Molt Stage Research Articles

Second Prebasic Molt of a Black-headed Gull at Anchorage, Alaska

A second-cycle Black-headed Gull (Chroicocephalus ridibundus), a rare to casual visitor in western North America, remained at Anchorage, Alaska, from 16 July through 17 September 2023, providing a unique opportunity to track and document most stages of its second prebasic molt into definitive (adult) basic plumage. I estimated that the Anchorage bird required approximately 3 to 3.5 months (early/mid-June through late September) to complete the molt of its primaries. This is notably longer than the “average” 2.5 months stated for second-cycle Black-headed Gulls in several publications, but consistent with the duration and calendar limits reported by others for the entire species (mid-May/June through September). The start and duration of rectrix molt relative to the stage of primary molt closely matched that described for gulls in general, and specifically for predefinitive Bonaparte’s (C. philadephia) and Western (Larus occidentalis) gulls. When first observed in mid-July, the Anchorage bird had a white tail, as in definitive plumage, a trait infrequent in first-cycle Black-headed Gulls; then it molted the tail again through August. Although Black-headed Gulls occurring in western North America have been presumed to originate from eastern Asia, the timing of the Anchorage bird’s primary molt closely matched that published for second-cycle Black-headed Gulls in Europe, which has reported to be earlier—though molt schedules of Charadriiformes at the population level are highly variable.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

Insights into sequence characteristics and evolutionary history of DGATs in arthropods

Triacylglycerol (TAG) is crucial in animal energy storage and membrane biogenesis. The conversion of diacylglycerol (DAG) to triacylglycerol (TAG) is catalyzed by diacylglycerol acyltransferase enzymes (DGATs), which are encoded by genes belonging to two distinct gene families. Although arthropods are known to possess DGATs activities and utilize the glycerol-3-phosphate pathway and MAG pathway for TAG biosynthesis, the sequence characterization and evolutionary history of DGATs in arthropods remains unclear. This study aimed to comparatively evaluate genomic analyses of DGATs in 13 arthropod species and 14 outgroup species. We found that arthropods lack SOAT2 genes within the DGAT1 family, while DGAT2, MOGAT3, AWAT1, and AWAT2 were absent from in DGAT2 family. Gene structure and phylogenetic analyses revealed that DGAT1 and DGAT2 genes come from different gene families. The expression patterns of these genes were further analyzed in crustaceans, demonstrating the importance of DGAT1 in TAG biosynthesis. Additionally, we identified the DGAT1 gene in Swimming crab (P. trituberculatus) undergoes a mutually exclusive alternative splicing event in the molt stages. Our newly determined DGAT inventory data provide a more complete scenario and insights into the evolutionary dynamics and functional diversification of DGATs in arthropods.

Analysis of the factors influencing the quality of farmed mud crabs in the Mekong Delta, Vietnam

Enhancing the quality of cultivated mud crabs is a crucial element for elevating productivity, quality, and economic effectiveness within the mud crab farming sector. This study investigates factors influencing the quality of farmed crabs in the Mekong Delta, Vietnam. The study employs descriptive statistical methods and synthesis approaches to analyse the current situation of crab farming in the Mekong Delta, Vietnam. Additionally, multivariate regression analysis was employed to identify factors affecting the quality of farmed crabs in the Mekong Delta, Vietnam. The survey results from 304 crab farmers in the Mekong Delta, Vietnam indicate that the total area of the Mekong Delta, Vietnam is 465 thousand hectares, with a production of 68 thousand tons. Among the provinces in the Mekong Delta, Vietnam, Kien Giang, Ca Mau, and Bac Lieu are the top three provinces with the highest area and production. Furthermore, factors influencing the quality of crab products during the farming process were analysed, and three main factors were identified: the decision to catch crabs at the fourth moult stage (Y4), the number of crab traps used, and the duration of the farming period. This research represents the first study on the quality of farmed crab in the Mekong Delta, Vietnam. Therefore, these findings provide guidelines for improving the quality of farmed crab and serve as a foundation for further research in this field

Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.

