Responses of digestive enzyme profiles to various scenarios of food availability in newly-hatched Stage I phyllosoma larvae of the tropical spiny lobster Panulirus ornatus.

Stage I phyllosoma larvae of the spiny lobster Panulirus ornatus hatch in tropical oceanic waters with limited and variable food resources. To better understand how these larvae cope with food deprivation, this study examined specific (mU mg-1 protein) and total (mU larva-1) activities of major digestive enzymes (i.e., α-amylase, non-specific esterase, trypsin-like protease) during a series of ex situ experiments. Specifically, temporal changes in enzyme profiles were examined in three experiments simulating scenarios in which phyllosoma hatch and begin development in an environment where (1) prey is either continuously present or absent; (2) prey is initially present, but subsequently absent; (3) prey is initially absent, but subsequently present. Results indicated that the accessibility of suitable prey provides an overarching influence on digestive enzyme activities and substrate utilisation in Stage I phyllosoma of P. ornatus, with enzymatic responses to both intermittent prey availability and food deprivation being influenced by their nutritional history. In the absence of prey, larval digestive enzyme activities (mU larva-1) initially remained static from hatch but eventually declined with extended food deprivation. When prey became available, enzyme activities increased, with delayed access to prey having minimal impact on this enzymatic response. Furthermore, phyllosoma were able to adjust α-amylase and non-specific esterase activities (within 8 h) in response to the disappearance of prey, demonstrating adaptive changes to endure periods of food deprivation that were based on their nutritional history. The ability of phyllosoma to regulate enzyme activities from hatch is an important physiological strategy allowing them to survive in an environment characterised by highly variable zooplankton biomass and abundance, and explains why diets that differ greatly in nutrient composition have consistently been found effective for rearing phyllosoma in captivity.

Insulin-like androgenic gland hormone 1 (IAG1) regulates sexual differentiation in a hermaphrodite shrimp through feedback to neuroendocrine factors.

Insulin-like androgenic gland hormone (IAG) is regarded as a key sexual differentiation regulator in gonochoristic crustaceans. However, until now the knowledge concerning its functions in hermaphroditic crustaceans is scanty. Herein, we investigated the function of IAG (Lvit-IAG1) in peppermint shrimp Lysmata vittata, a species that possesses protandric simultaneous hermaphroditism (PSH) reproductive system, which is rare among crustaceans. Lvit-IAG1 was exclusively expressed in the androgenic gland. The qRT-PCR demonstrated that its mRNA expression level was relatively high at the functional male phase but decreased sharply in the subsequent euhermaphrodite phase. Both the short-term and long-term silencing experiments showed that Lvit-IAG1 negatively regulated both the gonad-inhibiting hormone (Lvit-GIH) and crustacean female sex hormone (Lvit-CFSH) expressions in the eyestalk ganglion. Besides, Lvit-IAG1 gene knockdown induced a retarded development of the appendices masculinae (AM) and male gonopores while suppressing the germ cells at the primary spermatocyte stage. Also, Lvit-IAG1 gene silencing hindered ovarian development. This in turn led to small vitellogenic oocytes and decreased expression of vitellogenin and vitellogenin receptor genes in hepatopancreas and ovarian region, respectively. Generally, this study's findings imply that Lvit-IAG1 modulated the male sexual differentiation in PSH species L. vittata, and exhibited negative feedback on Lvit-GIH and Lvit-CFSH genes expression in the species' eyestalk ganglion.

Roles of gonad-inhibiting hormone in the protandric simultaneous hermaphrodite peppermint shrimp†.

To date, the molecular mechanisms of the unique gonadal development mode known as protandric simultaneous hermaphroditism (PSH) are unclear in crustaceans. In this study, cDNA of a gonad-inhibiting hormone (Lv-GIH1) was isolated from the PSH peppermint shrimp Lysmata vittata, and its expression was exclusively found in the eyestalk ganglion. Real-time quantitative polymerase chain reaction (qRT-PCR) revealed that the expression of Lv-GIH1 increased during gonadal development of the functional male stages but decreased significantly at subsequent simultaneous hermaphroditism stage. Further in vitro experiment showed that recombinant GIH1 protein (rGIH1) effectively inhibited Vg expression in the cultured hepatopancreas tissues while the short-term injection of GIH1-dsRNA resulted in reduced expression of Lv-GIH1 and upregulated expression of Vg in the hepatopancreas. Moreover, long-term rGIH1 injection led to significantly reduced expression of Lv-Vg, Lv-VgR, and Lv-CFSH1, subdued growth of oocytes, and feathery setae as a secondary sexual characteristic in females. Interestingly, while germ cells in testicular part were suppressed by rGIH1 injection, the expression of Lv-IAGs showed no significant difference; and long-term GIH1-dsRNA injection results were contrary to those of rGIH1 injection. Taken together, the results of this study indicate that Lv-GIH1 is involved in gonadal development and might also participate in controlling secondary sexual characteristic development in L. vittata by inhibiting Lv-CFSH1 expression.

