Molt Cycle Research Articles

High densities of large tuaki, the New Zealand cockle Austrovenus stutchburyi, provide a post-settlement predation refuge for conspecific juveniles

Bivalves are ecosystem engineers, and their effective management relies on a full understanding of the mechanisms that influence population dynamics. Juvenile tuaki (New Zealand cockle) Austrovenus stutchburyi, a numerically dominant and ecologically important bivalve in New Zealand estuaries, were found at higher densities when surrounded by high densities of large adults within Waitati Inlet, in southern New Zealand. Consequently, we hypothesised that adults provided a refuge from predation for juveniles, resulting in higher juvenile survival and retention in dense beds. The observed pattern was consistent with post-settlement Allee effects, where declines in density and truncated size distributions may have resulted in unsuccessful recruitment if a density threshold was reached. Field experiments were undertaken in 2020 and 2021 to test the potential sheltering effect of adults on retention and survival of juveniles, with and without the addition of organic loading of sediments. Presence of adults had significant effects on juvenile retention and interaction with organic loading in 2020, but not in 2021. To isolate the effects of adult presence on predation of juveniles, a series of laboratory experiments were carried out using the predatory crab Ovalipes catharus. Juvenile survivorship from crab predation was enhanced in the presence of large adults, consistent with the hypothesised sheltering effect. Further, predation rates increased significantly from winter to summer, which correlated with increasing water temperatures and the breeding and moult cycle of crabs. These findings across surveys, field and laboratory manipulations have significant implications for effective fisheries management and restoration of bed-forming bivalves.

Identifying the genes involved in the egg-carrying ovigerous hair development of the female blue crab Callinectes sapidus: transcriptomic and genomic expression analyses

BackgroundCrustacean female sex hormone (CFSH) controls gradually developing adult female-specific morphological features essential for mating and brood care. Specifically, ovigerous hairs are developed during the prepuberty molt cycle of the blue crab Callinectes sapidus that are essential for carrying the eggs until they finish development. Reduced CFSH transcripts by CFSH-dsRNA injections result in fewer and shorter ovigerous hairs than the control. This study aimed to identify the specific genes responsible for ovigerous hair formation using transcriptomic, genomic and expression analyses of the ovigerous setae at three stages: prepuberty at early (OE) and late premolt (OL), and adult (AO) stages.ResultsThe de novo Trinity assembly on filtered sequence reads produced 96,684 Trinity genes and 124,128 transcripts with an N50 of 1,615 bp. About 27.3% of the assembled Trinity genes are annotated to the public protein sequence databases (i.e., NR, Swiss-Prot, COG, KEGG, and GO databases). The OE vs. OL, OL vs. AO, and OE vs. AO comparisons resulted in 6,547, 7,793, and 7,481 differentially expressed genes, respectively, at a log2-fold difference. Specifically, the genes involved in the Wnt signaling and cell cycle pathways are positively associated with ovigerous hair development. Moreover, the transcripts of ten cuticle protein genes containing chitin-binding domains are most significantly changed by transcriptomic analysis and RT-qPCR assays, which shows a molt-stage specific, down-up-down mode across the OE-OL-AO stages. Furthermore, the expression of the cuticle genes with the chitin-binding domain, Rebers and Riddiford domain (RR)-1 appears at early premolt, followed by RR-2 at late premolt stage. Mapping these 10 cuticle protein sequences to the C. sapidus genome reveals that two scaffolds with a 549.5Kb region and 35 with a 1.19 Mb region harbor 21 RR1 and 20 RR2 cuticle protein genes, respectively. With these findings, a putative mode of CFSH action in decapod crustaceans is proposed.ConclusionsThe present study describes a first step in understanding the mechanism underlying ovigerous hair formation in C. sapidus at the molecular level. Overall, demonstrating the first transcriptome analysis of crustacean ovigerous setae, our results may facilitate future studies into the decapod female reproduction belonging to the suborder Pleocyemata.