MicroRNA let-7 targets BmCDK1 to regulate cell proliferation and endomitosis of silk gland in the silkworm, Bombyx mori.

MicroRNAs play critical roles in multiple developmental processes in insects. Our previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands, thereby improving the silk production capacity. In this study, we elucidate the molecular mechanism underlying of let-7 regulates growth. Identification of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication. let-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN. Dual-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7, with only 1 binding site on its 3'-untranslated region. RNA interference of BmCDK1 inhibited cell proliferation, but this effect was counteracted by co-transfection with let-7 antagomir. Moreover, let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues, and further induced endomitosis in the silk gland in vivo. Knock down of BmCDK1 resulted in abnormal formation of a new epidermis, and larval development was arrested at the 2nd or 3rd molt stage. Taken together, our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination, possessing potential for improving silk yield in silkworm.

Long-read sequencing reveals the shell microbiome of apparently healthy American lobsters Homarus americanus from Atlantic Canada.

The shell microbial community of lobsters-a key factor in the development of epizootic shell disease (ESD)-is still insufficiently researched in Atlantic Canada and many knowledge gaps remain. This study aimed to establish a baseline description and analysis of the shell microbiome of apparently healthy lobsters from four locations in the region. More than 180 lobster shell swab samples were collected from New Brunswick, Nova Scotia and Prince Edward Island (PEI). PacBio long-read 16S rDNA sequencing and bioinformatic analyses in QIIME2 identified the shell-associated bacteria. The shell microbiome of healthy lobsters consisted mainly of the bacterial classes Gammaproteobacteria, Saprospiria, Verrucomicrobiae, Alphaproteobacteria, Flavobacteriia, Acidimicrobiia and Planctomycetia. The microbial composition differed regionally and seasonally, with some classes showing decreased or increased relative abundances in the PEI samples as well as in the winter and spring samples in Nova Scotia. The core shell microbiome included potentially pathogenic as well as beneficial bacterial taxa, of which some were present only in certain regions. Bacterial taxa that have previously been associated with ESD were present on healthy lobsters in Atlantic Canada, but their frequency differed by location, sampling time, and moult stage. This study indicated that geographical and seasonal factors influenced the shell microbiome of apparently healthy lobsters more than host factors such as sex, size, and moult stage. Our results provide valuable reference microbial data from lobsters in a disease-free state.

Trawl-related mortality and injuries to the southern king crab Lithodes santolla of Patagonia

The catch and discard of non-target species could have detrimental effects on bycatch, species due, among others, to the increase in mortality and prevalence of injuries. In Argentina, the southern king crab (SKC), Lithodes santolla, supports a profitable trap fishery but is also caught as bycatch in trawl fisheries. The aims of this work were: i) to quantify the mortality and injuries in SKC caught as bycatch in the hake fishery in San Jorge Gulf (southwestern Atlantic Ocean), ii) to assess the factors that affect their mortality and injury occurrence, and iii) to estimate their postdiscard mortality.Immediate mortality (IM) and injuries in SKC were recorded in hauls of the hake fishery, and then related to operational and biological variables. SKC vitality was assessed using the reflex action mortality predictor (RAMP) test. Postdiscard mortality was analyzed in aquaria and wild (sea cages) conditions. The IM of SKC in the common hake fishery was 31%. The IM, as well as increased injuries and decreased vitality, were directly associated with the time and conditions of air exposure. Both the IM and injury occurrence were also affected by biological variables such as the sex,size and molt stage. Postdiscard mortality was lower in wild conditions than in the aquaria (1%). Our results suggest that SKC could have high resistance to handling and high recovery capacity after its return to the sea. In consequence, the quick return of SKC to the sea may have a positive effect on SKC survival.

