Molt Stage Research Articles

On the Recovery of Malformed Horseshoe Crabs Across Multiple Molting Stages

On the Recovery of Malformed Horseshoe Crabs Across Multiple Molting Stages

The effect of the molting cycle on the acoustic characteristics of clicks emitted by Litopenaeus vannamei

In penaeid shrimp, the mandibles are responsible for emitting click sounds when they collide during food intake, which has been used to assess feeding activity by passive acoustic monitoring. However, the acoustic parameters of the clicks could be affected by variations in the thickness of mandible cuticle during the molting cycle. Thus, the aim of this study was to evaluate the effects of the molting cycle on the acoustic characteristics of clicks emitted during the feeding activity of Litopenaeus vannamei fed commercial pelleted diet. Shrimp with an average weight of 8.75 ± 1.05 g were individually maintained in 24 tanks in a recirculating water system. A total of 12 animals had their feeding activity individually recorded soon after ecdysis (postmolt group), while another 12 were recorded in the middle of their molting cycle (intermolt group). Recordings took place in anechoic chambers with a hydrophone connected to a digital recorder (sampling rate of 192 kHz), and the click acoustic parameters were characterized in Raven® 1.5 Pro software. After the recordings, the shrimp had their mandibles removed for length measurement and histological analysis of the mandibular cuticle. Body (weight and length) and mandibular measurements did not differ between shrimp groups, but those in the postmolt stage showed significantly lower cuticle thickness. The acoustic characteristics of the clicks were affected by shrimp molting stages, with significantly lower maximum energy (dB) and maximum frequency (kHz) in postmolt shrimp, probably related to their lower mandibular cuticle thickness. These results may contribute to the assessment of shrimp feeding behavior associated with the molting cycle, as well as to the optimization of algorithms controlling acoustic automatic feeders in farming systems.

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.

Temporal changes in streamflow can predict parasitism levels in freshwater prawns better than host traits

Abstract Understanding how changes in the hydrological regime drive parasite loads and dynamics remains a challenging issue in ecological parasitology. Temporal changes in streamflow and rainfall are key hydrological factors that could alter interactions between the parasite and host and affect parasitism levels. However, to investigate the effect of streamflow, rainfall and its mechanisms, it is important to control host traits that can also influence parasitism levels in freshwater systems. Here, we used a cymothoid‐palaemonid prawn model to test the combined effects of streamflow and rainfall accounting for host traits to predict variations in parasite loads in stream ecosystems. We collected the palaemonid prawns monthly for 2 years and measured the variation of rainfall and streamflow (i.e. stream discharge) at the time of prawn sampling. Our best model showed that streamflow can predict parasitism levels in prawns better than host traits. We found a higher prevalence and abundance of parasites in reduced streamflow compared to increased ones. We also found higher ectoparasite loads in females rather than males and occurring in autumn and winter rather than in spring and summer. Our results also showed that ectoparasite loads also suffer an effect of host body size, sex and moult stage, but not host age. Our findings show that reduced streamflow can facilitate host finding that favours parasite transmission in dry seasons and increase parasitism levels in stream systems. Temporal variability in streamflow can have a strong influence on parasite loads and dynamics compared to host traits in stream ecosystems. Identifying how interactions between aquatic invertebrates and parasites respond to variability in the hydrological regime can help to better understand and predict disease outbreaks as habitat reduction and disturbance continue in stream ecosystems.

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.

Changing the focus: The need for cross-scale dynamic management in the Southern Ocean and implications for holistic conservation of Antarctic marine living resources

The Convention on the Conservation of Antarctic Marine Living Resources (CCAMLR) establishes the regulatory framework for fisheries management and the establishment of Marine Protected Areas (MPA) in the Southern Ocean. Identifying foraging areas regularly used by penguins provides valuable information for the small-scale management of the krill fishery and the design of MPAs, currently under discussion in CCAMLR. We used geolocators to identify the areas used by Adélie penguins from Ardley Island (South Shetlands Islands) during the non-breeding season and Stable Isotope Analysis (SIA) (δ13 C and δ15 N) on feathers to study the trophic niche. Adélie penguins depended on resources available in CCAMLR subareas 48.1 and 48.5 during the post-breeding and molting stage, the subarea 48.5 during the post-molting stage and subareas 48.1, 48.2 and 48.5 during the pre-breeding stage, critical moments of the species’ annual cycle. The Domain 1, Weddell Sea and SOISS MPA proposal areas were extensively used. SIA results show the use of coastal and off-shore foraging areas in 2022, but principally off-shore areas in 2021. Adélie penguins dependence on resources from different subareas in different times of the year emphasizes the need of incorporating the temporal dimension of resources utilization when designing conservation measures in the Southern Ocean. With an increasing pressure on Adélie penguin colonies in the western Antarctic Peninsula, its long-term protection might depend on both 1) well designed and managed MPAs in the Domain 1 and the Weddell Sea, and 2) small-scale fisheries management that simultaneously consider activities undertaken in different small-scale management units.

