Molting Process Research Articles

Chitin Deacetylase 1 Gene as an Optimal RNAi-Based Target for Controlling the Tomato Leaf Miner Tuta absoluta

Chitin is a critical component of both the exoskeleton and internal structures of insects, which can protect insects from mechanical damage, dehydration and pathogen infection, and plays a significant role in the molting process. Chitin deacetylases (CDAs), key enzymes involved in chitin metabolism, are widely distributed among arthropods and microorganisms. In this study, we identified a CDA gene, TaCDA1, in the invasive insect species Tuta absoluta (Meyrick). Sequence analysis demonstrated a high degree of similarity to CDAs in other insects, revealing the presence of three conserved domains. Quantitative analysis showed that the TaCDA1 gene exhibited peak expression during the pupal stage, particularly within the epidermis. The suppression of TaCDA1 expression through RNA interference in T. absoluta pupae significantly impacted the expression of genes associated with chitin metabolism, increasing mortality and developmental abnormalities during the pupa–adult transition and reducing the pupal weight. Furthermore, soaking gene-specific dsRNA resulted in elevated mortality rates during the larva–pupa transition, causing the inability to form new cuticles or undergo ecdysis, as confirmed by subsequent histological observations. The oral administration of dsTaCDA1 + sucrose solution did not significantly impact NtCDA1 expression or the mortality rate compared to the dsGFP + sucrose solution control in the non-target insect Nesidiocoris tenuis. This study demonstrated that TaCDA1 is a potential and safe target for pest control of T. absoluta.

RNAi of nuclear receptor E78 inhibits the cuticle formation in the molting process of spider mite, Tetranychus urticae.

The two-spotted spider mite, Tetranychus urticae, is an important pest mite in agriculture worldwide. E78, as a member of the nuclear receptor superfamily and a downstream responsive gene of ecdysteroids, plays a crucial role in regulating physiological behaviors such as development and reproduction in insects. However, its function in mites remains unclear. The aim of this study was to explore how E78 functions in the molting process of spider mites. In this study, reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments to analyze the expression pattern of TuE78 during the development of Tetranychus urticae, demonstrated that the expression level of TuE78 was higher during the molting state than that after the completion of molting, and it reached a peak expression level when the deutonymph mites entered the molting stage. RNA interference (RNAi)-mediated gene-silencing of TuE78 resulted in 95% deutonymph mite molt failure. A series of analysis under a light microscope, and scanning and transmission electron microscopy revealed that RNAi mites died within the exuvium without ecdysis, and that apolysis had started but the new cuticle was thin and the typical cuticular lamellae were absent, indicating blockage of the post-apolysial processes and explaining molt failure. Hence, transcriptome sequencing confirmed that the expression of cuticle protein and lipid metabolism-related genes was significantly affected after TuE78 silencing. This study demonstrated that TuE78 participates in the molting process of Tetranychus urticae by regulating the post-apolysial processes with the formation of new cuticle and successful ecdysis. This in turn suggests the potential of TuE78 as a target for pest mite control and provides a theoretical basis for further exploration of the molecular regulatory mechanism of spider mite molting. © 2024 Society of Chemical Industry. Published by John Wiley & Sons Ltd.

Identification and functional characterization of the chitinase and chitinase-like gene family in Myzus persicae (Sulzer) during molting.

The crucial role of insect chitinase in molting, pupation, and emergence renders it a promising target for pest control strategies. Despite the extensive investigation of chitinase genes in various pests, there is still a lack of systematic identification and functional analysis related to aphid chitinase. We systematically identified a total of nine chitinase/chitinase-like genes and one ENGase gene, which included eight Cht genes, one IDGF gene, and one ENGase gene. Through phylogenetic analysis, the chitinase proteins were classified into nine distinct groups (I, II, III, V, VI, VIII, X, other, and ENGase). The expression profile revealed that the epidermis exhibited relatively high expression levels for three chitinase genes: MpCht5, MpCht7, and MpCht10. Furthermore, transcriptional levels of nine chitinase genes were up-regulated following treatment with 20-hydroxyecdysone (20E) hormone. Silencing MpCht3, MpCht5, MpCht7, MpCht10, and MpCht11-2 via RNA interference (RNAi) during the molting stage resulted in nymph shrinking, hindering normal molting and leading to death. Additionally, it was observed that silencing of MpIDGF induced the body color of the aphids to change from reddish brown to colorless after molting, culminating in eventual mortality. Our findings suggest that chitinase/chitinase-like genes play a crucial role in the molting process of Myzus persicae. Utilizing RNAi technology, we aimed to elucidate the precise function of MpCht genes in the molting mechanism of M. persicae, this discovery establishes a significant theoretical foundation for future research on aphid control, with chitinase as the target. © 2024 Society of Chemical Industry.

