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.

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.

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.

Exposure of female lobsters to sediments spiked with emamectin benzoate: Effects on eggs and larvae early development.

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.

Behavioral events and functional analysis of bursicon signal during adult eclosion in the 28-spotted larger potato ladybird.

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 mlpt smORF gene is essential for digestive physiology and molting during nymphal stages in the kissing bug Rhodnius prolixus.

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.

Earliest evidence of avian primary feather moult.

Feather moulting is a crucial process in the avian life cycle, which evolved to maintain plumage functionality. However, moulting involves both energetic and functional costs. During moulting, plumage function temporarily decreases between the shedding of old feathers and the full growth of new ones. In flying taxa, a gradual and sequential replacement of flight feathers evolved to maintain aerodynamic capabilities during the moulting period. Little is known about the moult strategies of non-avian pennaraptoran dinosaurs and stem birds, before the emergence of crown lineage. Here, we report on two Early Cretaceous pygostylian birds from the Yixian Formation (125 mya), probably referable to Confuciusornithiformes, exhibiting morphological characteristics that suggest a gradual and sequential moult of wing flight feathers. Short primary feathers interpreted as immature are symmetrically present on both wings, as is typical among extant flying birds. Our survey of the enormous collection of the Tianyu Museum confirms previous findings that evidence of active moult in non-neornithine pennaraptorans is rare and likely indicates a moult cycle greater than one year. Documenting moult in Mesozoic feathered dinosaurs is critical for understanding their ecology, locomotor ability and the evolution of this important life-history process in birds.

The Metabolic Effort and Duration of Ecdysis in Timber Rattlesnakes: Implications for Time-Energy Budgets of Reptiles.

AbstractTemperate reptiles are often considered to be low-energy systems, with their discrete use of time and energy making them model systems for the study of time-energy budgets. However, the semifrequent replacement and sloughing of the epidermis is a ubiquitous feature of squamate reptiles that is often overlooked when accounting for time and energy budgets in these animals. We used open-flow respirometry to measure both the energetic effort of ecdysis and the duration of the associated metabolic upregulation (likely related to behavioral changes often reported for animals in shed) in wild-caught timber rattlesnakes (Crotalus horridus). We hypothesized that total effort of skin biosynthesis and physical removal would be related to body mass and expected the duration of the process to remain static across individuals at a fixed temperature (25°C). We provide both the first measurements of the cost of skin biosynthesis and physical removal in a reptile and the highest-resolution estimate of process duration recorded to date. We found that skin biosynthesis, but not the cost of physical removal of the epidermis, was related to body mass. Shed cycle duration was consistent across individuals, taking nearly 4 wk from process initiation to physical removal of the outermost epidermal layer. Total energetic effort of ecdysis was of sizeable magnitude, requiring ∼3% of the total annual energy budget of a timber rattlesnake. Energetic effort for a 500-g snake was equivalent to the amount of metabolizable energy acquired from the consumption of approximately two adult mice. Ecdysis is a significant part of the time-energy budgets of snakes, necessitating further attention in studies of reptilian energetics.

Complexity in the timing of the first postnatal ecdysis in snakes.

Lepidosaurian reptiles, particularly snakes, periodically shed the outer epidermal layers of their skin (ecdysis) to restore or enhance vital functions such as regulating water and gaseous exchange, growth, and protection against insult, infection or physical injury. Although many studies have focused on the nature and mechanisms of skin shedding, little attention has been paid to the timing of the first ecdysis in neonates following birth or hatching. A recent study investigated patterns of the time to first postnatal ecdysis in snakes based on a large dataset taken from the literature. The analysis demonstrated patterns in the time to first postnatal ecdysis related to phylogeny as well as several life history traits. While this assessment provides important advances in our knowledge of this topic, data on known biophysical drivers of ecdysis - temperature and humidity - were largely unavailable and were not evaluated. The first postnatal ecdysis of neonatal snakes can be viewed as an adaptive adjustment to the transition from the aqueous environment of the embryo to the aerial environment of the newborn. Hence, the timing of the first postnatal ecdysis is logically influenced by the aerial environment into which a newborn snake or hatchling finds itself. Therefore, in this Commentary, we first emphasize the putative plasticity of ecdysis with respect to epidermal lipids that structure the water permeability barrier and are established or renewed during ecdysis to reduce transepidermal evaporative water loss. We then discuss the likely importance of biophysical variables as influential covariates that need future investigation as potential co-determinants of the timing of first postnatal ecdysis.

Effects of sublethal methylmercury and food stress on songbird energetic performance: metabolic rates, molt and feather quality.

