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 providing a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.

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.

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.

To accommodate growth, insects must periodically shed their exoskeletons. In Manduca sexta, Drosophila melanogaster and Tribolium castaneum, Bursicon (Burs)/ Partner of bursicon (Pburs)-LGR2 signal is an indispensable component for the proper execution of ecdysis behavior during adult eclosion. Nevertheless, the behavioral events and the roles of bursicon signaling in other insects deserve further exploration. In the current paper, we found that the pupal-adult ecdysis in Henosepilachna vigintioctomaculata could be divided into three distinct stages, preecdysis, ecdysis and postecdysis. Preecdysis behavioral sequences included abdomen twitches, dorsal-ventral contractions and air filling that function to loosen the old cuticle. Ecdysis events began with anterior-posterior contractions that gradually split the old integument along the dorsal body midline, followed by freeing of legs and mouthparts, and culminated in detachment from pupal cuticle. Postecdysis behavioral processes contained three actions: perch selection and stretching of elytra and hindwings. RNA interference for HvBurs, HvPburs or Hvrk (encoding LGR2) strongly impaired wing expansion actions, and slightly influenced preecdysis and ecdysis behaviors. The RNAi beetles failed to extend their elytra and hindwings. In addition, injected with dsrk also caused kinked femurs and tibia. Our findings establish that bursicon pathway is involved in regulation of adult eclosion behavior, especially wing expansion motor programs. Given that wings facilitate food foraging, courtship, predator avoidance, dispersal and migration, our results provide a potential target for controlling H. vigintioctomaculata.

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.

The salmon aquaculture industry is an important economic activity established on both the west and east coast of Canada. To control sea lice infestations, in-feed products like emamectin benzoate (EMB) are widely used. Due to its low solubility and persistence EMB can accumulate in marine sediments and be potentially bioavailable to non-target organisms from months to years. The American lobster (Homarus americanus) is a key species in the Northwest Atlantic with high economic and ecological value. It may be exposed to therapeutants considering lobster habitats overlap with aquaculture locations requiring a better understanding of the potential impact of these therapeutants through varied pathways of exposure. In this study, we investigated the exposure of gravid female lobsters to EMB spiked sediment to mimic the likely presence of these females at aquaculture sites for a 10-day period. We completed testing by assessing EMB effects on adult molting and quality, embryo hatching rates, and larval offspring quality and larval molting. Our results show that a single, 10-day exposure of ovigerous females to EMB concentrations higher than environmentally relevant values did not affect females or their offspring.

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.

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-β 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, TGFβ2, 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.

Chagas disease affects around 8 million people globally, with Latin America bearing approximately 10,000 deaths each year. Combatting the disease relies heavily on vector control methods, necessitating the identification of new targets. Within insect genomes, genes harboring small open reading frames (smORFs - < 100 amino acids) present numerous potential candidates. In our investigation, we elucidate the pivotal role of the archetypal smORF-containing gene, mille-pattes/polished-rice/tarsalless (mlpt/pri/tal), in the post-embryonic development of the kissing bug Rhodnius prolixus. Injection of double-stranded RNA targeting mlpt (dsmlpt) during nymphal stages yields a spectrum of phenotypes hindering post-embryonic growth. Notably, fourth or fifth stage nymphs subjected to dsmlpt do not undergo molting. These dsmlpt nymphs display heightened mRNA levels of JHAMT-like and EPOX-like, enzymes putatively involved in the juvenile hormone (JH) pathway, alongside increased expression of the transcription factor Kr-h1, indicating changes in the hormonal control. Histological examination reveals structural alterations in the hindgut and external cuticle of dsmlpt nymphs compared to control (dsGFP) counterparts. Furthermore, significant changes in the vector\'s digestive physiology were observed, with elevated hemozoin and glucose levels in the posterior midgut of dsmlpt nymphs. Importantly, dsmlpt nymphs exhibit impaired metacyclogenesis of Trypanosoma cruzi, the causative agent of Chagas disease, underscoring the crucial role of proper gut organization in parasite differentiation. Thus, our findings constitute the first evidence of a smORF-containing gene\'s regulatory influence on vector physiology, parasitic cycle, and disease transmission.

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.