Genome-wide identification of Cytochrome P450 gene in Fall Armyworm (Spodoptera frugiperda) in response to insecticide resistance

The fall armyworm (FAW), Spodoptera frugiperda, poses a significant threat to maize, sorghum, and cotton crops, leading to substantial economic losses of up to 80 % in severe infestations. Despite its economic impact, the characterization of Cytochrome P450 (Cyp) genes, pivotal in regulatory metabolic processes, remains unexplored. This study identifies and investigates 33 Cyp-genes involved in critical metabolic pathways. These include fatty acid metabolism, resistance mechanisms, hormone regulation affecting moulting and developmental stages, response to phytotoxins, and detoxification of insecticides.Utilizing in-silico gene expression profiling, we pinpoint key Cyp-genes—Cyp306a1-like, Cyp9e2-like, Cyp6l1-like, Cyp12b1, and Cyp6B2-like—playing critical roles in conferring resistance against four commonly used insecticides: emamectin benzoate, tetrazolium, cyantraniliprole, and spinetoram. Our findings reveal that these identified genes are essential in detoxifying chemical treatments, thus contributing to the development of resistance in fall armyworm populations. In this investigation, key genes such as Cyp306a1-like, Cyp9e2-like, and Cyp6l1-like emerge as important regulatory genes. These genes play a role in resistance and detoxification when exposed to chemical stress. This in-silico study provides insights into the genetic mechanisms underlying resistance and regulatory genes in the fall armyworm, shedding light on potential targets for controlling the notorious agricultural pest. However, further comprehensive investigations are needed to elucidate the intricate resistance mechanisms governed by these key genes, paving the way for developing novel and effective strategies for fall armyworm management in agricultural ecosystems.

Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four LdKnk-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing LdKnk and LdKnk3-5' led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.

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.

Physicochemical Properties of Chitosan Extracted from Silkworm (Bombyx mori L.) Exuviae

This study investigated the chitosan content in various developmental stages of silkworms, specifically focusing on larval cuticle, moult exuviae and larval exuviae. The chitosan percentage varied significantly among different stages, with the highest content observed in bivoltine hybrid larval exuviae (19.37%) and the lowest in the 1st instar larval cuticle (9.93%). Chitosan content showed a consistent increase during moulting stages, reaching its peak in exuviae. The investigation extended to examine properties of chitosan, including moisture content, nitrogen content, ash content, degree of deacetylation (DD), solubility, viscosity and pH. Moisture content ranged from 5.80 to 7.84%, nitrogen content from 5.05 to 6.17%, ash content within 0.50 to 0.80% and DD from 85.10 to 92.71%. Solubility ranged from 92.25 to 98.25% and viscosity was measured between 47 and 58 cP. The pH values ranged from 6.72 to 7.49. These findings contribute valuable insights into the chitosan characteristics at different developmental stages of silkworms, providing a comprehensive understanding of its variations and potential applications.

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.

Broad Complex-Z2 directly activates BmMBF2 to inhibit the silk protein synthesis in the silkworm, Bombyx mori

Silk proteins, as natural macromolecules, have extensive applications in biomaterials and biomedicine. In the silkworm, the expression of silk protein genes is negatively associated with ecdysone during the molt stage, while it is positively correlated with juvenile hormone during the intermolt stage. In our previous study, overexpression of an isoform Z2 of Broad Complex (BmBrC-Z2), an ecdysone early response factor, significantly reduced the expression of silk protein genes. However, the underlying regulatory mechanism remains unclear. In this study, we conducted transcriptomic analysis and found that overexpressing BmBrC-Z2 significantly upregulated the expression level of multiprotein bridging factor 2 (BmMBF2), an inhibitor of fibroin heavy chain (FibH). Further investigations revealed that BmBrC-Z2 directly regulated BmMBF2 by binding to cis-regulatory elements, as demonstrated using Dual-Luciferase Reporter Gene Assay, EMSA, and ChIP-PCR assay. Additionally, when using the CRISPR/Cas9 system to knock out BmMBF2, silk protein genes were significantly upregulated during the molt stage of mutant larvae. These findings uncover the negative regulation of silk protein synthesis by the ecdysone signaling cascade, specifically through the manipulation of BmMBF2 expression during the molt stage. This study enhances to our understanding of the temporal regulatory mechanism governing silk protein synthesis and offers a potential strategy for improving silk yield.

