Microalgal Preference and Feeding Density of Selected Microalgae Diets by Blue Swimming Crab Portunus pelagicus (Linnaeus, 1758).

<b>Background and Objectives:</b> Blue swimming crab, <i>Portunus pelagicus</i> (Linnaeus, 1758) are economically important sources of soft-shell products for export demand. However, increasing over-exploitation and pollution towards the sea resources affected the production of the <i>P. pelagicus</i>. The study aimed to determine the microalgal preference and feeding density of the monospecific (<i>Nannochloropsis oculata</i>, <i>Chlorella vulgaris</i>, <i>Isochrysis galbana</i> and <i>Chaetoceros wighamii </i>) by individual <i>P. pelagicus </i>larvae. <b>Materials and Methods:</b> In the microalgal preference study, one polystyrene tray with 100 holes was set up in the experimental tank (200 L) to place the 50 mL centrifuges tube. For feeding density study, the survival rate, Specific Growth Rate (SGR), Body Weight (BW) and Larvae Stage Index (LSI) of each larvae stage were collected from Zoea 1 (Z₁) till megalopa. <b>Results:</b> Results for mean microalgal preference of individual larvae preferred most <i>Chlorella vulgaris</i> during Zoea 1 and 2, the larvae consumed the highest of <i>Nannochloropsis</i> <i>oculata</i>. For Zoea 3 and 4, the highest preference is <i>Isochrysis galbana</i>. For feeding regime, Zoea 1, larvae fed rotifer, <i>Artemia</i> and <i>Chlorella vulgaris</i> (T₃), was the highest survival rate, Zoea 2 was larvae fed rotifer, <i>Artemia</i> and <i>Nannochloropsis oculata</i> (T₅) and both Zoea 3 and Zoea 4, larvae fed rotifer, <i>Artemia</i> and <i>Isochrysis galbana</i> (T₄) was the highest survival rate. The highest SGR and LSI were obtained in T₄ cultured in 14 days. <b>Conclusion:</b> In conclusion, the microalgal preference and feeding density of the selected microalgae are different depending on each larvae stage.

Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material.

The complete larval development of Amphithrax hemphilli was described, illustrated, and compared with that of the previously described larvae of the genus Mithrax sensu lato. Specimens of A. hemphilli were sampled from the northeastern Brazilian coast. The larval development of A. hemphilli consisted of two zoeal stages and one megalopa. Amphithrax hemphilli shows morphological features in all stages of larval development that differ from those observed in other species of Amphithrax, Mithrax, and Maguimithrax. In the first larval stage, A. hemphilli was the only species with two aesthetascs on the antennule and the coxal endite of the maxillule with five setae. In the second larval stage, A. hemphilli was distinguished from the other species of the genus Mithrax sensu lato by the presence of five setae on the basial endite of the maxilla and 26 setae in the scaphognathite of the maxilla. Finally, the megalopa stage of A. hemphilli is characterized by the number of aesthetascs on the three-segmented exopod of the antennule, the setation of both the endopod of the third maxilliped, and pleonites 1-6. Based on the results obtained in the present study, we verified that the more advanced the larval stage, the more distinct characteristics are observed among species of the genus Mithrax sensu lato, facilitating the identification of species through later larval stages.

Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain).

Forkhead box protein L2 (Foxl2) is involved in multiple physiological processes, such as ovarian development, granulosa cell differentiation, ovarian follicle development, and oocyte growth. In this study, a Spfoxl2 gene encoded 530 amino acid protein with characteristic forkhead (FH) domain was identified from transcriptome data of mud crab Scylla paramamosain and validated the accuracy by PCR technology. Meanwhile, the orthologues of the Spfoxl2 gene in other 14 crustacean species were identified with the same method. Further multiple sequence alignment analysis revealed the Foxl2 was highly conserved, especially in the FH domain, even completely identical in several species. Besides, the semi-quantitative PCR (Sq-PCR) result showed Spfoxl2 gene was mainly expressed in the gonad (testis and ovary). Further quantitative real-time PCR (qRT-PCR) result demonstrated its expression level in the testis was significantly higher than that in the ovary (p < 0.01). In addition, the qRT-PCR result showed that in zoea V, megalopa, and larval I, the expression level of Spfoxl2 in megalopa is the highest. In addition, a putative Foxl2 binding site was identified on the promoter region of Spvtg, and knockdown of Spfoxl2 mediated by RNAi technology increased the expression of Spvtg in the ovary, suggesting Spfoxl2 might be the upstream negative regulator of Spvtg. Overall, this study provided new insights into the role of Spfoxl2 in ovary development through regulating Spvtg expression in S. paramamosain.