A Possible Role of Crustacean Cardioactive Peptide in Regulating Immune Response in Hepatopancreas of Mud Crab.

Crustacean cardioactive peptide (CCAP), a cyclic amidated non-apeptide, is widely found in arthropods. The functions of CCAP have been revealed to include regulation of heart rate, intestinal peristalsis, molting, and osmotic pressure. However, to date, there has not been any report on the possible involvement of CCAP in immunoregulation in crustaceans. In this study, a CCAP precursor (designated as Sp-CCAP) was identified in the commercially important mud crab Scylla paramamosain, which could be processed into four CCAP-associated peptides and one mature peptide (PFCNAFTGC-NH2). Bioinformatics analysis indicated that Sp-CCAP was highly conserved in crustaceans. RT-PCR results revealed that Sp-CCAP was expressed in nerve tissues and gonads, whereas the Sp-CCAP receptor gene (Sp-CCAPR) was expressed in 12 tissues of S. paramamosain, including hepatopancreas. In situ hybridization further showed that an Sp-CCAPR-positive signal is mainly localized in the F-cells of hepatopancreas. Moreover, the mRNA expression level of Sp-CCAPR in the hepatopancreas was significantly up-regulated after lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge. Meanwhile, the mRNA expression level of Sp-CCAPR, nuclear transcription factor NF-κB homologs (Sp-Dorsal and Sp-Relish), member of mitogen-activated protein kinase (MAPK) signaling pathway (Sp-P38), pro-inflammatory cytokines factor (Sp-TNFSF and Sp-IL16), and antimicrobial peptide (Sp-Lysozyme, Sp-ALF, Sp-ALF4, and Sp-ALF5) in the hepatopancreas were all up-regulated after the administration of synthetic Sp-CCAP mature peptide both in vivo and in vitro. The addition of synthetic Sp-CCAP mature peptide in vitro also led to an increase in nitric oxide (NO) concentration and an improved bacterial clearance ability in the hepatopancreas culture medium. The present study suggested that Sp-CCAP signaling system might be involved in the immune responses of S. paramamosain by activating immune molecules on the hepatopancreas. Collectively, our findings shed new light on neuroendocrine-immune regulatory system in arthropods and could potentially provide a new strategy for disease prevention and control for mud crab aquaculture.

Embryonic and early larval development of two marine angelfish, Centropyge bicolor and Centropyge bispinosa.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 µm for C. bicolor and 627.6 ± 7.8 µm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Morphological descriptions of the larval and first juvenile stages of the decorator crab Camposcia retusa (Latreille, 1829) from laboratory-reared material.

The complete larval and first crab stages of the decorator crab Camposcia retusa (Latreille, 1829) are described and illustrated based on laboratory-reared material for the first time. Specimens were obtained from larvae hatched from adult crabs collected from coral reefs of Queensland, Australia. Newly hatched larvae were successfully reared to settlement as the first-stage crabs. Larval development consisted of two zoeal stages and one megalopal stage. The morphology of each larval stage was compared with those available from a previous study using material from the Red Sea. Due to substantial differences in morphology of the second zoeal and megalopal stages between the two studies, we argue that these larval stages described by the earlier report may not be that of C. retusa. Finally, the morphological characters of both larval and first crab stages of C. retusa are also compared with the corresponding stages of previously reported Inachidae.

Profiles of calreticulin and Ca2+ concentration under low temperature and salinity stress in the mud crab, Scylla paramamosain.