Meta‐analytic assessment of physiological markers for decapod crustacean welfare

AbstractDecapod crustacean welfare is not only an ethical consideration for legislative purposes. Under culture and fishery settings, poor environmental conditions and operator practices can cause stress, which compromises the immune system and increases the risk of disease. For finfish, there are established environmental and animal (group, individual) welfare indicators. This is not the case for crabs, lobsters or shrimp. While environment‐based indicators like temperature, pH and oxygen levels are easily transferable, there is no consensus for a common measure of stress to monitor welfare in decapod crustaceans. To address this shortcoming, we conducted a meta‐analysis of the primary literature to test for relationships between haemolymph (blood) condition, for example, levels of glucose, L‐lactate, haemocytes (n = 8), and sources of physical stress, for example, handling, trawling, emersion and transport (n = 12). Despite variations in effect sizes (n = 459), generalised linear mixed models repeatedly ranked L‐lactate (followed by urea and glucose) as the most significant physiological predictor of stress in the haemolymph, with trawling followed closely by emersion as the most impactful stressors. Duration post (stress) exposure, sex (male, female), water temperature and moult stage (pre‐, inter‐, post‐moult) were not associated significantly with stress prediction using the selected biochemical and cellular parameters, however, moult status was undefined in ~48% of literature sources and should be interpreted with caution. We present evidence that quantitating L‐lactate levels in the haemolymph represents a physiologic operational welfare indicator of decapod crustaceans, which complements existing condition/vigour indices.

New data on biology and captures of <i>Erimacrus isenbeckii</i> (Decapoda: Cheiragonidae) in the Shelikhov Bay (northern Okhotsk Sea)

New data on biology of hair crab Erimacrus isenbeckii are presented and ecological and geographical variability of this species is discussed. Molting, growth, sex and size structure of the crab are considered on the data collected in the Shelikhov Bay of the Okhotsk Sea in September-October, 2022. The male : female ratio of E. isenbeckii in the trap catches was 14 : 1. Gender differences in their age, weight, and carapace width were not significant (p < 0.05). The maximum size of E. isenbeckii in the Shelikhov Bay was 104 mm that is less the size values at the western coast of Kamchatka, southward from Kamchatka, and in the Aniva Bay of Sakhalin Island, and much smaller the crabs from the waters of Hokkaido Island and Alaska Peninsula. Rather high portion of the males on the 3rd intermolting stage was detected (57 %). The age composition of males varied significantly in dependence on stage of molting (p < 0.05). A freshly molted female on the 1st intermolting stage with an unoccluded genital opening was caught together with its exuvia. After molting, within the crab trap, it had increased the width of carapace in 6 mm. This new information could be used in monitoring of the E. isenbeckii population in the Shelikhov Bay.

A molting chemical cue (N-acetylglucosamine-6-phosphate) contributes to cannibalism of Chinese mitten crab Eriocheir sinensis

Under high-density culture, cannibalism occurs frequently during the molting of the Chinese mitten crabs Eriocheir sinensis, resulting in a large reduction in production. We found that the leakage of molting fluid from sexually immature crabs informs conspecifics that they are in a molting process. This hypothesis was verified through metabolomics analyses combined with behavioral experiments. The GlcNAc-6-P was identified as a molting biomarker from the differential metabolites by non-targeted metabolomics. In addition, we found that the concentration of GlcNAc-6-P in the molting fluid was significantly higher than other molting metabolites at different molting stages, reaching 5.84 μmol L−1, indicating that the molting fluid was the source of GlcNAc-6-P. Moreover, the behavioral experiments showed that crabs were actively approached to high concentrations of GlcNAc-6-P (1 μmol L−1), but had no obvious choice tendency at different concentrations of UTP, 20-HE and low concentrations of GlcNAc-6-P (0.1 μmol L−1, 0.01 μmol L−1) compared with the control groups. In conclusion, that E. sinensis by sensing the concentration change of GlcNAc-6-P can locate the source of GlcNAc-6-P release and actively approach the high concentration GlcNAc-6-P area and attack the molting crab, causing cannibalism.Blocking the reception pathway of molting chemical cues in E. sinensis, thereby preventing the perception of signals originating from conspecifics' molting in the vicinity, could lead to a reduction in cannibalistic behavior and an increase in overall production. Additionally, this method presents a prospective solution for addressing cannibalism in other crustacean species where such behavior is prevalent.

