlated closely with the strength of nekl alleles and with their stage of arrest. Interestingly, transit through L2d rescued nekl-associated expression defects in suppressed worms, providing an example of how life-cycle decisions can impact subsequent developmental events. Given that NEKLs are implicated in the uptake of sterols by the epidermis, we propose that loss of NEKLs leads to a physiological reduction in steroid-hormone signaling and consequent defects in the transcription of genes required for molting.

Maintaining control: metabolism of molting Arctic seals in water and when hauled out.

Seals haul out of water for extended periods during the annual molt, when they shed and regrow their pelage. This behavior is believed to limit heat loss to the environment given increased peripheral blood flow to support tissue regeneration. The degree to which time in water, particularly during the molt, may affect thermoregulatory costs is poorly understood. We measured the resting metabolism of three spotted seals (Phoca largha), one ringed seal (Pusa hispida) and one bearded seal (Erignathus barbatus) during and outside the molting period, while resting in water and when hauled out. Metabolic rates were elevated in spotted and ringed seals during molt, but comparable in water and air for individuals of all species, regardless of molt status. Our data indicate that elevated metabolism during molt primarily reflects the cost of tissue regeneration, while increased haul out behavior is driven by the need to maintain elevated skin temperatures to support tissue regeneration.

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.

Functional transcriptome reveals hepatopancreatic lipid metabolism during the molting cycle of the Chinese mitten crab Eriocheir sinensis.

Crustacean molting is highly related to energy and lipid metabolism. This study was conducted to detect the changes of total lipids (TL), triacylglyceride (TAG), phospholipid (PL) and lipid droplets in hepatopancreas, and then to investigate the gene expression patterns related to hepatopancreatic lipid metabolism during the molting cycle of Chinese mitten crab Eriocheir sinensis. Hepatopancreatic TL and TAG increased significantly from post-molt stage to pre-molt stage, then decreased significantly from pre-molt stage to ecdysis stage, which is consistent to the changes of neutral lipid-rich adipocytes in hepatopancreas. By transcriptomic analysis, 65,325 transcripts were sequenced and assembled, and 28,033 transcripts were annotated. Most genes were related to energy metabolism, and the enriched genes were involved in carbohydrate and lipid metabolism and biosynthesis, especially in de novo synthesis of fatty acids and TAG, and ketone body production. Compared to the inter-molt stages, acetyl-CoA carboxylase, fatty acid synthase and other genes related to the synthesis of fatty acids were upregulated in the pre-molt stage. TAG synthesis related genes, including Glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferases, were upregulated in the post-molt stage compared to the inter-molt stage. The expression of ketone body-related genes had no significant changes during the molting cycle. Compared to the TAG synthetic pathway, ketone body biosynthesis may contribute less/secondarily to fatty acid metabolic processes, which could be involved in the other physiological processes or metabolism. In conclusion, these results showed that TAG is the major lipid deposition during inter- and pre-molt stages, and the most genes are related to the fatty acids and TAG metabolism in the hepatopancreas during the molting cycle of E. sinensis.

Molecular characterization of TRPA1 and its function in temperature preference in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species, and its growth and development are regulated by temperature, but the molecular mechanisms of the responses to temperature remain unclear. Herein, we identified TRPA1 from E. sinensis, a member of the TRP family of heat receptor potential channels, performed RACE cloning and bioinformatics analysis, and investigated the effect of TRPA1 on temperature responses and molting by real-time PCR and RNA interference (RNAi). The open reading frame of Es-TRPA1 is 3660 bp, and the encoded protein has a molecular weight of 136.91 kDa, and is expressed in embryos and juveniles. RNAi-mediated silencing decreased Es-TRPA1 expression in juvenile crabs, molting rate was decreased, mortality was increased, and crabs avoided cold areas (4 °C) much less than control juvenile crabs. The results suggest that Es-TRPA1 is involved in regulating temperature adaptation and molting processes in E. sinensis. The findings lay a foundation for further exploration of temperature regulation mechanisms in E. sinensis and other crustaceans.

SfDicer1 participates in the regulation of molting development and reproduction in the white-backed planthopper, Sogatella furcifera.

Dicer1 plays a vital role in the formation of mature miRNA and regulates the growth, development, and reproduction of insects. However, it remains to be clarified whether Dicer1 is involved in regulating the biological processes underlying molting and reproduction of Sogatella furcifera (Horváth). Herein, SfDicer1 expression fluctuated in all the developmental stages of S. furcifera and increased as molting progressed. SfDicer1 exhibited high expression in the integument, head, fat body, and ovary of the insects. SfDicer1 dsRNA injection into 1-day-old fourth instar nymphs of S. furcifera substantially decreased the survival rate and expression of the lethal phenotypes of wing malformation and molting defects and significantly inhibited the expression of four conserved miRNAs associated with molting development. Subsequently, following the knockdown of SfDicer1 in the newly emerged (1-12 h) females of S. furcifera, SfVg and SfVgR expression levels were decreased, thereby delaying ovarian development, decreasing the number of eggs, and considerably reducing the hatching rate compared with those of the control. Finally, after silencing SfDicer1 for 48 h, the comparative transcriptome analysis of differentially expressed genes revealed considerable enrichment of the Gene Ontology terms structural constituent of cuticle, structural molecule activity, chitin metabolic process, amino sugar metabolic process, and intracellular anatomical structure, indicating that SfDicer1 inhibition affects the transcription of genes associated with growth and development. Thus, our results suggest that SfDicer1 is essential in the molting, survival, ovarian development, and fecundity of S. furcifera and is a suitable target gene for developing an RNAi-based strategy targeting the most destructive rice insect pest.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus.

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.