The smaller, the better? First evaluation of growth and mortality in crayfish internally tagged with p-Chips

Abstract Small-bodied aquatic animals present a challenge to researchers seeking to uniquely mark individuals for scientific study. Microtransponder tags, such as p-Chips, represent the smallest electronic animal tags available to meet this need. The use of p-Chips to tag freshwater crayfishes, however, has not been explored. The goal of this study, therefore, was to determine the effects of p-Chip tagging on growth and survival of crayfishes in a controlled laboratory environment. We also investigated potential influences from variables such as sex, reproductive form, number of molt events, and crayfish size on mortality. We internally tagged individuals of the woodland crayfish Faxonius hylas (Faxon, 1890) (12.2–26.6 mm carapace length; CL) with either one p-Chip (single-tag) or one p-Chip and one visual implant elastomer tag (double-tagged) and observed the effects over a period of 90 days. Survival probability over time was not statistically different (P > 0.05) among tagging groups, sex, and reproductive form. Survival rates were similar across all tagging groups, with 75% in the control group, 77% in the double-tagged group, and 78% in the single-tag group. A strong correlation, however, was observed between survival and the number of molt events. Additionally, there was a negative correlation between survival and crayfish size (CL), indicating a higher risk of mortality for larger individuals. There were no statistically significant differences in growth in CL (P = 0.30) or mass (P = 0.19) among the tagging groups. We conclude that p-Chips are a viable tagging option for the study of crayfishes given their size, readability, and retention through molting cycles. We recommend that future studies repeat this experiment using smaller individuals to determine the minimum crayfish size compatible with p-Chip tagging. It is also important to test p-Chips with other crayfish species and compare the growth and mortality of crayfish tagged with p-Chips in natural habitats.

Avian nuptial plumage coloration changes differently when exposed to urban and rural areas

Avian plumage coloration could be altered by external factors like dust accumulation, but the effect of environmental conditions on such post-moult colour changes are poorly studied. Here, we investigated how exposure to the atmosphere in habitats of differing anthropogenic pressures modifies UV/blue structural plumage coloration — a sexually selected trait in eastern bluebirds Sialia sialis. We collected feather samples from live birds and distributed them in three types of habitats: (i) urban roads with low traffic, (ii) urban roads with high traffic, and (iii) rural roads with low traffic. After 4 weeks, the brightness of feathers decreased in all types of habitats-resulting in less-ornamented coloration. Changes in UV and blue chroma, however, varied with habitat type; chroma decreased in urban areas (becoming less ornamented), but increased in rural locations (becoming more ornamented). According to physiological models of avian colour vision, however, these changes in coloration tended to be below perceptual thresholds. Our results underscore the importance of how human activity can influence plumage colour of individual birds during the period between moult cycles which could have an impact on avian visual signalling. The study opens venues for further studies focusing on how the optical properties of specific airborne particles can influence coloration of wild-living animals.Significance statementPlumage coloration plays an important role in avian visual communication. In addition to moult-based change, coloration may change between moult cycles due to exposure to external factors like, for example, dust accumulation. Ecological factors modifying a passive plumage change, however, remains poorly studied. We collected samples of structurally coloured feather from a species wherein plumage colour is sexually selected and exposed them to outdoor conditions. We demonstrate that UV/blue chroma of coloration changes differently in urban and rural habitats. It increased in rural and decreased in urban areas. On the other hand, brightness decreased in both habitat types. Using physiological models of avian colour vision, however, we found that these colour changes were likely not perceptible to birds. Our study provides the first evidence that changes in coloration of fully grown feathers may be habitat dependent.

Molecular signature of the ontogenic development of the prawn Macrobrachium tenellum

The prawn Macrobrachium tenellum shows aquaculture potential due to its well-defined reproductive cycle linked to female nutritional requirements. Significant changes occur in egg composition during the 16 to 17-day embryo development. Understanding the ontogenic proteins is crucial for developmental insights and controlled reproduction. We employed free-label quantitative proteomics to analyze egg peptides at the initial and final stages of wild females. Using the emPAI protocol and Proteome Discoverer 2.0, we identified 89 differentially expressed proteins in M. tenellum eggs. Of these, 27 were exclusive to early-stage development and three to late-stage. Abundant proteins included Vitellogenin, glyceraldehyde-3-phosphate dehydrogenase, histone 4, beta-actin, and hemocyanin. Gene Ontology analysis revealed 518 terms across molecular functions, biological processes, and cellular components using the GoRetriever tool of AgBase and the CateGOrizer tool of the Animal Genome Research Program. Carbohydrate metabolism was significant in early-stage development, with glyceraldehyde-3-phosphate dehydrogenase being the second most abundant protein. Proteins involved in ATP synthesis and cytoplasmic proteins associated with catalytic and binding activities related to primary metabolism were also detected. Our study elucidates the role of Vitellogenin in lipid transport activity and its potential involvement in the juvenile hormone feedback pathway. This pathway includes farnesoic acid O-methyltransferase and juvenile hormone epoxide oxidase, regulating protein biosynthesis, molt cycles (including chitinase activity), and potentially influencing controlled reproduction. Our proteomic analysis provides insights into the molecular mechanisms driving Ontogenic development in Macrobrachium tenellum, with implications for controlled reproduction strategies and advancements in aquaculture practices.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Pattern and intensity of moult in White-capped/Shy Albatrosses Thalassarche steadi/cauta

