Physical And Behavioral Response Of Eriocheir Sinensis Broodstock, Early Developmental Stages To Salinity

Chinese mitten crab Eriocheir sinensis H. Milne Edwards,1853(Decapoda, Brachyura) is a diadromous freshwater crab. Adult crabs spend most of their lives growing in rivers and lakes but must migrate downstream towards the estuary or sea for reproduction after their puberty molt, where they reach maturity, complete mating and embryonic development as well as planktonic larvae release. Estuary features drastically temporal and spatial variation in salinity. Yangtze River estuary is the biggest and vital breeding ground of E. sinensis in China. Recently, saltwater intrudes the Yangtze River estuary more and more frequently, and it has become the focus of researches. Remarkable salinity change might exert a negatively potential effect on the reproduction and development of E. sinensis.To evaluate the possible adverse implications of salinity change to E. sinensis comprehensively, we monitored the salinity preference behavior and the behavioral response of Chinese mitten crab E. sinensis broodstock in different salinities, and low salinity preference behavior of megalopas. We investigated the osmotic and ionic regulation, variation of ion-transporting enzymes activities, haemolymph metabolic and immune parameters and variation of digestive enzyme activities in both females and males, when crabs were exposed to freshwater and acclimated to step-wise salinities medium ranging from7to35ppt. We also studied the effect of salinity on E. sinensis embryo and larva development, and determined the developmental changes of energy source, digestive enzymes and Na+/K7-ATPase activity, and CHH and Na+/K+-ATPase a-subunit expression in the eggs of E. sinensis under salinity stress. Results obtained from this study will enhance our comprehension of the ontogeny of crustaceas’osmoregulation, especially during embryo stage and reproductive stage. Furthermore, this information is also great important to protect natural resources and to propagate of Chinese mitten crab E. sinensis artificially. These results will also provide theoretical basis for evaluating the potential effect of salinity change on E. sinensis.1. Behavioral responses of female Chinese mitten crab Eriocheir sinensis broodstock to salinitiesBehavioral response of female E. sinensis broodstock were monitored after acclimating crabs to freshwater (0ppt) and brackish water (18ppt) and hyper-osmotic (increased salinity from18to30ppt) or hypo-osmotic (decreased salinity from18to 0ppt) environment abruptly, by the method of video recording analysis. Locomotor activity and the frequency of mouthparts movements increased after acclimating crabs to brackish water (P<0.05) and hypo-osmotic environment. However, the two kinds of behaviors decreased after hyper-osmotic stress. Behaviors that the antennules retracted into the carapace for periods of time and the mouthparts closed were discovered only in the control group of18ppt and hyper-osmotic stress group (P<0.05). There was a decrease of the times of antennae flicking with the increase of environmental salinity. Behavior of the antennae flicking frequently showed up after hypo-osmotic stress (P<0.05). Behavior of eyestalk movement decreased after acclimating crabs to brackish water and increased after salinity stress. E. sinensis cleaned the antennae and antennules with the palps of the third maxillipeds. This behavior increased after gradual increase of environmental salinity, comparing with the response of crabs maintained in freshwater (P<0.05). Behavior of cleaning the antennae and antennules decreased after hypo-hyper osmotic stress (P<0.05). The abdomen extension behavior was observed only after hypo-osmotic stress. The results suggested that the increase of salinity induced the locomotor activity and other related behaviors of E. sinensis broodstock. Abdomen extension under hypo-osmotic stress could directly expose the hindgut to the water, and function as an additional means of ion uptake in lower salinity. Behavior of closure reaction under hyper-osmotic stress could help the reduction of water absorption and salt loss in high salinity environment.2. Salinity preference behavior of Chinese mitten crab Eriocheir sinensis broodstockPreference chambers are widely used to measure the responses of aquatic organisms to different environmental gradients, such as salinity, temperature and pollutants. A six-chambered device was designed to investigate the salinity preference of downstream-migrating Chinese mitten crab, Eriocheir sinensis. Occurrence numbers and retention time within6h are as indicators for evaluating the preference of E. sinensis to salinity. Males did not show preference for salinity. However, the occurrence number was the least at28ppt salinity chamber while was the most at35ppt salinity chamber. Males spend slightly more time in the chambers of21ppt and35ppt than others salinity chambers. Females occurred in the salinity chambers more and more frequently with the increase of salinity at a