Transcriptional changes revealed water acidification leads to the immune response and ovary maturation delay in the Chinese mitten crab Eriocheir sinensis.

Nowadays, due to increasing carbon dioxide released, water acidification poses a series of serious impacts on aquatic organisms. To evaluate the effects of water acidification on crustaceans, we focused on the Chinese mitten crab Eriocheir sinensis, which is a spawning migration and farmed species in China. Based on histological and oocyte transparent liquid observation, we found that the acidified environment significantly delayed the ovarian maturation of E. sinensis. Moreover, RNA-seq was applied to obtain gene expression profile from the crab's gills and ovaries in response to acidified environment. Compared with control groups, a total of 5471 differentially expressed genes (DEGs) were identified in acidified gills and 485 DEGs were identified in acidified ovaries. Enrichment analysis indicated that some pathways also responded to the acidified environment, such as PI3K-Akt signaling pathway, Chemokine signaling pathway, apoptosis, and toll-like receptor signaling pathway. Subsequently, some DEGs involved in immune response (ALF, Cathepsin A, HSP70, HSP90, and catalase) and ovarian maturation (Cyclin B, Fem-1a, Fem-1b, and Fem-1c) were selected to further validate the influence of water acidification on gene expression by qRT-PCR. The results showed that the expression level of immune-related genes was significantly increased to response to the water acidification, while the ovarian maturation-related genes were significantly decreased. Overall, our data suggested that E. sinensis was sensitive to the reduced pH. This comparative transcriptome also provides valuable molecular information on the mechanisms of the crustaceans responding to acidified environment.

Identification, characterization and mRNA transcript abundance profiles of the carboxylesterase (CXE5) gene in Eriocheir sinensis suggest that it may play a role in methyl farnesoate degradation.

The sesquiterpenoid methyl farnesoate (MF) is a de-epoxidized form of insect juvenile hormone (JH) III in crustaceans, and its precise titer plays important roles in regulating many critical physiological processes, including reproduction and ovarian maturation. Understanding the synthetic and degradation pathways of MF is equally important for determining how to maintain MF titers at appropriate levels and thus for potential applications in crab aquaculture. Although the synthetic pathway of MF has been well established, little is known about MF degradation. Previous research proposed that specific carboxylesterases (CXEs) that degrade MF in crustaceans are conserved from those of JH III. In this study, we identified a novel Es-CXE5 gene from Eriocheir sinensis. The Es-CXE5 protein contains some conserved motifs, including catalytic triad and oxyanion hole, which are characteristics of the biologically active CXE family. The phylogenetic analysis showed that Es-CXE5 belongs to the hormone/semiochemical processing group of the CXE family. Moreover, Tissue and stage-specific expression results suggested that Es-CXE5 expression in hepatopancreas was highest and associated with the hemolymph MF titer. Furthermore, Es-CXE5 mRNA transcripts were detected in both in vitro and in vivo experiments and ESA experiment in the hepatopancreas and ovary. The results of this study showed that Es-CXE5 mRNA abundance in the hepatopancreas was notably induced by MF addition but had no effect on the ovary. Taken together, our results suggest that Es-CXE5 may degrade MF in the hepatopancreas and may thus be involved in ovarian development in E. sinensis.