Albatrosses typically only replace a subset of flight feathers each annual moult cycle. We scored the moult of 662 White-capped/Shy Albatrosses Thalassarche steadi/cauta, using birds that had been killed by longline fisheries off South Africa. Most adults and immatures alternated replacing the outer primaries (phase 1) and inner primaries (phase 2), but there was considerable variation among adults, with 0–10 primaries replaced each moult cycle. Birds in phase 1 moult replaced fewer primaries than birds in phase 2, but the proportion by mass was similar. Adults replaced more primaries than immatures as there was a disproportionate number of adults in phase 2 moult (82%), which might indicate that most adults off South Africa are in their first year after a successful breeding attempt, but a similar bias occurred among beached birds from New Zealand and Australia. The outer primaries were usually replaced outwards from P8, but the replacement sequence was more variable among the inner primaries. Secondary moult overlapped with primary moult, but not all secondaries were moulted each year. Adults had more active moult centres and replaced more secondaries at once than immatures. Tail moult started after primary moult, with 1–11 feathers growing from 1–6 active centres. Some birds replaced their rectrices in pairs, with often 2 or 3 pairs moulting at once, but others replaced alternate feathers or almost all rectrices at once. A few birds exhibited intense wing moult, but the norm was to replace only 1 or 2 primaries and 1–6 secondaries at once. Age- and sex-related differences in moult intensity presumably result from different time constraints. Adult White-capped/Shy Albatrosses take a year off between successful breeding attempts, allowing time for a more protracted moult. However, we cannot explain the intense moult in some immatures. Our findings support recent studies showing that albatross flight feather moult patterns are more varied than previously reported.

Walking leg regeneration in the sea spider Nymphon brevirostre Hodge, 1863 (Pycnogonida)

Regeneration is widespread across all animal taxa, but patterns of its distribution and key factors determining regeneration capabilities stay enigmatic. A comparative approach could shed light on the problem, but its efficacy is limited by the fact that data is only available on a few species from derived taxa. Pycnogonida are nested basally within the Chelicerata. They can shed and replace their walking legs and have a high regeneration capacity. In this work, we carried careful observation on leg appendotomy and regeneration processes in a sea spider under laboratory settings. The limb structure and in vivo observation reveal autotomy as the most likely appendotomy mechanism. High regeneration capabilities were ascertained: an anatomically normal but small leg appeared in a single molting cycle and the full functionality regained in 2–3 cycles. Wound closure after appendotomy in N. brevirostre primarily relies on hemolymph coagulation, which apparently differs from both xiphosurans and crustaceans. Regeneration is provided by proliferation in the leg cutpiece. Regenerative morphogenesis resembles the normal ontogenetic morphogenesis of a walking leg, but accelerated. Unlike in most arthropods, in N. brevirostre, regeneration does not necessarily correspond to the molting cycle, inferring a plesiomorphic state.

Expression pattern of AGPATs isoforms indicate different functions during the triacylglyceride synthesis in Chinese mitten crab, Eriocheir sinensis