range0-21ppt. Occurrence number of females in chamber with21ppt was highest while lowest with28ppt. Females spends less and less time in salinity chambers with the increase of salinity at a range of0-28ppt. However, they retained in35ppt salinity chamber for the longest time. Males changed their positions more frequently than females (P<0.05). After offering different salinities in six chambers, locomotor activities of the crabs were weakening. Different salinity preference behaviors presented between females and males might be related to the asynchronism in gonadal development and the effect of salinity to their gonadal development. It is indicated that other factors such as water depth, substrate could be also vital to control their distribution in estuary.3. Osmotic and ionic regulation and Na+/K+-ATPase, carbonic anhydrase activities in Chinese mitten crab Eriocheir sinensis broodstck exposed to different salinitiesChinese mitten crabs broodstock, are exposed to brackish water or seawater as an obligatory part of their reproductive migration. We investigated the changes in haemolymph osmolality, haemolymph Na+, Cl-, Ca2+and K+concentrations, as well as gill Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) activity in both females and males, when in freshwater (0ppt,8mOsm (kg H2O)-1) and after step-wise acclimation to salinities ranging from7to35ppt (175-1044mOsm (kg H2O)-1). Both females and males strongly hyper-regulated their haemolymph osmolality over salinity range of0-21ppt, the isosmotic point approaching28ppt, and slightly hypo-regulated at35ppt. Haemolymph Na+and Cl-levels correlated positively with haemolymph osmolality. Ca2+and K+levels, however, were maintained at a relatively narrow range and showed a contrary trend in their response to salinity. This indicated that Na+and Cl-are the main contributors to increasing haemolymph osmolality. Reduced Na+/K+-ATPase and carbonic anhydrase activity in the posterior gills at elevated salinity suggest a direct physiological response to a decrease of Na+intake. No sexual differences existed for all parameters except for significantly higher haemolymph Ca2+and K+levels and CA activity in females than in males at some salinity levels. It is suggested that Eriocheir sinensis broodstock is a strong hyperosmoregulator but a weak hypo-osmoregulator. No sexual differences appeared to exist in osmotic and ionic regulation of Eriocheir sinensis broodstock exposed to different salinities.4. Metabolic, immune and digestive response of Chinese mitten crab Eriocheir sinensis broodstock exposed to different salinitiesTo understand the metabolic, immune and digestive adjustments of Chinese mitten crab broodstock, Eriocheir sinensis, during their reproductive migration from freshwater to seawater, concentrations haemolymph metabolic variations (protein, glucose, triglycerides, cholesterol, urea, uric acid and creatinine) and activities of immune enzyme (alkaline phosphatase, superoxide dismutase and phenoloxidase) and digestive enzyme were determined after gradual acclimating of females and males from freshwater (0ppt) to different salinities (7,14,21,28,35ppt) medium. The results showed that increasing salinities caused a gradual decrease of hemolymph total protein and oxyhemocyanin contents of females and males. Some haemolymph metabolic variables reached a minimum at21ppt, and increased with both increase and decrease of the medium salinity (triglycerides, cholesterol, urea, creatinine in males and triglycerides, uric acid in females), or elevated either with the increase (urea in females) or decrease (uric acid in males) of medium salinities. Responses of haemolymph metabolic variations to salinity slightly differ between females and males. Hemolymph alkaline phosphatase activity increased slightly in7ppt; thereafter it dropped slowly until the end of experiment. Hemolymph phenoloxidase activity of females and males decreased gradually with the increase of environmental salinity. However, it maintained a stable level when the salinities were above21ppt in females. Hemolymph superoxide dismutase activity of males decreased with the increase of the medium salinity until21ppt and increased when the medium salinity above21ppt. All tested immune parameters except for SOD of females were significantly higher than those of males. Digestive enzyme activity except lipase of males were significantly higher than those of females at some salinities (P<0.05). Amylase, pepsin and trypsin activities of females reduced significantly when the salinity reached28ppt (P<0.05). Cellulase activity decreased when increasing the salinity to35(P<0.05). Remarkable decrease of males’digestive enzyme activities except lipase occurred at21and higher salinity, and an increase was shown in14ppt. The present results indicated that salinity of21ppt was the turning point of several biochemical parameters in E. sinensis broodstock. This salinity could be more’profitable’for E. sinensis broodstock from the energy-saving point of view. Immune and digestive enzyme activities of E. sinensis