Enhancing laying performance and immunity via probiotic and vitamin additives during induced molting.

Molting is induced in commercial laying hens to rejuvenate the reproductive system and increase egg production. However, this process causes stress and reduces bird health and performance. The experiment was conducted to study the effect of multi probiotics and vitamin additives on induced molting in 240 ISA Brown hens. Hens were randomly divided into four groups receiving probiotic and vitamin additives (I-IV) during different period of molting. During the whole molting process, the laying performance indexes such as egg laying rate, egg quality, ovary weight and oviduct lengths were measured, and the spleen index, serum immunoglobulin, immune response of NDV and AIV vaccine were monitored. Molted hens resumed 50% egg production in just 37 days, with 1.62% mortality. Egg quality such as egg weight, yolk color, Haugh unit, eggshell strength and protein height were significantly improved. After the second production peak, the reproductive organs and immune organs returned to normal, and the immune antibody titer of NDV vaccine increased significantly. Molting with probiotic and vitamin additives improve the laying performance and egg quality, reduce mortality, significantly improve immune function and vaccine titer, and help to enhance disease resistance and maintain production performance of aged laying hens.

Design and Synthesis of Novel Indole-Derived N-Methylcarbamoylguanidinyl Chitinase Inhibitors with Significantly Improved Insecticidal Activity.

Chitinases play an important role in the molting process of insects and are potential targets for the development of green insecticides. Based on the feature that the +1/+2 sites in OfChtI, OfChtII, and OfChi-h have tryptophan residues in mismatch-parallel position, a strategy to introduce indole scaffold into chitinase inhibitors was proposed, and multitarget chitinase inhibitors containing N-methylcarbamoylguanidinyl and indole scaffold were successfully synthesized. The inhibitory activity showed that compound 8u exhibited significant inhibitory activity against OfChtI, OfChtII, and OfChi-h, with IC50 values of 0.7, 0.79, and 0.58 μM, and Ki values of 0.05 ± 0.005, 0.065 ± 0.004, and 0.025 ± 0.006 μM, respectively. In vivo insecticidal activity showed that compounds 8a and 8g exhibited excellent insecticidal activity against Plutella xylostella and Mythimna separata, with LC50 values of 0.79 and 9.17 mg/L against P. xylostella, respectively, and 3.58 and 83.09 mg/L against M. separata, respectively, making them the most potent chitinase inhibitors with in vivo insecticidal activity discovered to date. The inhibition mechanism and binding free energy results suggested that N-methylcarbamoylguanidinyl binds to the -1 catalytic site, while additional interactions acquired by π-π stacking and hydrophobic interactions of the indole scaffold with tryptophan increase the binding affinity of the targets to chitinases. This work provides a new direction for the development of chitinase inhibitors with compounds 8a and 8g potentially serving as promising candidates for pesticide development.

Multiple endpoints analysis of the effects of diesel oil on a commercial species, Carcinus maenas

Fuel spills in marine environments pose significant threats to aquatic ecosystems, evidencing the intricate relationship between fuel utilization and its impact on benthic species of commercial value for human consumption. This interconnectedness of human, animal and environmental welfare falls within the One Health framework. The aim of the present study was to evaluate the toxicological effects of diesel oil on the green crab Carcinus maenas, and make a parallelism between tested concentrations and petrogenic hydrocarbon levels in natural environments. Mortality, locomotion and feeding behavior, molting, somatic growth, morphological malformations, stress biomarkers, and nutritional variables were analyzed in three different bioassays. In Bioassay 1, prepuberal females were exposed to diesel oil water accommodated fraction (WAF) to determine the median lethal concentration (LC50) at different periods. In Bioassay 2, prepuberal females were exposed to 168 h LC50 and LC25 of diesel oil WAF for 7 days, and were subsequently exposed to clean water. In Bioassay 3, prepuberal females were exposed to 168 h LC12 and LC6 of diesel oil WAF for 30 days. Petrogenic hydrocarbon levels in the field were quantified at a port and a nature reserve, with concentrations of aromatic hydrocarbons being 1.92 μg/g in the former and below 0.01 μg/g in the latter. In Bioassay 1, the 168 h LC50 was estimated to be 1.04 % of diesel oil. The results obtained in Bioassays 2 (LC50 and LC25) and Bioassays 3 (LC12 and LC6) suggest that environmental exposure to petrogenic hydrocarbons produces high mortality or interferes with the molting process of crabs, leading to reduced growth and developmental abnormalities. Such malformations were observed in chelipeds, pereiopods, gills chambers and eye peduncles, and affected feeding and locomotion behaviors. Overall, this could impact on population size and health, and consequently alter the ecological role and commercial exploitation of economically important species like C. maenas.