The significant sex-biased expression pattern of Sp-Wnt4 provides novel insights into the ovarian development of mud crab (Scylla Paramamosain).

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5'-UTR and a 672 bp 3'-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene.

A network analysis of crab metamorphosis and the hypothesis of development as a process of unfolding of an intensive complexity.

Development has intrigued humanity since ancient times. Today, the main paradigm in developmental biology and evolutionary developmental biology (evo-devo) is the genetic program, in which development is explained by the interplay and interaction of genes, that is, by the action of gene regulatory networks (GRNs). However, it is not even clear that a GRN, no matter how complex, can be translated into a form. Therefore, the fundamental enigma of development still remains: how is a complex organism formed from a single cell? This question unfolded the historical drama and the dialectical tension between preformation and epigenesis. In order to shed light on these issues, I studied the development of crabs (infraorder Brachyura), as representative of the subphylum Crustacea, using network theory. The external morphology of the different phases of brachyuran metamorphosis were modeled as networks and their main characteristics analyzed. As one could expect, the parameters usually regarded as indicative of network complexity, such as modularity and hierarchy, increased during development. However, when more sophisticated complexity measures were tested, it was evidenced that whereas a group of complexity measures increased during development, another group decreased. This led to consider that two kinds of complexities were being measured. I called them intensive and extensive complexity. In view of these results, I propose that crab development involves a passage from an intensive to an extensive complexity. In other words, crab development can be interpreted as a process of unfolding of an intensive, preexistent complexity.

Effects of environmental antidepressants on colour change and locomotor behaviour in juvenile shore crabs, Carcinus maenas.

Juvenile crabs of Carcinus maenas thrive in coastal waters reputed to be the receptacle of continental pollution. Amongst the many pollutants encountered, antidepressants, such as fluoxetine (FLX) and venlafaxine (VEN), often detected at the ng•L-1 range, are particularly worrying because of their action on the levels of monoamines, such as serotonin, noradrenaline and dopamine. In crustaceans, those monoamines are involved in colour change through their action on neuropeptide hormones. In addition, they are known to have a role in different behaviours, such as locomotion. Both colour change and locomotion are strategies used by juvenile crabs to hide and escape from predators. To investigate if the presence of antidepressants may alter behaviours of ecological importance, juvenile crabs were exposed to environmentally realistic concentrations of either 5 ng•L-1 of FLX alone or in combination with VEN at 5 ng•L-1. The ability to change colour depending on the environment and the locomotor activity of juvenile crabs were monitored weekly over 25 days. Animals exposed to antidepressants displayed a different pattern of colour change than the controls, especially those exposed to the combination of FLX and VEN at 5 ng•L-1 each, and were less efficient to adapt to their environment, i.e., they were not as pale and not as dark as controls or crabs exposed to FLX at 5 ng•L-1. Moreover, juvenile crabs exposed to the combination of antidepressants exhibited an enhanced locomotor activity throughout the exposure period with a higher velocity and distance moved as well as more time spend moving. The alteration of cryptic behaviours, such as colour change and locomotion by antidepressants persistently present in marine environment at low concentrations may have an impact on the survival of juvenile of C. maenas on the long term.

Gonadal development and biochemical composition of Chinese mitten crabs (Eriocheir sinensis) from four sources.

The gonadal development; edible tissue ratio; and proximate, fatty acid, and free amino acid composition were examined and compared among Eriocheir sinensis samples from four sources. The gonadosomatic index (GSI) of Heilongjiang (HLJ) crabs was significantly higher than that of the other crabs from September to October (P < 0.05). Of all the edible tissues of E. sinensis, HLJ crab had the highest linolenic acid (18:3n3, LNA) and arachidonic acid (20:4n6, ARA) contents (P < 0.05), while Qinghai (QH) crab had the highest eicosapentaenoic acid (20:5n3, EPA) content (P < 0.05). The highest docosahexaenoic acid (22:6n3, DHA) and DHA/EPA contents were observed in Shandong (SD) crabs (P < 0.05), and the highest linoleic acid (18:2n6, LA) content was found in Shanghai (SH) crabs (P < 0.05). In conclusion, E. sinensis inhabiting relatively low-temperature regions (northern China) can initiate gonadal development earlier, and the quality of E. sinensis living in paddies and lakes is better than that of crabs living in ponds.

