拟穴青蟹Cactus基因的cDNA克隆、序列及生物学功能

为获取拟穴青蟹Cactus基因cDNA全长、分析基本生物学信息,并初步探索其在病原物刺激下的免疫反应,实验采用RACE技术获得了拟穴青蟹Cactus(SpCactus)基因的cDNA全长序列,其cDNA全长为2035 bp,开放阅读框(ORF)1311 bp,编码436个氨基酸,分子量为46.01 ku。对蛋白理化性质进行预测发现,SpCactus为亲水性蛋白,等电点pI为4.91。经预测,SpCactus与其他物种的IκB蛋白具有相似的功能结构域。同源性比对结果显示,SpCactus与凡纳滨对虾和中国明对虾的Cactus蛋白同源性均高达62%,相似度为73%。系统进化树分析显示,SpCactus与甲壳动物聚为一支,与无脊椎动物聚为一大支。实时荧光定量PCR(RT-PCR)检测发现,SpCactus基因在拟穴青蟹不同组织中均有表达,肌肉中的表达量最高,其次为心脏、眼柄、血液和鳃,肝胰腺中的表达量最低。LPS和金黄色葡萄球菌刺激均能显著诱导SpCactus基因的表达。本实验成功扩增了SpCactus基因全长,并进行了生物信息学分析、理化性质预测,初步探讨了其生物学功能,为进一步研究其在免疫反应中的生物学功能提供参考依据。     

温度对拟穴青蟹循环水养殖系统微生物群落结构的影响

为探究温度对拟穴青蟹(Scylla paramamosain)循环养殖水体中微生物群落的影响,深入理解环境因子与养殖水体微生物群落之间的相关性。在水温分别为18℃、22℃、26℃、30℃和32℃的循环养殖系统中进行拟穴青蟹养殖,采用16S rRNA高通量测序技术对不同温度下的养殖水体微生物群落组成进行分析。结果表明:(1) Alpha多样性指数显示5组水样中细菌群落多样性指数大小为t26t22t30t32t18,物种丰度大小为t22t26t32t30t18。温度可以导致养殖水体中微生物群落多样性发生变化,但并未发现二者之间直接的相关性。(2) 5种温度下的养殖系统中细菌群落共有31门、43纲、94目、185科和351属,变形菌门(Proteobacteria)为循环养殖水体中绝对优势菌门。在养殖系统水温为26℃时,养殖水体中氮磷污染物含量相对较高,其水中脱氮除磷菌属的相对丰度也显著高于其他4组。(3)由花瓣图和UPGMA聚类分析图进行相似性分析可知,水体微生物群落对温度的变化有着良好的响应, 5组水样的细菌群落结构总体相似性较低,其中最高温度32℃和最低温度18℃的水体中细菌群落结构最为相似。(4)水体中微生物群落结构和与环境因子的相关性分析显示,氮磷营养盐中的总磷(TP)和硝酸盐(NO3–-N)对水中微生物群落特征具有显著性影响,而温度是导致养殖水体中氮磷营养盐差异化的主要原因。上述研究结果初步解释了温度对拟穴青蟹循环养殖水微生物群落的作用机制,为下一步深入研究拟穴青蟹循环水养殖系统中环境变化与微生物的相互关系提供参考。     

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20–33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.     

Acorn dispersal and predation patterns of four tree species by wood mice in abandoned cut-over land

We compared patterns of acorn dispersal and predation by wood mice among four tree species (Quercus serrata, Quercus crispula, Castanea crenata, and Juglans mandshurica var. sieboldiana) that are abundant in cool temperate woodlands. We devised an acorn dispersal experiment using 400 magnet-inserted acorns and a magnetic locator in a 1.8-ha study plot, which spanned a cut-over area and an adjacent deciduous forest. Ten wire mesh baskets, each containing 40 acorns (10 acorns per species), were placed on the border between these two habitat types. About 13.0% (n = 52) of the total acorns remained in the baskets, while 77.3% (n = 309) were dispersed throughout the study plot and subsequently retrieved using the magnetic locator. Microhabitat, distance, and burial depth of transported acorns were significantly different among species. In the cut-over area, J. mandshurica var. sieboldiana acorns were dispersed under fallen trees or branches and near stumps, and were buried deeply in the soil. Dispersal distances of J. mandshurica var. sieboldiana acorns were significantly greater than those of Q. serrata acorns. The number and microhabitat of transported acorns significantly differed between habitat types. J. mandshurica var. sieboldiana acorns were dispersed in the cut-over area rather than in the forest. For all four species, the numbers of acorns delivered to fallen trees or branches, stumps, and crumbled soil with overhang under any vegetation type were greater in the cut-over area than in the forest.     