The 1-acylglycerol-3-phosphate acyltransferase (AGPAT) acts as a crucial enzyme in the process of triacylglycerol (TAG) synthesis, enabling the acylation of lysophosphatidic acid (LPA) into phosphatidic acid (PA). In order to decode the distinctive roles of AGPAT isoforms in the TAG production pathway, three AGPAT isoforms were detected for the first time in the Chinese mitten crab Eriocheir sinensis (Es-agpat2, Es-agpat3, and Es-agpat4). The mRNA levels of Es-agpat2 and Es-agpat4 demonstrated a conspicuous presence in the hepatopancreas, with subsequent high levels in the heart, muscle, and thoracic ganglion. On the other hand, the thoracic ganglion exhibited abundant levels of Es-agpat3, while other tissues recorded relatively low expression levels. Observing the molting cycle of E. sinensis, the hepatopancreas showed minimum expression levels of Es-agpat2 and Es-agpat4 at stage A/B. A peak at stage C was noted, which was then followed by a gradual drop until stage E. For the ovarian development cycle, stage II witnessed the maximum expression level of Es-agpat2 and Es-agpat4, succeeded by a sharp fall in stage III. After this, there was an increasing trend from stage III up to stage V. Expression of Es-agpat3 in the hepatopancreas was consistently lower than Es-agpat2 and Es-agpat4 during either the molting or ovarian development. However, in terms of ovarian expression, Es-agpat3 outperformed Es-agpat2 and Es-agpat4. It exhibited a steep increase in expression, peaking at stage II and subsequently diminishing. In situ hybridization (ISH) revealed that in stages II and IV hepatopancreas, Es-agpat4-mRNA was primarily located in fibrillar cells (F cell) and resorptive cells (R cell), with no signal from Es-agpat3. During stage II of ovarian development, both Es-agpat3-mRNA and Es-agpat4-mRNA were located in the cytoplasm of previtellogenic oocyte (PRO) and endogenous vitellogenic oocyte (EN), with no expression at stage IV. Additionally, the silencing of Es-agpat2 and Es-agpat4 caused a downward trend in the expression levels of all subsequent genes in the E. sinensis TAG synthesis pathway. To sum up, these findings suggest that the three Es-agpats may have unique functions in TAG synthesis during either the molting process or ovarian maturation of E. sinensis.

Insights Into a Chitin Synthase of Kuruma Shrimp Penaeus japonicus and Its Role in Peritrophic Membrane and Cuticle Formation.

Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans.

The role of octopamine and crustacean hyperglycemic hormone (CHH) in branchial acid-base regulation in the European green crab, Carcinus maenas.

Crustaceans' endocrinology is a vastly understudied area of research. The major focus of the studies on this topic to date has been on the molting cycle (and in particular, the role of crustacean hyperglycemic hormone (CHH)), as well as the role of other hormones in facilitating physiological phenotypic adjustments to salinity changes. Additionally, while many recent studies have been conducted on the acclimation and adaptation capacity of crustaceans to a changing environment, only few have investigated internal hormonal balance especially with respect to an endocrine response to environmental challenges. Consequently, our study aimed to identify and characterize endocrine components of acid-base regulation in the European green crab, Carcinus maenas. We show that both the biogenic amine octopamine (OCT) and the CHH are regulatory components of branchial acid-base regulation. While OCT suppressed branchial proton excretion, CHH seemed to promote it. Both hormones were also capable of enhancing branchial ammonia excretion. Furthermore, mRNA abundance for branchial receptors (OCT-R), or G-protein receptor activated soluble guanylate cyclase (sGC1b), are affected by environmental change such as elevated pCO2 (hypercapnia) and high environmental ammonia (HEA). Our findings support a role for both OCT and CHH in the general maintenance of steady-state acid-base maintenance in the gill, as well as regulating the acid-base response to environmental challenges that C. maenas encounters on a regular basis in the habitats it dwells in and more so in the future ocean.

Zinc is incorporated into the exoskeleton during post-ecdysial mineralization and inhibits exoskeletal calcification in the blue crab, Callinectes sapidus

Zinc is an essential but toxic metal with both natural and anthropogenic sources. Zinc has been reported to be present in crustacean exoskeleton, but it has remained unknown as to when zinc is incorporated into the shell during the molting cycle and the effects zinc has on exoskeleton properties. This study was conducted to identify a period during the molting cycle, in which zinc is incorporated into the exoskeleton of the blue crab, Callinectes sapidus, and to identify deleterious effects of zinc incorporation on the exoskeleton. It was hypothesized that zinc would be incorporated into the shell during the mineralization phase using calcium transporters, which would inhibit exoskeletal calcification. Post-ecdysial blue crabs were given two injections of zinc in the form of zinc chloride dissolved in Pantin's crustacean saline at the arthrodial membrane at three treatment levels: 0.0, 1.0 and 5.0 µg Zn/g wet weight. Exoskeletal and hemolymph samples were then analyzed for zinc, calcium, and magnesium content. Gill, muscle, and hepatopancreas samples were analyzed for zinc only. Epidermis samples were analyzed for carbonic anhydrase activity. The results showed that the injection dose of 1.0 µg Zn/g wet weight resulted in significant accumulation of zinc in the exoskeleton. There was no significant accumulation of exoskeletal zinc following 5.0 µg Zn/g wet weight injections. A significant reduction in exoskeletal calcium content in crabs treated with 1.0 or 5.0 µg Zn/g wet weight was also observed. The hypothetical model explaining zinc's incorporation into the exoskeleton and inhibition of exoskeletal calcification is proposed. Additionally, for the soft tissues examined, significant zinc accumulation was only observed in the hepatopancreas following zinc treatment. Our data points to the existence of crustacean zinc transporter. This study is the first to present evidence that zinc is deposited to the exoskeleton during post-ecdysial mineralization and inhibits exoskeletal calcification in a crustacean.