Study on Pasir Lobster Enlargement (Panulirus homarus) to Improve Cultivation Productivity with Keong Bakau (Telescopium telescopium)

Abstract The potential of lobster in Indonesia is very high, the demand for lobster continues to increase every year, while the supply still utilizes the catch of fishermen. Lobster utilization in Indonesia is still low, therefore there must be sustainable cultivation in order to increase the quantity and quality of lobsters in Indonesia. One way to increase lobster enlargement is by feeding Keong Bakau. The purpose of this study is to determine and improve Lobster Pasir enlargement, including enlargement management, water quality management, and feed management with Keong Bakau. The study was conducted at the Lombok Marine Aquaculture Center, in Indonesia. The method used is a descriptive method with a quantitative approach. The feed used was Keong Bakau. The survival rate (SR) value reached 100%, the specific growth rate (SGR) value was 0.002%, and the FCR value was 23.82. The amount of feed given is sufficient for the needs of the lobster, the Keong Bakau used has chitosan which can help support the survival of the lobster. The average results of water quality include temperature (28.06oC), pH (7.07), DO (7.34 mg/l), Salinity (32.9 ppt), Turbidity (2.66 m), Phosphate (0.175 mg/l), Nitrite (0.002 mg/l), Nitrate (0.02 mg/l), and Ammonia (0.055 mg/l). DO values were slightly different from the optimal range due to the reduced intensity of vessels passing through the KJA. Keong Bakau has calcium which plays an important role in the formation of the exoskeleton in lobsters after the molting process.

Growth patterns of the invasive crab Cronius ruber (Brachyura, Portunidae) in the Canary Islands (Central-East Atlantic)

The growth pattern, including the moult process, relative growth, and estimation of von Bertalanffy growth parameters of the invasive non-indigenus species (NIS) Cronius ruber were studied in the Canary Islands. Despite being found in both, native and invaded areas, the life cycle and ecology of this species remain unknown. The study revealed a greater abundance of females than males, with males exhibiting larger carapace sizes and weights than females. Throughout ontogeny, relative growth was observed, with males reaching carapace widths (CW) between 54.5 and 57.8 mm and females reaching 49.9 mm CW. Allometry in the growth of the males’ chelae and females’ abdomen was consistently positive. Cronius ruber was found to be a fast-growing species, reaching the postpubertal growth phase (maturity) between 0.5 and 1 year of age in males and between 0.4 and 0.8 years in females. The intermoult period increased by more than 638 % in males and 443 % in females with age. Individuals undergo 13 moults during the first phase of life (until 30 mm CW) and then reduced to 6 moults until the end of life, concluding with a final terminal moult. The lifespan until the terminal moult is approximately 2.5 years, followed by a survival time over 1.5 years. von Bertalanffy growth parameters estimated using monthly CW-frequency data for males (L∞=97.4 mm CW, k=1.89 years−1, to=0.31 years), and for females (L∞=86.72 mm CW, k=1.91 years−1, to=0.29 years). Similar results were obtained using growth increment data.

Corazonin Stimulates Ecdysteroid Synthesis during the Molting Process of the Swimming Crab, Portunus trituberculatus.