Cannibalism in cultured juvenile lobster Panulirus ornatus and contributing biological factors

Cannibalism is a major limitation to communally culturing the spiny lobster, Panulirus ornatus, and although frequently reported in research it is yet to be thoroughly investigated. To support continued innovation in culture, we must understand the contribution of cannibalism to juvenile mortality and identify influencing factors. This was accomplished through 900 h of time-lapse photography of three populations of juvenile lobsters over 30 d, including all ecdysis and cannibalism events to inspect individual weight, tank biomass, moult-stage, sex, and injury, as possible contributing factors. Total mortality was over 50% and attributed to the attack and cannibalism of moulting lobsters. Between 20 and 25% of moulting events in P. ornatus resulted in cannibalism of the moulting lobster. Population control and biomass stabilisation appear to be consequences of cannibalism, determined by fluctuations in tank biomass. Additionally, the moult stage of killer cannibals may suggest a nutritional benefit for lobsters in the latter half of the moult cycle. An increase in the average locomotor activity within a population during the hour before cannibalism occurs indicates that the factors driving cannibalism are present up to an hour before a moult. Here we demonstrate the flaws of previous assumptions related to the type and frequency of cannibalism in cultured P. ornatus systems and highlight contributing factors which warrant further study. Building from this research we may identify drivers of cannibalism among juvenile P. ornatus, with the aim of mitigating this behaviour in research and industry culture.

Growth, biochemical indices and transcriptomic profile of Chinese mitten crab (Eriocheir sinensis) respond to different ratios of dietary carbohydrates to lipids

IntroductionAlthough carbohydrates and lipids are important energy substances for Chinese mitten crab (Eriocheir sinensis), little is known about their synergistic effect on the growth, energy utilization characteristics and mechanisms involved in this process.MethodsA 58-d feeding experiment was conducted to investigate the effects of dietary carbohydrate to lipid ratio (C/L) on the growth performance, biochemical indices, and metabolism-related differential gene expression of juvenile E. sinensis in both intermolt (InM) and premolt (PrM) stages. Five experimental diets were formulated with increasing dietary C/L (1.34, 2.39, 3.59, 5.52 and 9.42).ResultsThe results showed that the weight growth rate of juvenile E. sinensis was highest in dietary C/L3.59 group, which was significantly higher than that in the other groups. As dietary C/L increased, the hepatic glycogen contents increased, but triglyceride contents decreased in the hepatopancreas of E. sinensis in the InM. In both two molting stages, the activities of glycogen synthase and fatty acid synthase paralleled with their contents, respectively. Crabs in the InM showed higher contents of triglyceride and the activities of glycolytic rate-limiting enzymes but lower contents of hepatic glycogen than those in the PrM, especially in the C/L 1.34 and C/L 3.59 groups. In all dietary groups, the activities and transcription of gluconeogenesis and fatty acid synthesis related enzymes were significantly higher in the InM than those in the PrM. KEGG analysis showed that differential genes were enriched in fatty acid biosynthesis, fatty acid metabolism, oxidative phosphorylation pathway, pentose phosphate pathway, pyruvate metabolism and steroid biosynthesis between different dietary groups and molting stages.DiscussionTo conclude, the optimal dietary C/L was estimated to be 3.59 for juvenile E. sinensis based on the survival and growth performance. Compared to PrM, E. sinensis in the InM was more active in the carbohydrate metabolism (glycolysis and gluconeogenesis) and fatty acid synthesis, with more triglyceride and less glycogen accumulated in the hepatopancreas.This study could contribute to better understanding the carbohydrate and lipid metabolism between different molting stages, and optimizing the precise feed formulation for juvenile E. sinensis.