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.

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.

Functional transcriptome reveals hepatopancreatic lipid metabolism during the molting cycle of the Chinese mitten crab Eriocheir sinensis

Crustacean molting is highly related to energy and lipid metabolism. This study was conducted to detect the changes of total lipids (TL), triacylglyceride (TAG), phospholipid (PL) and lipid droplets in hepatopancreas, and then to investigate the gene expression patterns related to hepatopancreatic lipid metabolism during the molting cycle of Chinese mitten crab Eriocheir sinensis. Hepatopancreatic TL and TAG increased significantly from post-molt stage to pre-molt stage, then decreased significantly from pre-molt stage to ecdysis stage, which is consistent to the changes of neutral lipid-rich adipocytes in hepatopancreas. By transcriptomic analysis, 65,325 transcripts were sequenced and assembled, and 28,033 transcripts were annotated. Most genes were related to energy metabolism, and the enriched genes were involved in carbohydrate and lipid metabolism and biosynthesis, especially in de novo synthesis of fatty acids and TAG, and ketone body production. Compared to the inter-molt stages, acetyl-CoA carboxylase, fatty acid synthase and other genes related to the synthesis of fatty acids were upregulated in the pre-molt stage. TAG synthesis related genes, including Glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferases, were upregulated in the post-molt stage compared to the inter-molt stage. The expression of ketone body-related genes had no significant changes during the molting cycle. Compared to the TAG synthetic pathway, ketone body biosynthesis may contribute less/secondarily to fatty acid metabolic processes, which could be involved in the other physiological processes or metabolism. In conclusion, these results showed that TAG is the major lipid deposition during inter- and pre-molt stages, and the most genes are related to the fatty acids and TAG metabolism in the hepatopancreas during the molting cycle of E. sinensis.

Disruption of oogenesis and molting by methoprene and glyphosate in Gammarus fossarum: involvement of retinoic acid?

In the last decade, the freshwater amphipod Gammarus fossarum proved to be a promising sentinel species in active biomonitoring programs to assess the effects of environmental contamination on non-target organisms. Given that the highly conserved retinoid (RETs) metabolism supports many biological functions and is perturbed by xenobiotics and used as biomarker for vertebrates, we explored the RETs functions in the crustacean model Gammarus fossarum. More specifically, we studied the implication of all -trans retinoic acid (atRA) in the reproduction (embryo, oocyte, and juvenile production) and development (success and delay of molting) by exposing G. fossarum females to atRA and citral (CIT), a known inhibitor of RA synthesis. In parallel, we exposed gammarids to methoprene (MET) and glyphosate (GLY), two pesticides suspected to interfere with atRA metabolism and signaling and frequently found in water systems. After 14days of exposure, atRA, CIT, and MET reduced the number of oocytes, whereas only MET caused a reduced number of embryos. After 44days, MET and GLY showed a tendency to decrease juvenile production. The duration of the molting cycle increased following the exposures to atRA and MET, while the treatment with CIT caused a typical endocrine disruptive inverted U-shaped curve. The exposure to GLY led to increased duration of the molting cycle at the lowest concentrations and lowered molting success at the highest concentration tested. This study highlights for the first time the implication of RA in the oogenesis and molting of G. fossarum and suggests that it may be a potential mediator of MET-induced effects on these processes. This study adds to the comprehension of the reproductive and developmental control in G. fossarum and opens new research avenues to study the effects of xenobiotics on the RET system in this sentinel species. Ultimately, our study will drive the development of RET-based biomarkers for non-target aquatic invertebrates exposed to xenobiotics.