Calreticulin (CRT) is an important molecular chaperon crucial to survival of organisms under adverse conditions. In this study, the potential roles of CRT in the mud crab, Scylla paramamosain, were investigated. Firstly, SpCRT gene expression was detected in various tissues of S. paramamosain with the highest expression found in the hepatopancreas. To evaluate potential role of SpCRT in cold adaption, sub-adult crabs were subjected to temperatures of 10, 15, 20 and 25°C and the profiles of SpCRT gene were determined in the hepatopancreas, chela muscle and gills. The results showed that the expressions of SpCRT mRNA in these tissues were significantly higher for those crabs exposed to low temperatures of 10 and 15°C as compared to those exposed to the higher temperatures, indicating SpCRT was involved in cold adaptation-probably through facilitating protein folding. When low temperature 10°C or 15°C was further combined with high and low salinity stress, the expression of SpCRT mRNA at low salinity (10 ppt) was in most cases significantly higher than that at high salinity (35 ppt), suggesting that under low temperatures, low salinity may represents a more stressful condition to the crab than high salinity. It was also shown that when crabs challenged by 10°C, Ca2+ concentration increased rapidly in the hepatopancreas and an in vitro experiment further showed that the expression of SpCRT mRNA increased concurrently with added Ca2+ concentration; these results together imply that Ca2+ probably plays a major role in low temperature signaling, which induces expression of genes related to cold adaption, such as CRT.

Salinity can change the lipid composition of adult Chinese mitten crab after long-term salinity adaptation.

The Chinese mitten crab (Eriocheir sinensis) is an euryhaline crustacean, whose adults migrate downstream to estuaries for reproduction. Lipids are believed to be involved in salinity adaptation during migration. This study investigated the effects of different salinities (0, 6, 12, and 18‰) on the total lipids, neutral lipids, and polar lipids contents, and fatty acid profiles in the gonads, hepatopancreas, and muscles of adult E. sinensis after 40 days of salinity adaptation. The results showed that the males and females from 12‰ treatment had the highest contents of total lipids and neutral lipids in their hepatopancreas and total lipids in the muscles. Notably, salinity had a greater effect on the fatty acid profiles in the hepatopancreas compared to that in the gonads and muscles. The male hepatopancreas treated with 18‰ salinity had the highest percentage of total n-6 polyunsaturated fatty acid (∑n-6PUFA) in both neutral lipids and polar lipids, while the percentage of total n-3 polyunsaturated fatty acid (∑n-3PUFA) in neutral lipids and polar lipids decreased significantly with increasing salinity in males. In females, the 0‰ treatment had the highest percentages of total saturated fatty acids in neutral lipids and polar lipids in the hepatopancreas, while the highest ∑n-3PUFA and ∑n-6PUFA in neutral lipids and polar lipids were detected in the 12‰ treatment group. In conclusion, brackish water could promote the accumulations of total lipids and neutral lipids in the hepatopancreas and change the fatty acid profiles of adult E. sinensis, particularly in the hepatopancreas after long-term salinity adaptation.

Tamoxifen affects the histology and hepatopancreatic lipid metabolism of swimming crab Portunus trituberculatus.

Tamoxifen (TAM) is an antiestrogenic agent and can enter the aquatic environment in wastewater. It has been reported that TAM can induce hepatic steatosis in vertebrates, however, the effects of TAM exposure on lipid metabolism of hepatopancreas in crustaceans remains unclear. In this study, four TAM concentrations (0, 6.7, 13.4 and 20 µg g-1 crab body weight) were injected into the swimming-leg of swimming crabs Portunus trituberculatus, as a means of evaluating the effects of TAM on the expression levels of lipid metabolism-related genes, lipid composition, and hepatopancreas histology. The results showed that the mRNA levels of three lipogenic related genes (diacylglycerol acyltransferase 1 (DGAT1), acetyl-CoA carboxylase (ACC) and fatty acyl desaturase (FAD)) decreased significantly in the 6.7 µg g-1 and 20 µg g-1 TAM treatments compare to the control. The mRNA levels of fatty acid synthase (FAS) decreased significantly in a dose-dependent manner as TAM concentration increased. The mRNA levels of two lipid catabolism-related genes (acyl-CoA oxidase (ACOX) and fatty acid transport protein (FATP)) were down-regulated among the three TAM treatments, while the enzyme activity and mRNA level of carnitine palmitoyltransferase I (CPT-I) was up-regulated by TAM treatments. Compared to the control, the lowest levels of total lipids and phospholipids were detected in the 6.7 µg g-1 TAM treatment, while the 20 µg g-1 TAM treatment had the lowest free fatty acids concentration. The 6.7 µg g-1 TAM treatment had the lowest percentages of 16:1n-7, 18:1n-9, 18:1n-7 and total monounsaturated fatty acids (∑MUFA), whilst simultaneously recording the highest percentages of 18:2n-6 and 20:2n-6 in this treatment. Moreover, histological observations indicated that TAM caused the walls of the hepatopancreatic tubules to become brittle, with a concurrent increase in the number of blister-like cells. These results suggest that TAM damages the hepatopancreas and leads to a reduction in hepatopancreatic lipid deposition in P. trituberculatus.

