Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

Effects of the complete replacement of fish oil with linseed oil on growth,fatty acid composition,and protein expression in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir sinensis</Emphasis>)

Background

The finite marine resources make it difficult for us to obtain enough fish oil (FO) used in aquatic feeds. Another sustainable ingredients should be found to substitute FO. The effects of replacing FO with vegetable oil have been studied in a variety of crustaceans, but most studies have focused on the phenotypic effects. Little is known about the mechanisms of the effects.

Methods

To understand the molecular responses during the replacement of FO in Eriocheir sinensis, we investigated the effects of feeding FO or linseed oil (LO) on growth performance, digestive enzyme activity, fatty acid composition and protein expression in E. sinensis. Twenty-four juvenile crabs were fed diets containing FO or LO for 112 days. Weight, carapace length and width were recorded. Fatty acid composition of the diets and the hepatopancreas and protein expression in the hepatopancreas were analyzed.

Results

Growth performance and molting interval were unchanged by diet. Crabs fed FO and LO had same activity of lipase and amylase, but comparing with crabs fed LO, crabs fed FO had higher trypsin activity and lower pepsin activity. Hepatopancreas fatty acid composition changed to reflect the fatty acid composition of the diets. In total, 194 proteins were differentially expressed in the hepatopancreas between the diets. Expression of heat shock proteins was higher in crabs fed LO. Expression of fatty acid synthase, long-chain fatty acid transport protein 4, acyl-CoA delta-9 desaturase, and fatty acid-binding protein 1, was higher in crabs fed FO.

Conclusions

The substitution of FO with LO didn’t have any effects on the growth and molting of mitten crab, but could significantly decrease the ability of mitten crab to cope with stress. The high content of HUFAs in the hepatopancreas of mitten crab fed FO is due to the high abundance of the proteins relative to the transport of the HUFAs. These findings provide a reason of the high content of EPA and DHA in crabs fed with FO, and provide new information for the replacement of FO in diets of mitten crab.

     

Invasion biology of the Chinese mitten crab Eriochier sinensis: A brief review

The Chinese mitten crab Eriocheir sinensis is a native of freshwater and estuarine habitats along the east coast of Asia. Invasive populations have existed in northern Europe since the early 20th century, and more recently a breeding population has become established in the San Francisco Bay system along the west coast of North America. Ballast water is the most probable vector for both invasions, although there is also potential for escape from ethnic markets and from the ornamental aquarium industry. Invasive populations of mitten crabs have caused millions of dollars in economic and ecological damage. Economic impacts center largely on the burrowing activity of the crabs, which damages stream banks and levees, and the annual spawning migration, which interferes with fishing activities and irrigation projects. Chinese mitten crabs have recently appeared in the Chesapeake and Delaware Bays on the east coast of the USA, and there are confirmed reports of breeding females in both estuaries. The potential for large populations of mitten crabs in these estuaries has not been determined. This paper presents a review of the biology and ecology of native and invasive populations of the species and provides recommendations for research relevant to the prediction of future mitten crab invasions.     

Myosin Va Participates in Acrosomal Formation and Nuclear Morphogenesis during Spermatogenesis of Chinese Mitten Crab Eriocheir sinensis

Background

The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis.

Methodology/Principal Findings

Western blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus.

Conclusions/Significance

Our study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis.     

Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis

Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48 to 51%. CatA mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

Sexual cycle properties and changes in carotenoid levels in ovary of Japanese mitten crab <Emphasis Type="Italic">Eriocheir japonica</Emphasis> from rivers of Primorye

The sexual cycle of Japanese mitten crab Eriocheir japonica (de Haan, 1835) in its northern range in water bodies of the Primorye proved to have specific properties including changed seasonal dynamics of gonad maturity stages, delayed spawning period, and reduced breeding season. The ovary color regularly changed during the gonad growth in females, which was accompanied by an increase in total carotenoid content from 1.4 to 22.2 mg/100 g tissue and by changes in the proportion of major pigments. The relative content of the carotene fraction notably decreased and that of astaxanthin slightly decreased during gonad maturation. The revealed changes in carotenoid levels in the ovary point to the activity and significance of pigment metabolism in the reproductive cycle of E. japonica.     