broodstock will be affected when the salinity was near to or above28ppt, which could exert a negative effect on reproduction. Metabolic and immune parameters in the haemolymph of females were slightly higher than those of males at some salinity levels, this suggestes higher metabolic level and greater immunity. The initial salinity that induced the decrease of digestive enzyme activity in males was lower than that in females; this result indicated that females were more tolerant elevated salinities than males in the digestive ability. This study might help understand some aspects of the metabolic, digestive and immune adjustments in euryhaline crabs related to osmoregulation.5. Survival and development of Chinese mitten crab, Eriocheir sinensis embryo and Zoea in different salinitiesEmbryo and larval development of Chinese mitten crab. Eriocheir sinensis, is completed in the estuary, where they are often exposed to extreme salinity conditions. To test whether these salinity conditions have negative impacts on early development of embryo and zoea or not, we determined hatching rate, development time and survival rate when E. sinensis embryo for the gastrula, eyespot and pre-hatching stage were exposed to a salinity range of1-35ppt, and we also detected the survival rate of E. sinensis zoea I within192h later exposing them to different salinities. Reduced salinities (1and5ppt) accelerated embryonic development and increased hatching rate of eyespot and pre-hatching stage, but reduced salinities did not exert any effects on the gastrula stage development. Hatching rate significantly declined when the medium salinity reached at and above25ppt. No zoea I were hatched when eyespot stages embryos were exposed to30ppt and three stages embryos were exposed to35ppt. Higher survival rate of initial zoea I were presented when eyespot and pre-hatching stage embryos were exposed to salinity ranges of5-25ppt. However, survival rate of initial zoea I significantly declined when embryos were exposed to1and30ppt. All zoeas hardly survived at35ppt. Nearly40%zoeas I still survived until192h later during the range10-20ppt of salinity. However, all zoea I died24h later after exposure to salinity1and40ppt while died192h later at salinity range25-35and5ppt. The results showed that optimal salinity range was5-20ppt, when embryos began to be exposed to different salinities from gastrula stage and pre-hatching stage, while the salinity range became narrower (10-20ppt) for eyepot satge. The optimal salinity range was10-20ppt for zoea development. Out of above salinity ranges, will influence the development of embryo and larva, and then affect the wild population’ reproduction.6. Developmental changes of biochemical composition and digestive enzyme activity in the eggs of Chinese mitten crab, Eriocheir sinensis, under osmotic stressBiochemical compositions (total carbohydrate, soluble protein, lipid and water content) contents, digestive enzymes (amylase, lipase, pepsin and trypsin) activity and embryo volume (egg diameter) have been determined in three developmental stages (gastrula, eyespot and pre-hatching stage) of E. sinensis eggs under hyper or hypo-osmotic stress. Increase of water contents (P<0.05), eggs diameter and pepsin and trypsin activities and decrease of soluble protein (P<0.05) and lipid contents and lipase activity (P<0.05) in eggs were noticed during the development of embryo. Total carbohydrate content and amylase activity increased at the eyespot stage (P<0.05), whereas they reduced during the prehatching stages (P<0.05). Hyper-osmotic (P<0.05) and hypo-osmotic stress reduced water content of eggs at each developmental stage. Although hyper-osmotic stress reduced the water content of eggs, significant effect presented only at eyespot stage (P<0.05). Soluble protein content of three developmental stages eggs decreased under hyper-osmotic stress while increased under hypo-osmotic stress (P<0.05). Total lipid content was unaffected by hyper-osmotic stress at any developmental stage, whereas it reduced significantly at pre-hatching stages under hypo-osmotic stress (P<0.05). Total carbohydrate contents did not show any significant variation after osmotic stress. Both hypo-osmotic and hyper-osmotic stress induced the decrease of egg diameters during the gastrula stage (P<0.05), however, no significant effect on eyespot stage. Egg diameter decreased before hatching, only under the hyper-osmotic stress (P<0.05). Among the four digestive enzymes assayed, amylase activities were the highest while lipase activities were the lowest. Hyper-osmotic stress reduced enzymes activities of the three developmental stage eggs, while hypo-osmotic stress enhanced the activities of digestive enzymes with the exception of reducing the amylase activities in eyespot stage (P<0.05) and trypsin activities in pre-hatching stage (P>0.05). Effect of salinity change on digestive enzymes is statically significant only during gastrula stage (P<0.05) except for lipase. It is suggested that salinity stress changed the activity of digestive