Ontogenetic development of gonads and external sexual characters of the protandric simultaneous hermaphrodite peppermint shrimp, Lysmata vittata (Caridea: Hippolytidae).

The peppermint shrimp Lysmata vittata (Caridea: Hippolytidae) is a marine caridean shrimp popular in marine aquarium trade. The species is known to display the sexual system of protandric simultaneous hermaphrodite. In this study, based on captive bred specimens, the complete ontogenetic gonad development of L. vittata was studied both morphologically and histologically, from newly settled juveniles until they reached euhermaphrodite phase. It was found that in all specimens examined (carapace length: 1.8-8.5 mm), including the newly settled juveniles, possessed ovotestes, which comprised of an anterior ovarian and a posterior testicular part. Based on both morphological (e.g., size, color and shape) and histological features (e.g., oogenesis and spermatogenesis), four gonadal development stages were defined and described for L. vittata. From Stage I to III, the testicular part of the gonad became gradually mature but the ovarian part was still immature, which is defined as the male phase. At the male phase, cincinulli (5-8 hooks) presented at the tips of the appendix interna on the first pair of pleopods while appendices masculinae (AM), in a form of a stick structure with spines, presented at the inner edge of the appendix interna (AI) on the second pair of pleopods. At Stage IV, both the testicular part and the ovarian part were mature and hence is defined as euhermaphrodite phase. At the euhermaphrodite phase, most individuals lacked cincinulli and appendices masculinae on the first and second pair of pleopods respectively. This is the first time that complete ontogenetic gonadal and external sexual character development have been described and staged for a species from the genus Lysmata from newly settled juveniles to euhermaphrodite phase.

Effect of estradiol on hepatopancreatic lipid metabolism in the swimming crab, Portunus trituberculatus.

Estradiol is an important sex steroid hormone that involved in regulation of animal lipid metabolism. However, the effect of estradiol on lipid metabolism in swimming crab (Portunus trituberculatus) is unclear. The present study investigated the effect of four concentrations of exogenous estradiol (0, 0.01, 0.1 and 1 µg g-1 crab weight) on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in the P. trituberculatus. The results showed that the mRNA levels of carnitine palmitoyltransferase I and II (CPT-I and CPT-II) increased significantly at the low concentrations (0.01 µg g-1 and 0.1 µg g-1), while decreased significantly in the highest concentration (1 µg g-1). The mRNA levels of acyl-CoA oxidase (ACOX), fatty acid transport protein (FATP), fatty acid-binding protein (FABP), diacylglycerol acyltransferase 1 (DGAT1) and acetyl-CoA carboxylase (ACC) were significantly down-regulated. The transcripts of fatty acid synthase (FAS) and fatty acyl desaturase (FAD) decreased significantly only in 1 µg g-1 treatment. All estradiol treatments (0.01, 0.1 and 1 µg g-1) had significantly higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of total monounsaturated fatty acids and polar lipids than the control treatment (0 µg g-1). Histological observations indicated the size of B cell became larger under estradiol treatment. The results indicated that estradiol promoted lipid catabolism in the hepatopancreas of P. trituberculatus.

A novel crustacean hyperglycemic hormone (CHH) from the mud crab Scylla paramamosain regulating carbohydrate metabolism.

