Habitat Preference And Marking Techniques Of Ark Shell Anadara Broughtonii In Stock Enhancement

The ark shell Anadara broughtonii is a commercially important bivalve species in China, Japan, Korea, Philippines and Southeastast of Russia which burrows shallowly in sandy mud or muddy bottoms. In recent years, the natural populations of this species are decreasing quickly and dramatically due to the deterioration of the coastal environment and over exploitation. Consequently, enhancement methods and techniques have been developed to recover the ark shell resources. But there are still have some problems in the stock enhancement, such as the lack of researches in releasing and marking techniques. With an attempt to provide useful release strategies and to estimate the effectiveness of the stock enhancement strategies correctly, environment choice, sulfide metabolic mechanism in molecular level, and marking methods for juvenile A. broughtonii were investigated. The results are as follows:1. The effects of temperature on burrowing ability of juvenile ark shell（Anadara broughtonii） and predation rate on juvenile ark shell by sea star（Asterias amurensis）Experiments were carried out to investigate the burrowing ability of juvenile ark shell Anadara broughtonii （20-30 mm shell length）, and the predation rate on juveniles by sea star （Asterias amurensis） at different seawater temperatures （10,15,20,25 and 30℃, respectively）. The results showed that A broughtonii had the longest burrowing time （28.87 min） and the lowest burrowing rate （21.25%） at 10℃, which were significantly different from other temperature treatments （P<0.05）. The shortest burrowing time （7.49 min） and the highest burrowing rate （55.63%） were observed at 20℃, suggesting that 20℃ was the suitable burrowing temperature. The predation rate at 20℃ （5.28 ind/d） was significantly higher than those at 10 and 25℃ （P<0.05）. The optimum predation temperature was between 15 and 20℃ and the Q10 coefficient for A. amurensis was 2.22 at that range. The overlap of the optimum temperature range was observed for A. broughtonii burrowing and preyed by A. amurensis, suggesting that protective strategies for juvenile A. broughtonii are needed when the water temperature is close to 20℃.2. Substrate preference and burrowing ability assessment of the juvenile Anadara broughtoniiBehavioral preference of juvenile Anadara broughtonii with different shell length （10-15 mm,15-20 mm,20-25 mm, and 25-30 mm） to four kinds of substrates （granule gravel, coarse sand, medium sand, and fine sand） were under laboratory conditions studiedand then the burrowing ability of juvenile A. broughtonii （20-30 mm shell length） to five kinds of substrates （add mud group） were investigated. The results showed that juveniles （10-15 mm shell length） have a developed byssi and mostly in sessile life with the highest distribution rate at the junction between substrates and percentage of hanging on the tank walls. Also, these significantly attached on coarse sand （P<0.05）. The juveniles （15-30 mm shell length） change into infaunal life and grain sizes preferred by them increased as the increase of shell length, accompanying a decrease in the percentage of laying in joint and percentage of hanging on the tank walls.The juveniles （15-20 mm shell length） preferred medium sand significantly （P<0.05） with a distribution rate of 50.5%, then the fine sand. The juveniles （20-25 mm shell length） have well adaption for all kinds of substrates. The juveniles （25-30 mm shell length） preferred coarse sand significantly （P<0.05） with a distribution rate of 65%, then the medium sand. A. broughtonii with 20-30 mm shell length had the shortest burrowing time （2.74 min） at mud, then the fine sand and medium sand. The highest percent of burrowing rate （85%） were attained in the fine sand, then in the medium sand. Our data indicated that shell length 20-30 mm and sea bottom mainly with medium and fine sand are fit for stock enhancement.3. Transcriptomic response to sulfide exposure in the ark shell, Anadara broughtoniiSulfide exists as aggregate of H2S, HS-, and S2-, is harmful to aerobic organisms in variety of ways. To better understand the interaction between toxic damage and detoxification processes, digital gene expression profile （DGE） analysis was performed in the gills of A. broughtonii in control group （GC）, anoxia group （GO）, low sulfide group （GO_LS） and high sulfide group （GO_HS）, using the RNA-seq technology. The top 10 expressed different express genes is relate to hemoglobin gene and immune gene （Rh-related protein, beat shock protein and baculovirus polyhedron envelope protein）. GO functional enrichment analysis results showed some differentially expressed genes were significant enriched in crucial biological processes related to aminoacyl-tRNA in GO_LS vs. GC group, and biological regulation, regulation of biological process and regulation of cellular process in GO_HS vs. GC group. KEGG pathway enrichment analysis results showed that aminoacyl-tRNA biosynthesis and protein processing in endoplasmic reticulum pathway significant enriched in GO_LS vs. GC group, and TNF signaling pathway and MAPK signaling pathway significant enriched in GO_HS vs. GC group. The result showed that ARS gene in aminoacyl-tRNA biosynthesis pathway and HSP、OST、PDI genes in protein processing in endoplasmic reticulum pathway were almost up-regulated in low sulfide expose were probably related to maintain the genome integrity and cause immune response such as protein repair and proteolysis. In high sulfide group （GO_HS）, BIRC2₃ in TNF pathway and a large number of MAPK kinase and HSP genes in MAPK pathway up-regulated, analysis that high concentration sulfide are probably cause great number of protein repair, proteolysis and immune responses. Our data will provide clues for further probing of the molecular mechanism of sulfide metabolism.4. The fluorescent staining and CLIP tagging for ark shell, Anadara broughtoniiThe growth, survival, and tag retention were investigated in juvenile ark shell Anadara broughtonii with initial shell length of 27.24±1.12 mm stained by alizarin red S （ARS） and calcein （CAL） at the concentration of 200 mg/L and 300 mg/L and marked by stainless steel CLIP tags. Then the marked ark shell juveniles were reared in cages suspended in culture rafts in Qingdao, China for 1 year when the growth performance, survival and CLIP retention were monitored 15 d,30d,60d,90 d,120 d,160 d after treatment, and the fluorescent markings were monitored for 1 year （add 270 d,365 d）. The results showed that there were no significant differences in survival and shell growth in the juveniles stained by the two dyes at 200 mg/Land 300 mg/L, with survival of 100% in 1-year culture. The fluorescent stain marking （≥ grade 3） was observed in the juveniles stained by the two dyes under a microscope, and the fluorescent stain marking （≥ grade 4） in some samples even by naked eye, the brighter stain marking by ARS than by CAL and more easy to be detected at 300 mg/L of ARS. As the ark shell grew, the CLIP tags shown to be incorporated into the shell thereby preventing disengagement of the tag from the shell portion, without significant difference in the survival and daily growth （P>0.05）, and with retention rate of 64.25% in the 160 d tag, indicating that the tags were wrapped by nacreous layer secreted by the ark shell. The findings reveal that the fluorescent stain marking can be used for the large-scale releasing of ark shell A. broughtonii, and that CLIP tags can provide additional individual information, and is more suitable for accurate scientific research.