Field-based body temperatures reveal behavioral thermoregulation strategies of the Atlantic marsh fiddler crab Minuca pugnax.

Behavioral thermoregulation is an important defense against the negative impacts of climate change for ectotherms. In this study we examined the use of burrows by a common intertidal crab, Minuca pugnax, to control body temperature. To understand how body temperatures respond to changes in the surface temperature and explore how efficiently crabs exploit the cooling potential of burrows to thermoregulate, we measured body, surface, and burrow temperatures during low tide on Sapelo Island, GA in March, May, August, and September of 2019. We found that an increase in 1°C in the surface temperature led to a 0.70-0.71°C increase in body temperature for females and an increase in 0.75-0.77°C in body temperature for males. Body temperatures of small females were 0.3°C warmer than large females for the same surface temperature. Female crabs used burrows more efficiently for thermoregulation compared to the males. Specifically, an increase of 1°C in the cooling capacity (the difference between the burrow temperature and the surface temperature) led to an increase of 0.42-0.50°C for females and 0.34-0.35°C for males in the thermoregulation capacity (the difference between body temperature and surface temperature). The body temperature that crabs began to use burrows to thermoregulate was estimated to be around 24°C, which is far below the critical body temperatures that could lead to death. Many crabs experience body temperatures of 24°C early in the reproductive season, several months before the hottest days of the year. Because the use of burrows involves fitness trade-offs, these results suggest that warming temperatures could begin to impact crabs far earlier in the year than expected.

Effects of dietary icariin supplementation on the ovary development-related transcriptome of Chinese mitten crab (Eriocheir sinensis).

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China, with distinct differences in ovarian maturation status between crabs fed with natural diets and artificial diets during the listing period, thus, leading to selling price differentiation. Our previous study showed that dietary supplementation with 100 mg/kg icariin can effectively promote ovarian development of E. sinensis. However, the internal molecular mechanism has not yet been elucidated because of a lack of comprehensive genome sequence information. We compared the ovary transcriptomes of E. sinensis fed with two diets containing 0 and 100 mg/kg ICA using the BGISEQ-500 platform. This yielded 12.54 Gb clean bases and 54,794 unigenes, 13,832 of which were found to be differentially expressed after icariin exposure. Twenty pathways closely related to gonadal development were selected through KEGG analysis. Seven differentially expressed genes relevant to vitellogenesis and oocyte maturation (serine/threonine-protein kinase mos-like, Eg2, cytoplasmic polyadenylation element-binding protein, cyclin B, vitellogenin 1, cathepsin D, and juvenile hormone esterase-like carboxylesterase 1) were validated by qRT-PCR, and four proteins (MEK1/2, ERK1/2, Cyclin B and Cdc2) associated with the progesterone mediated oocyte maturation pathway (i.e., MAPK/MPF pathway) were analyzed by western-blot. The results showed that icariin could promote the synthesis, processing and deposition of vitellogenin in oocytes, and that it also has the potential to promote oocyte maturation (resumption of Meiosis I) by altering the expression of the relevant genes and proteins.

Dietary lipid and n-3 long-chain PUFA levels impact growth performance and lipid metabolism of juvenile mud crab, Scylla paramamosain.