Crustacean hyperglycemic hormone (CHH) plays a crucial role in regulating carbohydrate metabolism in crustaceans. In this study, a new cDNA encoding type I CHH peptide, termed Sp-CHH3, was isolated from the mud crab Scylla paramamosain and its potential functions were investigated. The full length cDNA of Sp-CHH3 was identified as encoding a 127-aa precursor composed of a 27-aa signal peptide, a 23-aa CHH precursor-related peptide and a 75-aa mature peptide with a typical motif of CHH. Phylogenic analysis suggested that, Sp-CHH3 is a previously unreported CHH from S. paramamosain. Tissue distribution analysis showed that Sp-CHH3 was mainly expressed in the eyestalk ganglia, thoracic ganglia, stomach and the ovary. A RNA interference experiments showed that after injection of Sp-CHH3-targeted dsRNA, both the level of Sp-CHH3 expression in the eyestalk ganglia and hemolymph glucose level decreased significantly. A further short-term starvation experiments demonstrated that, the level of Sp-CHH3 detected in the eyestalk ganglia was significantly up-regulated at the 12th h of starvation, it then fell back at the 24th h of starvation and subsequently remained relative stability between the 24th to 96th h of starvation. The hemolymph glucose level decreased significantly (P < .05) at each sampling time during the 96 h starvation duration when compared to that of 0 h (prior to starvation) and the overall trend was largely correlated with the level of Sp-CHH3 expression in the eyestalk ganglia. In summary, the results suggest that Sp-CHH3 plays a functional role in regulating carbohydrate metabolism in S. paramamosain.

Identifying neuropeptide GPCRs in the mud crab, Scylla paramamosain, by combinatorial bioinformatics analysis.

Neuropeptides, ubiquitous signaling molecules, commonly achieve their signaling function via interaction with cell membrane-spanning G-protein coupled receptors (GPCRs). In recent years, in the midst of the rapid development of next-generation sequencing technology, the amount of available information on encoded neuropeptides and their GPCRs sequences have increased dramatically. The repertoire of neuropeptides has been determined in many crustaceans, including the commercially important mud crab, Scylla paramamosain; however, determination of GPCRs is known to be more difficult and usually requires in vitro binding tests. In this study, we adopted a combinatorial bioinformatics analysis to identify S. paramamosain neuropeptide GPCRs. A total of 65 assembled GPCR sequences were collected from the transcriptome database. Subsequently these GPCRs were identified by comparison to known neuropeptide GPCRs based on the sequence-similarity-based clustering and phylogenetic analysis, which showed that many of them are closely related to insect GPCR families. Of these GPCRs, most of them were detected in various tissues of the mud crab and some of them showed differential expression by gender, suggesting they are involved in different physiological processes, such as sex differentiation. By employing ligand-receptor binding tests, we demonstrated that the predicted crustacean cardioactive peptide (CCAP) receptor was activated by CCAP peptide in a dose-dependent manner. This is the first CCAP receptor that has been functionally defined in crustaceans. In summary, the present study shortlists candidate neuropeptide GPCRs for ligand-receptor binding tests, and provides information for subsequent future research on the neuropeptide/GPCR signaling pathway in S. paramamosain.

Comparative study on the effects of two diatoms as diets on planktonic calanoid and benthic harpacticoid copepods.

Two diatoms, Chaetoceros muelleri and Nitzschia closterium f. minutissima, were used for rearing nauplii to adults of Tigriopus japonicus, Acartia pacifica, and Pseudodiaptomus annandalei at different concentrations. The survival rates to adults and activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) of the adult copepods were measured. The results showed that when fed on N. closterium f. minutissima, A. pacifica was unable to complete naupliar development; while SOD and GST activities of P. annandalei fed N. closterium f. minutissima were significantly higher than those fed the control algae Isochrysis galbana (Prymnesiod), suggesting this diatom species is harmful to these calanoid copepods. The survival rates of T. japonicus were very high when fed N. closterium f. minutissima, indicating that T. japonicus has high acceptance to diatoms. To evaluate whether calanoid copepod had the capacity to acclimatize to diatoms, P. annandalei was cultured for four generations on the two diatoms at 1.7 µgC/ml and survival rates as well as SOD and GST activities were determined for each generation. It was shown that starting at the second generation, P. annandalei exhibited adaption to the diatoms with improved survival. When fed on N. closterium f. minutissima, both SOD and GST activities of P. annandalei showed a trend of decreasing with the progress of generation, whereas no significant difference was found among four generations on C. muelleri. This study demonstrates that whether a diatom species is considered harmful to copepods, which is largely species specific, and diatom concentration is an important influencing factor. Additionally, the capacity of copepod adaption to diatoms over time should not be neglected.

Effects of salinity on gonadal development, osmoregulation and metabolism of adult male Chinese mitten crab, Eriocheir sinensis.