The neuropeptide corazonin (Crz) exerts diverse physiological effects in insects, yet its role in crustaceans remains elusive. The abundant expression of Crz receptor (CrzR) in the Y-organs of several crustaceans suggests a potential involvement of Crz in regulating ecdysteroid synthesis. In this study, we examined the effects of PtCrz on ecdysteroid synthesis during the molting period of Portunus trituberculatus through PtCrz treatments and PtCrzR silencing. Our results showed that PtCrz peptide stimulates ecdysteroid levels and the gene expression involved in ecdysteroidogenesis both in vitro and in vivo, whereas dsPtCrzR treatments had opposite effects on ecdysteroid levels and associated gene expression. Thus, our study suggests that PtCrz may modulate ecdysteroid synthesis via Y-organ-expressed PtCrzR. Furthermore, we also discovered the involvement of PtCrz/PtCrzR signaling in regulating PtETH expression. Notably, the inhibitory effect of dsPtCrzR on ecdysteroid synthesis or PtETH expression can be reversed by PtCrz treatment, suggesting the potential existence of multiple receptors for PtCrz. This study provides new insights into the function of crustacean Crz and, for the first time, elucidates the presence of a neuropeptide that can stimulate ecdysteroid synthesis in crustaceans.

An experimental study on attitude control of a tailless hummingbird-mimetic flapping-wing robot with defective wings

Hummingbirds demonstrate remarkable robustness to wing defects by maintaining stable hovering during the molting process. However, mimicking these capabilities in flapping-wing robots presents significant challenges. To investigate the effectiveness of various controllers in maintaining the attitude of a hummingbird-mimetic flapping-wing robot against external disturbances under different wing defects, this study implemented a PD controller, a PID controller, and a three-loop feedback controller with a disturbance observer (3L-DOB controller). The flapping-wing robot features a pair of wings and controls its body’s yaw, pitch, and roll rotations by modulating the tension of wing membranes and the neutral positions of wing torsion. The performance of these controllers was evaluated through semi-tethered experiments on a gimbal, employing three Wing Sets: intact wings, one wing was defective, and both wings were defective. The defective wing emulated hummingbird wing during molting, in which the wing area was cropped by 14.1%. As a result, the 3L-DOB controller showed the best performance in terms of responsiveness and accuracy across all Wing Sets, while the PID controller also achieved comparable performance.

The involvement of tumor necrosis factor receptor-associated factor 6 in regulating immune response by NF-κB at pre-molt stage of Chinese mitten crab (Eriocheir sinensis)

Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.

Yorkie negatively regulates the Crustin expression during molting in Chinese mitten crab, Eriocheir sinensis

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

Insights into the activation mechanism of Bm-CPA: Implications for insect molting regulation

Carboxypeptidase A has been found across various animal species, yet its activation mechanism during the insect molting process remains elusive. Our study specifically delved into the activation mechanism of carboxypeptidase A (Bm-CPA), identified in Bombyx mori's molting fluid during metamorphosis. Initially, western blotting identified two forms of Bm-CPA, 65 kDa and 54 kDa, in the epidermis of silkworms during the molting stage. Expressing the complete Bm-CPA sequence in Pichia pastoris allowed the identification, via mass spectrometry analysis, of a 75-amino-acid propeptide for the initial hydrolysis process. Subsequently, a 35 kDa form of Bm-CPA emerged in the molting fluid, confirmed as the active form through in vitro assays, demonstrating potent carboxypeptidase A activity and faint carboxypeptidase B activity. Four potential activation sites (including Lys158/Arg159 and Arg177/Arg178) were identified through mass spectrometry and amino acid mutation analysis. RNAi of Bm-CPA indicates its critical role in molting. Finally, the carboxypeptidase inhibitor (Bm-CPI) from silkworm molting fluid was expressed to explore its role in regulating Bm-CPA activity, demonstrating a direct interaction with the 35 kDa Bm-CPA. Our research implies Bm-CPA's potential involvement in the silkworm molting process, suggesting diverse regulatory roles. These findings highlight intricate protein regulation patterns during insect metamorphosis and development.

Effectiveness of Green Mussel Chitosan Supplement in Feed on the Growth of Vaname Shrimp (Litopenaeus vannamei)