An 8-week feeding trial was conducted to evaluate the effects of dietary n-3 LC-PUFA levels on growth performance, tissue fatty acid profiles and relative expression of genes involved in the lipid metabolism of mud crab (Scylla paramamosain). Ten isonitrogenous diets were formulated to contain five n-3 LC-PUFA levels at 7 and 12 % dietary lipid levels. The highest weight gain and specific growth rate were observed in crabs fed the diets with 19·8 and 13·2 mg/g n-3 LC-PUFA at 7 and 12 % lipid, respectively. Moisture and lipid contents in hepatopancreas and muscle were significantly influenced by dietary n-3 LC-PUFA at the two lipid levels. The DHA, EPA, n-3 LC-PUFA contents and n-3:n-6 PUFA ratio in hepatopancreas and muscle significantly increased as dietary n-3 LC-PUFA levels increased at both lipid levels. The expression levels of -6 fatty acyl desaturase and acyl-CoA oxidase in hepatopancreas increased significantly, and expression levels of fatty acid synthase, carnitine palmitoyltransferase I and hormone-sensitive TAG lipase were down-regulated, with increased dietary n-3 LC-PUFA regardless of lipid level. Based on weight gain, n-3 LC-PUFA requirements of S. paramamosain were estimated to be 20·1 and 12·7 mg/g of diet at 7 and 12 % dietary lipid, respectively. Overall, dietary lipid level influenced lipid metabolism, and purified, high-lipid diets rich in palmitic acid reduced the n-3 LC-PUFA requirement of juvenile mud crab.

High Plasticity of the Gut Microbiome and Muscle Metabolome of Chinese Mitten Crab (Eriocheir sinensis) in Diverse Environments.

Phenotypic plasticity is a rapid response mechanism that enables organisms to acclimate and survive in changing environments. The Chinese mitten crab (Eriocheir sinensis) survives and thrives in different and even introduced habitats, thereby indicating its high phenotypic plasticity. However, the underpinnings of the high plasticity of E. sinensis have not been comprehensively investigated. In this study, we conducted an integrated gut microbiome and muscle metabolome analysis on E. sinensis collected from three different environments, namely, an artificial pond, Yangcheng Lake, and Yangtze River, to uncover the mechanism of its high phenotypic plasticity. Our study presents three divergent gut microbiotas and muscle metabolic profiles that corresponded to the three environments. The composition and diversity of the core gut microbiota (Proteobacteria, Bacteroidetes, Tenericutes, and Firmicutes) varied among the different environments while the metabolites associated with amino acids, fatty acids, and terpene compounds displayed significantly different concentration levels. The results revealed that the gut microbiome community and muscle metabolome were significantly affected by the habitat environments. Our findings indicate the high phenotypic plasticity in terms of gut microbiome and muscle metabolome of E. sinensis when it faces environmental changes, which would also facilitate its acclimation and adaptation to diverse and even introduced environments.

Baseline growth of the Trinidad freshwater crab Poppiana dentata (Randall, 1840) under laboratory conditions / Crescimento do caranguejo de água doce de Trinidad, Poppiana dentata (Randall, 1840), em condições laboratoriais

Poppiana dentata (Randall, 1840) is widely distributed throughout riverine habitats in Trinidad. However, there is a scarcity of information on the biology of this species. This study provides the first baseline examination that describes growth aspects for P. dentata. Juvenile crabs were obtained from berried females collected in northwest Trinidad. Carapace width (CW), length (CL), moult incident, intermoult period and qualitative aspects were recorded for crabs (N = 23) over 9 months. CW, CL and intermoult period were used to derive percentage size increment, specific growth rate (SGR) and size at structural maturity for both sexes. Growth curves and logistic equations were also generated for each sex. Hatched crabs (< 5 mm CW) underwent rapid hardening after their first moult, indicating a fast turnover of moult cycles. SGR and CW percent increment were also the highest for this initial moult (P < 0.05). CW, CL, intermoult period, size increment and SGR did not differ between sexes (P > 0.05), with logistic equations expressed as CW = 32.81 (1+exp (1.481 ̵ 0.031t))-1 for males and CW = 34.07 (1+exp (1.516 ̵ 0.027t))-1 for females. Yet, breakpoint analyses indicated dissimilar sizes for structural maturity (male: 28.40 mm CW; female: 16.84 mm CW). These patterns reflect a shorter life span for this species in comparison to what has been reported for other trichodactylid relatives. This can have implications for P. dentata populations residing in anthropogenically disturbed habitats; thus, highlighting the need for conservation strategies to ensure preservation of native wild stock.