As a catadromous species, salinity is a key parameter that affects gonadal development of Chinese mitten crab Eriocheir sinensis during reproductive migration. It is however unclear the effects of salinity on the gonadal development of male E. sinensis as well as their physiological responses to salinity during reproductive migration. This study investigated the effects of four salinities (0 ‰, 6 ‰, 12 ‰ and 18 ‰) on gonadal development, osmoregulation and metabolism of adult male E. sinensis over a 40-day period. The results showed that elevating salinity promote gonadal development, increase hemolymph osmolality and K+ and Mg2+ concentrations (P < 0.05). The 12 ‰ salinity resulted in the highest contents of taurine and arginine in the hemolymph while the highest contents of threonine, phenylalanine, lysine, ß-alanine, tryptophan, ornithine and total free amino acids were found for 0 ‰ treatment (P < 0.05). A decreasing trend was detected for the Na+/K+-ATPase activity and its mRNA expression level in the posterior gills with salinity (P < 0.05). Total saturated fatty acids in the anterior gills decreased with increasing salinity (P < 0.05); the 0 ‰ treatment had the highest total polyunsaturated fatty acids in the posterior gills while total n-6 polyunsaturated fatty acids increased with salinity (P < 0.05). The hemolymph glucose and uric acid showed a decreasing trend as salinity while an increasing trend was found for the hemolymph triglyceride and high-density lipoprotein cholesterol (P < 0.05). The 12 ‰ treatment had the highest levels of hemolymph malonaldehyde and hepatopancreatic γ-glutamyltranspeptidase (P < 0.05). In conclusion, these results suggested that the brackish water promote gonadal development of male E. sinensis, and increase osmolality and ionic concentrations in hemolymph while reduced the activity of Na+ /K+- ATPase and its mRNA expression in the posterior gills as well as metabolism.

Toxic effects of polyethylene terephthalate microparticles and Di(2-ethylhexyl)phthalate on the calanoid copepod, Parvocalanus crassirostris.

Large amounts of plastic end up in the oceans every year where they fragment into microplastics over time. During this process, microplastics and their associated plasticizers become available for ingestion by different organisms. This study assessed the effects of microplastics (Polyethylene terephthalate; PET) and one plasticizer (Di(2-ethylhexyl)phthalate; DEHP) on mortality, productivity, population sizes and gene expression of the calanoid copepod Parvocalanus crassirostris. Copepods were exposed to DEHP for 48h to assess toxicity. Adults were very healthy following chemical exposure (up to 5120µg L-1), whereas nauplii were severely affected at very low concentrations (48h LC50value of 1.04 ng L-1). Adults exposed to sub-lethal concentrations of DEHP (0.1-0.3µg L-1) or microplastics (10,000-80,000 particles mL-1) exhibited substantial reductions in egg production. Populations were exposed to either microplastics or DEHP for 6 days with 18 days of recovery or for 24 days. Populations exposed to microplastics for 24 days significantly depleted in population size (60±4.1%, p<0.001) relative to controls, whilst populations exposed for only 6 days (with 18 days of recovery) experienced less severe depletions (75±6.0% of control, p<0.05). Populations exposed to DEHP, however, exhibited no recovery and both treatments (6 and 24 days) yielded the same average population size at the termination of the experiment (59±4.9% and 59±3.4% compared to control; p<0.001). These results suggest that DEHP may induce reproductive disorders that can be inherited by subsequent generations. Histone 3 (H3) was significantly (p<0.05) upregulated in both plastic and DEHP treatments after 6 days of exposure, but not after 18 days of recovery. Hsp70-like expression showed to be unresponsive to either DEHP or microplastic exposure. Clearly, microplastics and plasticizers pose a serious threat to zooplankton and potentially to higher trophic levels.

Physiological Responses and Ovarian Development of Female Chinese Mitten Crab Eriocheir sinensis Subjected to Different Salinity Conditions.