Anatomical and molecular insights into the antennal gland of the giant freshwater prawn Macrobrachium rosenbergii

In this study, the complex organization of the AnG in the giant freshwater prawn Macrobrachium rosenbergii was revealed using various techniques, including conventional histology, histochemistry, scanning electron microscopy, and X-ray tomography. The results showed the diversity of cells in the AnG and the detailed organization of the labyrinth’s tubule into four radiated areas from the central to peripheral zones. The study also demonstrated the expression of some vertebrate kidney-associated homolog genes, aquaporin (AQP), solute carrier family 22 (SLC-22), nephrin, and uromodulin, in the AnG by qPCR. The result of in situ hybridization further showed the localization of SLC-22 and AQP transcript in the bladder and labyrinth’s epithelium, specifically in regions 2, 3, and 4. Additionally, the study revealed neuropeptide expressions in the AnG by qPCR and in situ hybridization, i.e., crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH), implying that the AnG may have a role in hormone production. Moreover, male and female prawns exhibited different levels of AQP, SLC-22, nephrin, and CHH expressions during the premolt and intermolt stages, suggesting a crucial role relevant to the molting stages. In conclusion, this study clarified the complex structure of the AnG in M. rosenbergii and demonstrated for the first time the expression of vertebrate kidney-associated genes and the possible endocrine role of the AnG. Further investigation is needed to clarify the role of these genes, particularly during ecdysis. The implications of these findings could significantly advance our understanding of the AnG in decapod crustaceans.

Sensing Pre- and Post-Ecdysis of Tropical Rock Lobsters, Panulirus ornatus, Using a Low-Cost Novel Spectral Camera

Tropical rock lobsters (Panulirus ornatus) are a highly cannibalistic species with intermoult animals predominantly attacking animals during ecdysis (moulting). Rapid, positive characterisation of pre-ecdysis lobsters may open a pathway to disrupt cannibalism. Ecdysial suture line development is considered for pre-ecdysis recognition with suture line definition compared for intermoult and pre-ecdysis lobsters emerged and immerged, using white, near ultraviolet (365 nm), near infrared (850 nm), and specialty SFH 4737 broadband IR LEDs against a reference of intermoult lobsters with no suture line development. Difficulties in acquiring suture line images prompted research into pre-ecdysis characterisation from the lobster’s dorsal carapace, due to its accessibility through a culture vessel’s surface. In this study, a novel low-cost spectral camera was developed by coordinating an IMX219 image sensor, an AS7265x spectral sensor, and four SFH 4737 broadband infrared LEDs through a single-board computer. Images and spectral data from the lobster’s dorsal carapace were acquired from intermoult, pre-ecdysis, and post-ecdysis lobsters. For the first time, suture line definition was found to be enhanced under 850 nm, 365 nm, and SFH 4737 LEDs for immerged lobsters, while the 850 nm LED achieved the best suture line definition of emerged lobsters. Although the spectral camera was unable to characterise pre-ecdysis, its development was validated when a least squares regression for binary classification decision boundary successfully separated 86.7% of post-ecdysis lobsters. Achieving post-ecdysis characterisation is the first time the dorsal carapace surface has been used to characterise a moult stage for palinurid lobsters.

Developmental and nuclear proteomic signatures characterize the temporal regulation of fibroin synthesis during the last molting-feeding transition of silkworm

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a “CTCF-FOX-HOX-SOX” association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated “closed” and “open” conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in silkworms.

Changes in body surface temperature reveal the thermal challenge associated with catastrophic moult in captive gentoo penguins.