Deciphering Molecular Mechanisms Governing the Reproductive Molt of Macrobrachium nipponense: A Transcriptome Analysis of Ovaries across Various Molting Stages

The relationship between molting and reproduction has received more attention in economically important crustacean decapods. Molting and reproduction are synergistic events in Macrobrachium nipponense, but the molecular regulatory mechanisms behind them are unclear. In the current study, we performed Illumina sequencing for the ovaries of M. nipponense during the molt cycle (pre-molting, Prm; mid-molting, Mm; and post-molting, Pom). A total of 66.57 Gb of transcriptome data were generated through sequencing, resulting in the identification of 105,149 unigenes whose alignment ratio with the reference genome exceeded 87.57%. Differentially expressed genes (DEGs) were annotated through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for gene classification and pathway analysis. A total of twenty-six molt-related DEGs were found, and their expression patterns were examined across various molting stages. The KEGG enrichment analysis revealed that the key pathways involved in regulating the molting process of M. nipponense primarily include the mTOR, insect hormone biosynthesis, TGF-beta, and Wnt signaling pathways. Our transcriptomic data suggest that these pathways crosstalk with each other to regulate the synthesis and degradation of ecdysone throughout the molt cycle. The current study has deepened our understanding of the molecular mechanisms of crustacean molting and will serve as a basis for future studies of crustaceans and other molting animals.

Sex- and age-dependent breeding plumage acquisition in monomorphic species, the Black-headed Gull Chroicocephalus ridibundus

In monomorphic species, like in the Black-headed Gull, both sexes look alike in breeding plumage. With large sets of data on captured and photographed birds and using the Underhill–Zucchini moult model, we provided a detailed pattern of breeding plumage development in this species by age and sex. This study, similar to other studies, documented first adults with the initial stage of head moult at the beginning of January, yet the mean start date of nuptial moult in adults was the end of February. Half of adults acquired full breeding plumage about mid-March and almost all of them before mid-April. The start date of nuptial moult was more variable in immatures. The mean start of head moult in immatures was 19th April, which is 52 days later than in adults, and the majority, i.e., 70%, did not complete moult until the end of May. We showed for the first-time sex-dependent breeding plumage acquisition in monomorphic species. According to the Underhill–Zucchini moult model, males started to moult on average 7 days earlier than females and their moult lasted 7 days longer. Hence, the final date of completed head moult was the same in both sexes. A fully developed hood is an important part of the status signalling during pairing; therefore, completing the moult before mating is important for both sexes.

Role of the Hox Genes, Sex combs reduced, Fushi tarazu and Antennapedia, in Leg Development of the Spider Mite Tetranychus urticae.

Mites, the second largest arthropod group, exhibit rich phenotypic diversity in the development of appendages (legs). For example, the fourth pair of legs (L4) does not form until the second postembryonic developmental stage, namely the protonymph stage. These leg developmental diversities drive body plan diversity in mites. However, little is known about the mechanisms of leg development in mites. Hox genes, homeotic genes, can regulate the development of appendages in arthropods. Three Hox genes, Sex combs reduced (Scr), Fushi tarazu (Ftz) and Antennapedia (Antp), have previously been shown to be expressed in the leg segments of mites. Here, the quantitative real-time reverse transcription PCR shows that three Hox genes are significantly increased in the first molt stage. RNA interference results in a set of abnormalities, including L3 curl and L4 loss. These results suggest that these Hox genes are required for normal leg development. Furthermore, the loss of single Hox genes results in downregulating the expression of the appendage marker Distal-less (Dll), suggesting that the three Hox genes can work together with Dll to maintain leg development in Tetranychus urticae. This study will be essential to understanding the diversity of leg development in mites and changes in Hox gene function.

Metabolomic and transcriptomic analysis reveals immune and hormone modulation at the molting stage of juvenile mud crabs challenged with Staphylococcus aureus and Vibrio alginolyticus