Salinity plays a key role affecting ovarian development, osmoregulation and metabolism of female Chinese mitten crab, Eriocheir sinensis during reproductive migration. In this study, female E. sinensis after their puberty molt were subjected to four salinities of 0, 6, 12, and 18‰ for 40 days to investigate the salinity effects on their ovarian development as well as a range of important physiological parameters. Elevated salinity accelerated the ovarian development with ovigerous crabs found at salinity treatments of 12 and 18‰ despite no copulation had occurred. Meanwhile the survival rate of female crabs showed a decreasing trend with increasing salinity. Higher salinity also led to increased hemolymph Na+, K+, Ca2+, Cl-, and Mg2+ concentrations. The 6‰ treatment had the highest contents of hemolymph total and major free amino acids while the Na+/K+ -ATPase activity in the posterior gills was the lowest among treatments. Total n-3 polyunsaturated fatty acids (∑n-3PUFA) and n-3/n-6 PUFA ratio in the anterior gills showed a decreasing trend with salinity while 18‰ had the highest ∑PUFA and ∑n-6PUFA. The ∑n-3PUFA content and n-3/n-6 PUFA ratio of the posterior gills showed a fluctuating pattern and the highest value was detected at 0‰, while an increasing trend was found for the ∑n-6PUFA with increasing salinity. The hemolymph glucose showed a decreasing trend with increasing salinity and the highest total cholesterol in hemolymph was detected at 12‰. The 18‰ treatment had the highest levels of hemolymph γ-glutamyltransferase, alkaline phosphatase and acid phosphatase, as well as glucose, urea and acid phosphatase in hepatopancreas while the highest hemolymph superoxide dismutase and malondialdehyde were detected at 0‰. Overall, the results showed that salinity increase from freshwater to brackish conditions led to lower metabolism, accelerated ovarian development, and the appearance of ovigerous crabs without copulation in female E. sinensis post puberty molt.

The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway.

In arthropods, retinoid X receptor (RXR) is a highly conserved nuclear hormone receptor. By forming a heterodimeric complex with the ecdysone receptor (EcR), RXR is known to be vital importance for various physiological processes. However, in comparison to EcR, the RXR signaling pathway and its roles in crustacean reproduction are poorly understood. In the present study, the RXR mRNA was detected in the ovarian follicular cells of mud crab Scylla paramamosain (SpRXR) and during ovarian maturation, its expression level was found to increase significantly. In vitro experiment showed that both SpRXR and vitellogenin (SpVg) mRNA in the ovarian explants were significantly induced by 20-hydroxyecdysone (20E) but not methyl farnesoate (MF). However, differing from the in vitro experiment, injection of MF in in vivo experiment significantly stimulated the expressions of SpRXR and SpVg in female crabs at early vitellogenic stage, but the ecdysone and insect juvenile hormone (JH) signaling pathway genes were not induced. The results together suggest that both MF and SpRXR play significant roles in regulating the expression of SpVg and ovarian development of S. paramamosain through their own specific signaling pathway rather than sharing with the ecdysone or the insect JH.

Characterization of CCTα and evaluating its expression in the mud crab Scylla paramamosain when challenged by low temperatures alone and in combination with high and low salinity.

Chaperonin containing the T-complex polypeptide-1 (CCT), which is known to be involved in intracellular assembly and folding of proteins, is a class of chaperonin omnipresent in all forms of life. Previous studies showed that CCT played a vital role in cold hardiness of various animals. In order to understand the response of the polypeptide complex to low temperature challenge and other environmental stresses, a subunit of CCT (CCTα) was cloned from the mud crab Scylla paramamosain by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA SpCCTα was of 1972 bp and contained a 1668 bp open reading frame (ORF) encoding a polypeptide of 555 amino acids with four conserved motifs. The messenger ribonucleic acid (mRNA) levels of SpCCTα in ten tissues of adult S. paramamosain was subsequently examined and the highest expression was found in muscle, followed by gill, hepatopancreas, thoracic ganglion, hemocyte, heart, cerebral ganglion, stomach, eyestalk ganglion, and epidermis. The expressions of SpCCTα in the muscle of sub-adult crabs (pre-acclimated to 28 °C) subjected to the challenges of both lower temperatures (25, 20, 15, and 10 °C) alone and low temperatures (15 and 10 °C) in combination with salinity of 35 and 10 were further investigated by fluorescent quantitative real-time PCR (qPCR). It was revealed that when exposed to lower temperatures alone, the mRNA transcripts of the SpCCTα gene in the muscle were generally induced for significant higher expression at 10 °C treatment than the 25, 20, and 15 °C treatments; meanwhile, exposure to 15 °C also frequently led to significantly higher expression than those at 20 and 25 °C. This finding indicated that the up-regulation of SpCCTα was closely related to the cold hardiness of S. paramamosain. The results of an additional experiment challenging the sub-adult crabs with various combinations of low temperatures with different salinity conditions generally demonstrated that at both 10 and 15 °C, the expression of SpCCTα under the high salinity of 35 was significantly lower than that at low salinity of 10, implying that the damages caused by low temperatures with high salinity were less than that under low salinity.