enzymes which further affectes the utilizing of yolk substances, consequently, affects embryonic development. Dramatical variation of salinity in estuary ought to be having potential effect on embryonic development, especially induced by salt water encroachment.7. Developmental changes of mRNA expression and activity of Na+/K+-ATPase, and expression of crustacean hyperglycemic hormone (CHH) gene in Chinese mitten crab Eriocheir sinensis eggs under osmotic stressWe examined ontogenetic and salinity-induced changes of Na+/K+-ATPase activity and mRNA expression of the a-subunit. We also detected the expression of crustacean hyperglycemic hormone (CHH) neuropeptides. which involved directly or indirectly in ionic and osmotic regulation, in the eyespot stage and pre-hatching stage. All activities and mRNA expression were determined24h later after directly exposeing embryos to1,15(control group), and35ppt. NKA activities were low in gastrula stage, and the enzyme activity increased6.53times in pre-hatching stage. Na+/K+-ATPase activity significantly decreased in gastrula and eyespot stages (P<0.05), and slightly reduced in pre-hatching stage at1ppt compared with15ppt, whereas the activity in the embryo of gastrula and pre-hatching stages was increased at salinity30ppt than at15and1ppt. However, increasing salinity reduced the enzyme activity (P<0.05) at eyespot stage, compared with the control group. The Na+/K+-ATPase a-subunit gene was expressed and elevated1.17times from gastrula stage to pre-hatching stage. There was no significant difference of Na+/K+-ATPase a-subunit gene expression between gastrula and eyespot stages, but expression increased remarkably before hatching (P<0.05). The expression of Na+/K+-ATPase a-subunit gene increased at30and1ppt compared with15ppt, but significant difference occurred only at eyepot stage under hyper salinity stress (P<0.05). Expression of CHH gene significantly increased with the development of embryo from eyespot stage to pre-hatching stage at all salinities (P<0.05), the expression remarkably elevated before hatching under hyper salinity stress while significantly decreased at eyespot stage under hypo salinity stress. It is suggested that although transcription and protein synthesis of Na+/K+-ATPase increased with the development of embryo, no consistent pattern between them under salinity stress. Neuroendocrine hormone CHH might be play important regulatory function in osmoregulation of embryo under osmotic stress.8. Low salinity preference behavior of Chinese mitten crab Eriocheir sinensis megalopaeUp-estuary migration of crab larvae to adult habitats is thought to be started after molt to megalopae. However, the mechanisms used by crab megalopae for reinvasion of the estuary are unclear. We investigated the salinity preference behavior of E. sinensis megalopae in a seven choice-chambers device, to determine whether they showed preference behavior for the low salinity of the water or not. If low-salinity preference exists, this might be one of the bebavioral mechanisms for reinvasion estuary. Notable salinity gradients can be available in the seven choice-chambers device. Results revealed that most megalopa (60%-89%and69%-80%in two replicates, respectively) selected nominal0ppt as their final preference during9:00-12:00am. However, this behavioral weaken slightly (29%-60%and31%-57%megalopa prefer to nominal0ppt in two replicates, respectively) during14:00-17:00pm. Our findings reveal that E. sinensis megapolas obviously prefer low salinities, which associated with their strong osmoregulatory ability. Low-salinity preference behavior might be an important mechanism for megalopa’s up-estuary migration. However, the extent of preference behavioral seems to be influenced by light. We speculated that the low-salinity preference behavior of megalopa is a hereditary behavioral while unaffected by environmental change. The importance of low-salinity preferences in relation to the megapola’s up-estuary migration will be discussed in this study.9. Effect of salinity on molt and Na+/K+-ATPase activity of E. sinensis megalopaWe determinted the percentage of megalopa molt to first juvenile instars under the condition of salinity range of1-40ppt, and detected the Na+/K+-ATPase activities of total megalopas after directly exposing them to1,15ppt (control group), and30ppt salinity. The result showed that nearly all the megalopas molted to the first juvenile instars at salinities range of1-40ppt on the fifth day of experiment, whereas molt delayed significantly at40ppt, compared with other salinities. Na+/K+-ATPase activity increased remarkably either at low salinity (1ppt) or at high salinity (30ppt) medium, compared with control group. The results suggest that Eriocheir sinensis megalopas possess moderate hyper-/hypo-regulating ability, and the osmoregulatory ability was also exhibited by the change of Na+/K+-ATPase activity under salinity stress. The results of this study provide physical evidence for comprehensing the up-estuary migration of crab larvae to adult habitats.