The mud crab (Scylla paramamosain) is an important economic breeding species along the southeast coast of China. As a crustacean, molting is critical for growth and reproduction. At each molting, a new external cuticle of mud crab is inherently regenerated, but along with molting the crabs are easily susceptible to pathogenic infection that might result in a low survival rate particularly at juvenile stages. To elucidate the potential response mechanism of juvenile mud crabs infected with bacteria in the molting process, integrated transcriptomic and metabolomic techniques were applied and the hormones, associated-hormone genes or immune genes modulated by challenge with Staphylococcus aureus or Vibrio alginolyticus were analyzed. The omic analysis showed that S. aureus or V. alginolyticus infection caused decreases in steroid hormones (cortisol, cortodoxone, cortisone, deoxycorticosterone) and fatty acid-derived hormones (prostaglandin A2, Δ17–6-keto prostaglandin F1α, 13,14-Dihydro-15-keto-tetranor prostaglandin F1α, prostaglandin B2, 2,3-Dinor-11β-prostaglandin F2α, thromboxane B2, and 11-Dehydro thromboxane B2). Correspondingly, transcriptomic analysis revealed that both S. aureus and V. alginolyticus infection downregulated the expression levels of fatty acid-derived hormone synthases. In addition, both bacterial infections caused upregulation in Toll, IMD, the Jak/Stat pathway, antimicrobial peptide, and the prophenoloxidase system. The study provides a new insight into the effects of bacteria on the juvenile crab molting process in vivo that lead to the modulation of both immune-associated genes and hormones.

Identification and profiling of long non-coding RNAs during molt cycle: An involvement of lnc1182 in the molt of white shrimp, Litopenaeus vannamei

Molt is an essential physiological process for crustacean growth. It is known to be controlled by factors such as hormones and steroids, but the role of long non-coding RNA (lncRNA), a potential regulatory molecule, has not been studied. In this study, we investigated the role of lncRNA in the molt process of white shrimp, Litopenaeus vannamei. The 9437 of 58,142 unigenes of de novo assembled transcriptome were identified as putative lncRNAs, and 768 lncRNAs were differentially expressed during molt cycles. Five differentially expressed lncRNAs were selected to determine their expressions in different tissues and molt stages. Most were expressed more in gills and epidermis than other tissues. Expressions of lnc1182 and lnc336 significantly decreased during premolt stages and increased at postmolt in gills and epidermis, while lnc179 expression increased during premolt stages. RNA interference was used to investigate the role of lnc1182 in the molt process. The molt of lnc1182 knockdown shrimp progressed to early premolt faster than shrimp injected with dsGFP, and also shortened molt duration. In addition, total sugar level in hemolymph was higher in the lnc1182 knockdown shrimp than the control shrimp, whereas gill Na+/K+ ATPase activity, total hemocyte count, and expression of molt-inhibiting hormone (MIH) were not changed. These suggested that lnc1182 is likely involved in the control of sugar metabolism during molting but not in ion exchange activity and MIH expression. Furthermore, lnc1182 and 20-HE probably regulated each other. RNA sequencing was used to investigate the set of genes associated with lnc1182. Fifty-five transcripts were differentially expressed in gills of lnc1182 knockdown shrimp. Expressions of candidate metabolism and immune-related genes were validated for their differential expressions in lnc1182 knockdown shrimp. Our work produced lncRNA profiles and indicated possible functions of lnc1182 in metabolism and immunity during the molt cycle in shrimp.

First occurrence of caryocaridids (Crustacea, Phyllocarida) in the Ordovician of North China

Although caryocaridids experienced a significant biodiversity boost during the Ordovician and spread over broad trans-equatorial areas, they have been rarely reported from Siberia, Tarim and North China. Herein, we describe a new species of caryocaridids, Soomicaris ordosensis sp. nov., from the Upper Ordovician of western Inner Mongolia, China, which represents the first report of caryocaridids from the North China craton and extends the palaeogeographical distribution of caryocaridid phyllocarids to the northwestern margin of the Palaeo-Tethys. Detailed morphological and morphometric studies show that these specimens can be ascribed to different ontogenetic stages. After close inspection, we infer that Soomicaris ordosensis sp. nov. should have experienced a long pre-adult time, with several moulting stages, during which time the posterior margin of carapace gradually extends backward to form the posterodorsal spine, and the posterior margin of carapace gradually transitions from convex, linear, and finally to a rightward-sloping sigmoid curve. The taphonomic features of caryocaridids from the Upper Ordovician in the western Inner Mongolia suggest in-situ and parautochthonous preservation. The carapaces have experienced weak phosphatic mineralization during diagenesis. The Ordovician caryocaridids were most likely stenothermic which preferred to inhabit the cool- to cold-water environments from the upper mesopelagic to the lower epipelagic zones, and showed the polar emergence phenomenon.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.