White shrimp (Litopenaeus vannamei) is an important aquaculture commodity with production demand that continues to increase every year. The addition of chitosan from green mussel shells in feed can increase calcium availability, support the moulting process, and increase shrimp growth and survival. This study aims to examine the effectiveness of green mussel chitosan supplements on the growth and survival of vaname shrimp. This research consists of three stages, namely making chitosan nanoparticles from green mussel shells, making feed with various doses of chitosan nanoparticles (0%/A, 10%/B, 15%/C, and 20%/D), and cultivating vaname shrimp. Feed is made by mixing chitosan nanoparticles with commercial feed, then drying and crushing it into small crumbs. White vaname shrimp are kept in concrete ponds for 80 days, with water quality monitoring and regular measurements of growth in weight and length. Data shows that C treatment with a dose of 15% chitosan nanoparticles resulted in the highest growth in weight and length of vanamei shrimp, reaching an average weight of 12.58 grams and a length of 14.07 cm on the 80th day. Treatment C provides optimal growth compared to other treatments because chitosan accelerates the moulting process and exoskeleton mineralization. Water quality during the study, including a temperature of 29.99°C, pH 7.23, and ammonia content of 0.25 ppm, was within the tolerable range for shrimp growth.

Molting in early Cambrian armored lobopodians

Lobopodians represent a key step in the early history of ecdysozoans since they were the first animals to evolve legs within this clade. Their Cambrian representatives share a similar body plan with a typically cylindrical annulated trunk and a series of non-jointed legs. However, they do not form a monophyletic group and likely include ancestors of the three extant panarthropod lineages (Tardigrada, Onychophora, Euarthropoda). Some species display astonishing protective devices such as cuticular plates and spines. We describe here the armor and molting process of Microdictyon from the early Cambrian of China. Microdictyon secreted ovoid paired cuticular sclerites that were duplicated in a non-synchronous way along the animal’s body. The reticulated pattern and cuticular architecture of these sclerites have similarities to extant armored tardigrades that recently served in hypothesizing that tardigrades are possibly miniaturized lobopodians. Ecdysis and hard cuticular protection are now well documented in the whole spectrum of early Cambrian ecdysozoans such as soft-bodied scalidophorans, lobopodians and fully articulated euarthropods. We hypothesize that the secretion of sclerotized cuticular elements periodically renewed via ecdysis was a key innovation that opened large-scale evolutionary opportunities to invertebrate animal life, specifically ecdysozoans, both in terms of anatomical functionalities and ecological success.

Effect of Adding Calcium Dolomite (CaMg(CO3)) and Tohor (CaO) in Cultivation Media to Increase Moulting of Litopenaeus vannamei

Vaname shrimp (Litopenaeus vannamei) is a fishery commodity that is the largest contributor to exports compared to other commodities. The success of shrimp survival and growth cannot be separated from the molting process, which requires calcium. The calcium that absorbed from the cultivation media can play a greater role in the process of exoskeleton formation. Dolomite lime (CaMg(CO3)) and tohor (CaO) can be sources of calcium that can be added to cultivation media. The research method used in this study was a completely randomized design (CRD) consisting of 5 different treatments (A: without additional lime, B: 0.8 gr dolomite, C: 0.8 gr tohor, D: 0.3 gr dolomite + 0.5 gr of tohor, E: 0.5 gr of dolomite + 0.3 gr of tohor) with 3 repetitions. The results showed that the addition of lime at different doses provided a molting frequency ranging from 2.07 – 3.23 times during the 60 days rearing period, where treatment E was the optimum treatment. The absolute weight growth obtained ranged from 2.52 gr – 5.03 gr, with an absolute length of 5.25 cm – 9.11 cm. The highest survival rate during maintenance in treatment E reached 72%. The water quality conditions measured during the activity were alkalinity 135 – 146 ppm, Ca hardness 132.74 – 172.39 ppm, temperature 29.9 – 30.3°C, DO 6.3 – 6.5 mg/l, pH 7 .9 – 8.3 and salinity 32 -34 ppt.

Sinuous Is a Claudin Required for Locust Molt in Locusta migratoria.

The epidermal cells of insects are polarized epithelial cells that play a pivotal role in the insect's molting process. Sinuous, a pivotal structural protein involved in the formation of septate junctions among epithelial cells, is essential for its physiological function. In this study, to determine whether sinuous participates in the regulation of insect molting, we identified the sinuous gene, Lmsinu, in Locusta migratoria, which encodes a protein belonging to the claudin family and shares 62.6% identity with Drosophila's sinuous protein. Lmsinu is expressed in multiple tissues, and its expression level in the integument significantly increases prior to molting. Knockdown of Lmsinu in L. migratoria results in larval mortality during molting. Furthermore, hematoxylin and eosin and chitin staining demonstrate that the downregulation of Lmsinu led to a prolonged degradation process of the old cuticle during the molting process. Electron microscopy analysis further revealed that knockdown of Lmsinu disrupts the formation of septate junctions among epidermal cells, which are a monolayer of polarized epithelial cells, which may hinder the functionality of epidermal cells during the process of molting. In summary, these findings suggest that Lmsinu plays a role in nymph molting by regulating the formation of septate junctions among epidermal cells.