An Experimental Study on the Gait Patterns and Kinematics of Chinese Mitten Crabs

Despite the many studies on eight-legged animals and the importance of their mechanics of terrestrial locomotion, the mechanical energy of crabs in voluntary locomotion on uneven, unpredictable terrain surfaces has received little attention thus far. In this paper, motion video images of Chinese mitten crab (Eriocheir sinensis Milne-Edwards) locomotion on five types of terrains were recorded using a high-speed three-dimensional (3D) recording video system. The typical variables of locomotion such as gait patterns, duty factor, mechanical energy of the mass center, mass-specific rate of the total mechanical power of the mass center, and percentage recovery, were analyzed. Results show that the Chinese mitten crab uses random gaits instead of the alternating tetrapod gait with the increasing terrain roughness. The duty factors of the rows of the leading legs are greater for all terrains than those of the rows of the trailing legs. On smooth terrain, the duty factors of the rows of the trailing legs are greater than that on rough terrains. Kinematic measurements and calculations reveal that similar to mammals, birds, and arthropods, the Chinese mitten crab uses two fundamental gaits to save mechanical energy: the inverted pendulum gait and the bouncing gait. The bouncing gait is the main pattern of mechanical energy conservation. The low probability of injury and energy expenditure due to adaptations to various terrains induce the Chinese mitten crab to modify the mass-specific rate of the total mechanical power of the mass center. The statistical results of percentage recovery also reveal that the Chinese mitten crab has lower energy recovery efficiency over rough terrains compared with smooth terrains.     

Symbiotic Bacteria in Gills and Guts of Chinese Mitten Crab (Eriocheir sinensis) Differ from the Free-Living Bacteria in Water

Aquatic animals have a close relationship with water, but differences in their symbiotic bacteria and the bacterial composition in water remains unclear. Wild or domestic Chinese mitten crabs (Eriocheir sinensis) and the water in which they live were collected from four sampling sites in Jiangsu and Shanghai, China. Bacterial composition in water, gills or guts of E. sinensis, were compared by high-throughput sequencing using 16S rRNA genes. Analysis of >660,000 sequences indicated that bacterial diversity was higher in water than in gills or guts. Tenericutes and Proteobacteria were dominant phyla in guts, while Actinobacteria, Proteobacteria and Bacteroidetes were dominant in gills and water. Non-metric multidimensional scaling analysis indicated that microbiota from gills, guts or water clearly separated into three groups, suggesting that crabs harbor a more specific microbial community than the water in which they live. The dominant OTUs in crab gut were related to Mycoplasmataceae, which were low in abundance in gills, showing that, like mammals, crabs have body-site specific microbiota. OTUs related to Ilumatobacter and Albimonas, which are commonly present in sediment and seawater, were dominant in gills but almost absent from the sampled water. Considering E. sinensis are bottom-dwelling crustacean and they mate in saline water or seawater, behavior and life cycle of crabs may play an important role in shaping the symbiotic bacterial pattern. This study revealed the relationship between the symbiotic bacteria of Chinese mitten crab and their habitat, affording information on the assembly factors of commensal bacteria in aquatic animals.     

Effect of natural disturbance on migrations and population dynamics of a catadromous species, Eriocheir japonicus, along the Lo-Mei stream in northern Taiwan

Japanese mitten crab, Eriocheir japonicus (de Haan), migrates between stream and sea and breeds in shallow coastal areas, exhibiting a catadromous life cycle. This study investigated how the crabs adjust reproductive strategy and migratory behavior to climate fluctuation and what kind of life history strategy individual crabs adopt in a disturbed environment. We conducted a 14-year monitoring study from 1998 to 2011 in Lo-Mei Stream in northern Taiwan. Streamflow and flood timing are controlled by precipitation and typhoons. Abundance of crabs was lower after disturbances by several typhoons and by lower temperatures associated with cold winters in 2006 and 2007. Samples from 2003 showed a reversal in reproductive trends, with increasing size in the same pattern of oocytes, compared to reproductive biology from 1999 to 2005. The life history of E. japonicus can be divided into three stages: spawning migration, growth period, and upstream migration of juveniles to compensate for downstream migration of mature crabs. Reproductively, in E. japonicus the combination of semelparity and synchronous breeding can increase reproductive potential and increase survival in a less disturbed environment at the optimum time. Environmental cues play an important role as major triggers in the persistence of populations.