O Poppiana dentata (Randall, 1840) está amplamente distribuído nos habitats fluviais de Trinidad. Existem, porém, poucas informações sobre a biologia dessa espécie. Este estudo fornece a primeira análise de referência que descreve aspectos do crescimento do P. dentata. Caranguejos jovens foram obtidos a partir de fêmeas em desova na região noroeste de Trinidad. A largura da carapaça (LC), o comprimento da carapaça (CC), a incidência de muda, o período de intermuda e aspectos qualitativos foram registrados para caranguejos (N = 23) ao longo de mais de nove meses. A LC, o CC e o período de intermuda foram utilizados para obter o aumento porcentual, a taxa específica de crescimento (TEC), e o tamanho na maturidade estrutural para os dois sexos. Curvas de crescimento e equações logísticas foram também geradas para cada sexo. Os caranguejos que nasceram (com largura de carapaça < 5 mm) apresentaram um endurecimento rápido depois de sua primeira muda, o que indica uma rápida rotação dos ciclos de muda. A TEC e o aumento da porcentual da LC foram também os mais altos para esta muda inicial (P < 0,05). Não houve variação da LC, CC, do período de intermuda, aumento do tamanho e da TEC entre os sexos (P > 0,05), e as equações logísticas foram expressas como: LC = 32,81 (1+exp (1,481 ̵ 0,031t))-1 para machos, e LC = 34,07 (1+exp (1,516 ̵ 0,027t))-1 para fêmeas. Porém, as análises de quebra indicaram tamanhos diferentes para maturidade estrutural (LC para macho: 28,40; para fêmea: 16,84 mm). Esses padrões refletem um período de vida mais curto para esta espécie em comparação com o que tem sido registrado para outras famílias de Trichodactylidae. Isso pode ter implicações para as populações de P. dentata que residem em habitats modificados antropogenicamente; destaca-se, assim, a necessidade de estratégias de conservação para assegurar a preservação das populações selvagens nativas.

Portunid Crab Breeding and Larval Rearing, Paving the Way for Sustainable Aquaculture.

This mini-review focus primarily on the current knowledge and techniques of portunid breeding and larva rearing. Topics include; (i) natural breeding in captivity and artificial breeding for breeding techniques and (ii) environmental conditions, diet requirements and developing factors for larval rearing. Specific knowledge of portunid crab breeding and larval rearing essential for optimizing breeding and culture conditions are shared. On the whole, this work allows for a better understanding of the portunid crab breeding and larval rearing and helps pave the way for future sustainable aquaculture of portunid crabs, among commercially important crustacean family in the world.

Assessing Intertidal Populations of the Invasive European Green Crab.

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one of the world's 100 worst invaders by the IUCN. Observations of spatiotemporal green crab population dynamics are essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab population dynamics in the rocky intertidal zone of New England and Atlantic Canada. The protocol was designed to be accessible to multiple users including researchers, educators, students, and citizen scientists. Although it was designed for surveying crab populations, this protocol is easy to adapt and could be used for any number of intertidal species. The resulting data collected using this protocol has a wide range of uses, including to inform ecological research, conservation efforts, mitigation strategies, and fishery development, as well as for educational outreach purposes.

Stimulatory roles of epidermal growth factor receptor (EGFR) in ovarian development of mud crab Scylla paramamosain.