ROLE OF PITYROGRAMMA CALOMELANOS (L.) LINK IN DIETARY AND NUTRITIONAL INDICES OF SPODOPTERA LITURA (F.) (LEPIDOPTERA: NOCTUIDAE)

The polyphagous, ubiquitous, destructive pest, Spodoptera litura(F.) affects 150 host plant species and notably their larval forms arenotorious. Extensive feeding and rapid reproduction cycles led the farmers andindustrialists to seek the effective chemical and synthetic pesticides. Randomand inappropriate usages of pesticides imposed a negative impact on non-targetorganisms, environment and abiotic factors and also pest resurgence to theparticular pesticides. To overcome this unnatural incidence, biopesticides arepracticed as an alternative source. Plant botanicals play a huge role as insectgrowth regulators (IGRs) by inhibiting the regular moulting cycle, developmentand survival of insect pests. Also, the silverback fern, Pityrogramma calomelanosare reported with several secondary metabolites which are known for theirpesticidal properties. Hence, the present work was framed to study the dietaryresponses as well as nutritional indices of S. litura treated with crudeextract of P. calomelanos. The treated larvae showed dietary andpost ingestion responses prior to moulting and death. On the 4th day oftreatment, reduction in food intake (522.89 mg), larval weight gain (41.02 mg),ECI (56.25 %), ECD (46.30 %), AD (47.21 %) and RGR (3.06 mg/mg/day) wasobserved as dose dependent manner except the feacal production (378.18 mg) ascompared to control. Therefore, the ethanolic extract of a fern, P. calomelanoscan be recommended as a biopesticides for its post ingestion andanti-nutritional effects on S. litura larvae.

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.

Dual roles of CYP302A1 in regulating ovarian maturation and molting in Macrobrachium nipponense

CYP302A1 is a member of the Halloween genes in the cytochrome P450 supergene family, which play an important regulatory role in the synthesis of 20-hydroxyecdysone (20E) in crustaceans and insects. In this study, we found that the Mn-CYP302A1sequence included typical CYP450 conserved domains. Phylogenic showed that it is closely related to crustaceans and insects. q-PCR analysis indicated that Mn-CYP302A1 was highly expressed in the ovaries and peaked before ovarian maturation. Mn-CYP302A1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization indicated that Mn-CYP302A1 was mainly distributed in the nucleus, yolk granules, cell membrane and cytoplasm To further establish the function of CYP302A1, a 21-day RNA interference experiment was conducted. On day 16, the Gonad Somatic Index of the control group and the experimental group showed significant differences, with GSI of 11.72% in the control group and 3.21% in the experimental group. The cumulative proportion of the second entry into stage O-Ⅲ was 100% in the control group, while it was 41.67% in the experimental group on day 21. The ecdysone content was 8.91nmol/L in the control group and 6.11nmol/L in the experimental group on day 9. A significant difference in the molting proportion between the control group and the experimental group was also observed (49% in the control group and 34% in the experimental group) on day 16. Statistical results showed that the average molting cycle of the control group was 14.5 days, while that of the experimental group was 16.5 days. However, the morphological structure of ovarian tissue did not abnormal change. Therefore, the results of this study suggest that Mn-CYP302A1 can promote ovarian maturation and molting in female M. nipponense.

Influence of Moult Cycles on Digestive Enzyme Activities during Early Larval Stages of Panulirus ornatus

The tropical spiny lobster, Panulirus ornatus, has a complex life cycle characterised by a series of moults that occur throughout pelagic larval stages. Significant morphological, physiological, and biochemical changes commonly coincide with moulting and can have dietary implications when culturing this species. Digestive enzyme activities respond to nutritional requirements and have provided useful insight into nutrient use dynamics associated with first-feeding in P. ornatus. Beyond first-feeding, however, information on digestive enzyme activities in P. ornatus is scarce. Greater knowledge of fluctuations in digestive enzyme activities during moult cycles should facilitate better formulation of feeds and more efficient feeding regimens. As an initial step towards this goal, the present study evaluated the influence of moult cycles on digestive enzyme activities during early larval stages of P. ornatus. The investigation focused exclusively on early larval stages (stages I–III) when delivery of appropriate feeds and nutrition can dramatically affect subsequent growth and survival.