Organisms regularly adjust their physiology and energy balance in response to predictable seasonal environmental changes. Stressors and contaminants have the potential to disrupt these critical seasonal transitions. No studies have investigated how simultaneous exposure to the ubiquitous toxin methylmercury (MeHg) and food stress affects birds' physiological performance across seasons. We quantified several aspects of energetic performance in song sparrows, Melospiza melodia, exposed or not to unpredictable food stress and MeHg in a 2×2 experimental design, over 3 months during the breeding season, followed by 3 months post-exposure. Birds exposed to food stress had reduced basal metabolic rate and non-significant higher factorial metabolic scope during the exposure period, and had a greater increase in lean mass throughout most of the experimental period. Birds exposed to MeHg had increased molt duration, and increased mass:length ratio of some of their primary feathers. Birds exposed to the combined food stress and MeHg treatment often had responses similar to the stress-only or MeHg-only exposure groups, suggesting these treatments affected physiological performance through different mechanisms and resulted in compensatory or independent effects. Because the MeHg and stress variables were selected in candidate models with a ΔAICc lower than 2 but the 95% confidence interval of these variables overlapped zero, we found weak support for MeHg effects on all measures except basal metabolic rate, and for food stress effects on maximum metabolic rate, factorial metabolic scope and feather mass:length ratio. This suggests that MeHg and food stress effects on these measures are statistically identified but not simple and/or were too weak to be detected via linear regression. Overall, combined exposure to ecologically relevant MeHg and unpredictable food stress during the breeding season does not appear to induce extra energetic costs for songbirds in the post-exposure period. However, MeHg effects on molt duration could carry over across multiple annual cycle stages.

NinaB and BCO Collaboratively Participate in the ß-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 ß, ß-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 ß-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 ß-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 ß-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 ß-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically ß-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.

NPC Intracellular Cholesterol Transporter 1 Regulates Ovarian Maturation and Molting in Female Macrobrachium nipponense.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

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.

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.

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.

Effects of pyriproxyfen on development and hormone of the aphis, Aphis craccivora (Hemiptera: Aphididae).

Pyriproxyfen (PPF) has been shown to affect the pupal stage and ecdysone levels in holometabolous insects, such as silkworms and mealworms. It remains unknown whether it affects hemimetabolous insects with their hormone levels in insects lacking a pupal stage. In this laboratory study, bioassays were conducted to investigate the effects of varying doses of PPF on Aphis craccivora Koch (Hemiptera: Aphididae). Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the types and titers of juvenile hormone (JH) and 20-hydroxyecdysone (20E). Additionally, the effects of PPF on A. craccivora reproduction and molting, as well as its influence on relevant gene expression, were examined. The results revealed LC50 and LC90 values of 3.84 and 7.49 mg/l for PPF, respectively, after 48 h of exposure. The results demonstrated a significant reduction in the titer of JH III and a significant increase in the titer of 20E following treatment with PPF. However, there was no significant decrease observed in the titer of JH III skipped bisepoxide (JH SB3). A sublethal concentration of PPF was found to inhibit Krüppel homolog 1 (kr-h1) gene expression and reduce aphid reproduction, but it did not significantly impact ecdysone receptor expression and aphid molting. The results of this study demonstrate that PPF exhibits a lethal effect on aphids, thereby providing an effective means of control. Additionally, sublethal concentrations of PPF have been found to inhibit the JH in aphids, resulting in a decline in their reproductive ability and achieving the desired control objectives.

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.

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.

Temporal Transcriptomic Profiling Reveals Dynamic Changes in Gene Expression of Giant Freshwater Prawn upon Acute Saline-Alkaline Stresses.

Bicarbonate and sulfate are among two primary ion constituents of saline-alkaline water, with excessive levels potentially causing metabolic disorders in crustaceans, affecting their molting and interrupting development. As an economically important crustacean species, the molecular adaptive mechanism of giant freshwater prawn Macrobrachium rosenbergii in response to the stress of bicarbonate and sulfate remains unexplored. To investigate the mechanism underlying NaHCO3, Na2SO4, and mixed NaHCO3, Na2SO4 stresses, M. rosenbergii larvae were exposed to the above three stress conditions, followed by total RNA extraction and high-throughput sequencing at eight distinct time points (0, 4, 8, 12, 24, 48, 72, and 96 h). Subsequent analysis revealed 13, 16, and 13 consistently identified differentially expressed genes (DEGs) across eight time points under three stress conditions. These consistently identified DEGs were significantly involved in the Gene Ontology (GO) terms of chitin-based cuticle development, protein-carbohydrate complex, structural constituent of cuticle, carnitine biosynthetic process, extracellular matrix, and polysaccharide catabolic process, indicating that alkaline stresses might potentially impact the energy metabolism, growth, and molting of M. rosenbergii larvae. Particularly, the transcriptome data revealed that DEGs associated with energy metabolism, immunity, and amino acid metabolism were enriched across multiple time points under three stress conditions. These DEGs are linked to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including glycolysis/glucogenesis, amino sugar and nucleotide sugar metabolism, and lysine degradation. Consistent enrichment findings across the three stress conditions support conclusions above. Together, these insights are instrumental in enhancing our understanding of the molecular mechanisms underlying the alkaline response in M. rosenbergii larvae. Additionally, they offer valuable perspectives on the regulatory mechanisms of freshwater crustaceans amid saline-alkaline water development.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.