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, to date little is known about its functions in crustaceans. In this study, we successfully identified SpEGFR from mud crab Scylla paramamosain. RT-PCR result showed that SpEGFR was widely distributed in all tested tissues and highly expressed in ovary. In situ hybridization revealed that SpEGFR mainly localized in oocyte perinuclear region with notably obvious signals. In vitro experiments showed that the expression of SpVgR and SpCyclin B in ovary explants from late vitellogenic stage crabs (summer) were significantly increased when treated with 1 nM human EGF (hEGF) for 1 h, while there was no obvious change towards SpEGFR. Interestingly, as for winter crab at the same vitellogenic stage, the expression of SpVgR and SpCyclin B in ovary explants did not show significant increase until treated with higher concentration of 10 nM hEGF and longer incubation time of 12 h. In addition, the hEGF-induced effect could be suppressed by pre-treated with EGFR inhibitor AG1478 and PD153035, respectively, which further indicated that EGF-EGFR pathway played a vital role in ovarian development in mud crab. In conclusion, SpEGFR might promote ovarian development by stimulating the expression of SpVgR and SpCyclin B under hEGF-induced treatment. The different physiological response to hEGF in the same vitellogenic stage crabs between summer and winter might be attributed to the changes in metabolism and physiological sensitivity.

Integrative Proteomic and MicroRNA Analysis: Insights Into Mechanisms of Eyestalk Ablation-Induced Ovarian Maturation in the Swimming Crab Portunus trituberculatus.

Eyestalk ablation is the most common method to induce ovarian maturation in decapod crustacean aquaculture, but it jeopardizes broodstock survival and larvae production. It is important to understand the molecular basis underlying the maturation triggered by ablation and thereby develop an alternative measure for maturation manipulation. In this study, we investigate alterations of ovarian proteome and miRNA profile after ablation in a commercially important marine crab Portunus trituberculatus. Quantitative proteomic analysis using iTRAQ reveals that 163 proteins are differentially expressed following ablation, and modulation of methyl farnesoate metabolism and activation of calcium signaling may play important roles in the ovarian maturation induced by ablation. miRNA expression profiling identifies 31 miRNAs that show statistically significant changes. Integration of miRNA and proteome expression data with miRNA target prediction algorithms generates a potential regulatory network consisting of 26 miRNAs and 30 proteins linked by 71 possible functional associations. The miRNA-protein network analysis suggests that miRNAs are involved in promoting ovarian maturation by controlling expression of proteins related to methyl farnesoate synthesis, calcium signals, and energy metabolism. Experimental validation and temporal expression analysis indicate multiple miRNAs can act synergistically to regulate expression of Farnesoic acid O-methyltransferase and Calmodulin. Our findings provide new insights for elucidating the mechanisms underlying eyestalk ablation-induced ovarian maturation and could be useful for devising an alternative technique for manipulating reproduction in P. trituberculatus and other decapods.

Molecular insights into information processing and developmental and immune regulation of Eriocheir sinensis megalopa under hyposaline stress.

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis.

Hepatopancreas immune response during molt cycle in the mud crab, Scylla paramamosain.

Molt is a critical developmental process in crustaceans. Recent studies have shown that the hepatopancreas is an important source of innate immune molecules, yet hepatopancreatic patterns of gene expression during the molt cycle which may underlie changes in immune mechanism are unknown. In this study, we performed Illumina sequencing for the hepatopancreas of the mud crab, Scylla paramamosain during molt cycle (pre-molt stage, post-molt stage, and inter-molt stage). A total of 44.55 Gb high-quality reads were obtained from the normalized cDNA of hepatopancreas. A total of 70,591 transcripts were assembled; 55,167 unigenes were identified. Transcriptomic comparison revealed 948 differentially expressed genes (DEGs) in the hepatopancreas from the three molt stages. We found that genes associated with immune response patterns changed in expression during the molt cycle. Antimicrobial peptide genes, inflammatory response genes, Toll signaling pathway factors, the phenoloxidase system, antioxidant enzymes, metal-binding proteins and other immune related genes are significantly up-regulated at the post-molt stage and inter-molt stage compared with the pre-molt stage, respectively. These genes are either not expressed or are expressed at low levels at the pre-molt stage. To our knowledge, this is the first systematic transcriptome analysis of genes capable of mobilizing a hepatopancreas immune response during the molt cycle in crustaceans, and this study will contribute to a better understanding of the hepatopancreas immune system and mud crab prophylactic immune mechanisms at the post-molt stage.