Once a year, penguins undergo a catastrophic moult, replacing their entire plumage during a fasting period on land or on sea-ice during which time individuals can lose 45% of their body mass. In penguins, new feather synthesis precedes the loss of old feathers, leading to an accumulation of two feather layers (double coat) before the old plumage is shed. We hypothesized that the combination of the high metabolism required for new feather synthesis and the potentially high thermal insulation linked to the double coat could lead to a thermal challenge requiring additional peripheral circulation to thermal windows to dissipate the extra heat. To test this hypothesis, we measured the surface temperature of different body regions of captive gentoo penguins (Pygoscelis papua) throughout the moult under constant environmental conditions. The surface temperature of the main body trunk decreased during the initial stages of the moult, suggesting greater thermal insulation. In contrast, the periorbital region, a potential proxy of core temperature in birds, increased during these same early moulting stages. The surface temperature of the bill, flipper and foot (thermal windows) tended to initially increase during the moult, highlighting the likely need for extra heat dissipation in moulting penguins. These results raise questions regarding the thermoregulatory capacities of penguins in the wild during the challenging period of moulting on land in the current context of global warming.

NinaB and BCO Collaboratively Participate in the β-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and β, β-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro β-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with β-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its β-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the β-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically β-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

A Potential Multitarget Insect Growth Regulator Candidate: Design, Synthesis, and Biological Activity of Novel Acetamido Derivatives Containing Hexacyclic Pyrazole Carboxamides.

Insect growth regulators (IGRs) are important green insecticides that disrupt normal growth and development in insects to reduce the harm caused by pests to crops. The ecdysone receptor (EcR) and three chitinases OfChtI, OfChtII, and OfChi-h are closely associated with the molting stage of insects. Thus, they are considered promising targets for the development of novel insecticides such as IGRs. Our previous work identified a dual-target compound 6j, which could act simultaneously on both EcR and OfChtI. In the present study, 6j was first found to have inhibitory activities against OfChtII and OfChi-h, too. Subsequently, taking 6j as a lead compound, 19 novel acetamido derivatives were rationally designed and synthesized by introducing an acetamido moiety into the amide bridge based on the flexibility of the binding cavities of 6j with EcR and three chitinases. Then, their insecticidal activities against Plutella xylostella (P. xylostella), Ostrinia furnacalis (O. furnacalis), and Spodoptera frugiperda (S. frugiperda) were carried out. The bioassay results revealed that most of these acetamido derivatives possessed moderate to good larvicidal activities against three lepidopteran pests. Especially, compound I-17 displayed excellent insecticidal activities against P. xylostella (LC50, 93.32 mg/L), O. furnacalis (LC50, 114.79 mg/L), and S. frugiperda (86.1% mortality at 500 mg/L), significantly better than that of 6j. In addition, further protein validation and molecular docking demonstrated that I-17 could act simultaneously on EcR (17.7% binding activity at 8 mg/L), OfChtI (69.2% inhibitory rate at 50 μM), OfChtII (71.5% inhibitory rate at 50 μM), and OfChi-h (73.9% inhibitory rate at 50 μM), indicating that I-17 is a potential lead candidate for novel multitarget IGRs. This work provides a promising starting point for the development of novel types of IGRs as pest management agents.

Differences in diversity and community composition of the shell microbiome of apparently healthy lobsters Homarus americanus across Atlantic Canada.

Host-microbe dynamics are of increasing interest in marine research due to their role in host health and productivity. Changes in the shell microbiome of American lobsters have been associated with epizootic shell disease, a syndrome that is spreading northwards across the eastern U.S. and Canadian Atlantic coast. This study analyzed differences in alpha and beta diversity, as well as differentially abundant taxa, in the shell-associated bacterial community of apparently healthy lobsters from four lobster fishing areas (LFAs) in Atlantic Canada. Over 180 lobsters from New Brunswick, Nova Scotia and Prince Edward Island (PEI) were sampled during seven sampling events over four sampling months. The bacterial community was identified using novel PacBio long-read sequencing, while alpha and beta diversity parameters were analyzed using linear regression models and weighted UniFrac distances. The bacterial richness, diversity and evenness differed by sampling location, sampling month, and molt stage, but not by lobster sex or size, nor sampling depth. Similarly, based on LFA, sampling month, year and lobster molt stage, the shell microbiome differed in microbial community composition with up to 34 out of 162 taxa differing significantly in abundance between sampling groups. This large-scale microbial survey suggests that the shell microbial diversity of apparently healthy lobsters is influenced by spatial and temporal factors such as geographic location, as well as the length of time the carapace is exposed to the surrounding seawater.