Conserved microRNAs miR-8-3p and miR-2a-3 targeting chitin biosynthesis to regulate the molting process of Sogatella furcifera (Horváth)(Hemiptera: Delphacidae).

Molting is a key solution to growth restriction in insects. The periodic synthesis and degradation of chitin, one of the major components of the insect epidermis, is necessary for insect growth. MicroRNA (miRNA) have been implicated in molting regulation, yet their involvement in the interplay interaction between the chitin synthesis pathway and 20-hydroxyecdysone signaling remains poorly understood. In this study, soluble trehalase (Tre1) and phosphoacetylglucosamine mutase (PAGM) were identified as targets of conserved miR-8-3p and miR-2a-3, respectively. The expression profiles of miR-8-3p-SfTre1 and miR-2a-3-SfPAGM exhibited an opposite pattern during the different developmental stages, indicating a negative regulatory relationship between them. This relationship was confirmed by an in vitro dual-luciferase reporter system. Overexpression of miR-8-3p and miR-2a-3 by injection of mimics inhibited the expression of their respective target genes and increased mortality, leading to death in the pre-molting, and molting death phenomena. They also caused a decrease in chitin content and expression levels of key genes in the chitin synthesis pathway (SfTre1, SfTre2, SfHK, SfG6PI, SfGFAT, SfGNA, SfPAGM, SfUAP, SfCHS1, SfCHS1a, and SfCHS1b). Conversely, the injection of miRNA inhibitors resulted in the upregulation of the expression levels of these genes. Following 20E treatment, the expression levels of miR-8-3p and miR-2a-3 decreased significantly, while their corresponding target genes increased significantly. These results indicate that miR-8-3p and miR-2a-3 play a regulatory role in the molting of Sogatella furcifera by targeting SfTre1 and SfPAGM, respectively. These findings provide new potential targets for the development of subsequent new control strategies.

Transcriptome response of a marine copepod in response to environmentally-relevant concentrations of saxitoxin

Paralytic shellfish toxins (PSTs) can pose a serious threat to human health. Among them, saxitoxin (STX) is one of the most potent natural neurotoxins. Here, the copepod Tigriopus japonicus, was exposed to environmentally relevant concentrations (2.5 and 25 μg/L) STX for 48 h. Although no lethal effects were observed at both concentrations, the transcriptome was significantly altered, and displayed a concentration-dependent response. STX exposure decreased the copepod's metabolism and compromised immune defense and detoxification. Additionally, STX disturbed signal transduction, which might affect other cellular processes. STX exposure could inhibit the copepod's chitin metabolism, disrupting its molting process. Also, the processes related to damage repair and protection were up-regulated to fight against high concentration exposure. Collectively, this study has provided an early warning of PSTs for coastal ecosystem not only because of their potent toxicity effect but also their bioaccumulation that can transfer up the food chain after ingestion by copepods.

The chitinase genes TuCht4 and TuCht10 are indispensable for molting and survival of Tetranychus urticae

Insect chitinases (Chts) play a crucial role in the molting process, enabling continuous growth through sequential developmental stages. Based on their high homology to insect Chts, TuCht1 (group II), TuCht4 (group I) and TuCht10 (group IV) were identified, and their roles during molting process were investigated. TuCht1 was mainly expressed in the deutonymphal stage, while TuCht4 was mainly expressed in the nymphal stage and the highest expression level of TuCht10 was observed in the larvae. Feeding RNAi assays have shown that group I TuCht4 and group Ⅳ TuCht10 are involved in mite molting. Suppression of TuCht4 or TuCht10 resulted in high mortality, molting abnormalities and the absence of distinct electron dense layers of chitinous horizontal laminae in the cuticle, as demonstrated by scanning electron microscopy and transmission electron microscopy. The nanocarrier mediated RNAi had significantly higher RNAi efficiency and caused higher mortality. The results of the present study suggest that chitinase genes TuCht4 and TuCht10 are potential targets for dietary RNAi, and demonstrates a nanocarrier-mediated delivery system to enhance the bioactivity of dsRNA, providing a potential technology for green pest management.