拟穴青蟹幼蟹耗氧率和窒息点的研究

在盐度20、温度28.0±0.2℃条件下,采用密封流水方法进行了拟穴青蟹幼蟹耗氧率和窒息点的研究。结果表明:拟穴青蟹Ⅴ期幼蟹的平均耗氧率高于Ⅵ期幼蟹,Ⅴ期幼蟹的窒息点也高于Ⅵ期幼蟹。其中,当蟹苗体质量为1.00±0.22 g时,耗氧率为0.437 2±0.083 mg/(g.h),耗氧量0.423 7±0.080 mg/(ind.h),窒息点为0.716 0±0.017 mg/L;当蟹苗体质量为1.81±0.30 g时,耗氧率为0.381 6±0.081 mg/(g.h),耗氧量0.671 7±0.140 mg/(ind.h),窒息点为0.698 7±0.011 mg/L。实验发现,拟穴青蟹幼蟹的耗氧率和耗氧量具有比较明显的昼夜变化规律,其峰值出现在20∶00及02∶00左右,谷值出现在08∶00及16∶00。研究结果可为拟穴青蟹养殖生产中制定合理的苗种放养密度和养殖工艺提供参考依据。     

Mud crab pen culture: replacement of fish feed requirement and impacts on mangrove community structure

Brackishwater pond culture has been a major factor in mangrove loss in Southeast Asia, hence, the need to develop environment‐friendly technologies such as mud crab Scylla (Portunidae) culture in mangrove pens exists. This study evaluated the effects of mud crab netpen systems in central Philippines on mangrove macroflora, and the replacement of dietary fish with low‐cost pellets. Wild or hatchery‐sourced Scylla olivacea and Scylla serrata were stocked at 0.5–0.8 m⁻² in 167–200 m² nylon netpens (2.3 cm stretched mesh) in Avicennia‐dominated mangrove habitats. The feeding treatments were: (A) Zarraga: (1) no feeding (natural productivity), (2) no feeding for 1 month+supplementary feeding, (3) fish biomass and (4) low‐cost pellets, and (B) Batan: (1) fish biomass and (2) pellets+fish biomass. Feeds were given ad libitum twice daily. Growth and survival rates of S. olivacea in Zarraga pens were not significantly different among treatments, although crabs fed fish biomass had the highest survival, body weight and production. Similarly, growth and survival of S. serrata were not significantly different between the Batan treatments. Economic analysis of the latter gave a 38.5% return on investment (ROI) and 2.6 years payback period (PP) for pellets+fish biomass treatment compared with 27.5% ROI and 3.6 years PP for fish alone. Sensitivity analysis showed an improved economic performance of the pellets+fish biomass treatment by increasing the survival rate. Evaluation of mangrove community structure showed that crab culture reduced species diversity, numbers and biomass of seedlings and saplings, but not of mangrove trees. Therefore, mud crab pen culture is recommended for mangrove sites with mature trees, but not seedlings and saplings, and low‐cost pellets can reduce dependence on fish biomass.     

锯缘青蟹血清凝集素的特性和盐析提取研究

对锯缘青蟹(Scylla serrate)血清凝集素的凝集特性进行了研究。结果显示:锯缘青蟹血清凝集素对人、兔、鼠、鸽子、中华鳖、鲫红细胞均有凝集作用,其中对小鼠红细胞凝集活性最强,血凝活性可达29;凝集素活性最适pH 6.0~7.4;盐度对凝集活力有较大影响,NaC l浓度在0.4~0.5 mol/L时基本失活;未纯化血清凝集素对Ca2+、Mg2+未表现出依赖性;凝集素活性能被N-乙酰葡萄糖胺特异性抑制。对青蟹血清凝集素进行硫酸铵分级沉淀分离后,发现凝集素活性主要分布在25%饱和度硫酸铵沉淀区,该组分凝集比活比血清高27.44倍,SDS-PAGE结果表明主要蛋白带型为79 ku、75 ku、72 ku。     

锯缘青蟹死亡的组织病理学初步观察

对2008年8月福建省泉州市某海水场养殖锯缘青蟹中发生的死亡现象，开展锯缘青蟹的组织病理学研究。解剖可见肌肉明显坏死、溶解；头胸甲储积大量暗黄色体液，经福尔马林固定成固态物质；光镜下发现病蟹的中肠腺上皮细胞坏死、细胞质中出现大型的包涵体；电镜下发现病蟹的细胞质中有长约为500hm、宽约为200hm杆状结构的疑似杆状病毒存在。     

锯缘青蟹Sox基因的PCR扩增

SRY基因(sex-determining region of the Y chromosome)是人类及哺乳动物睾丸决定因子TDF(testisdetermining factor)的最佳候选基因。SRY蛋白含有204个氨基酸,其中1个约79个氨基酸区域即HMG盒(high mobility group box)是SRY基因编码蛋白质的唯一功能区。由于SRY的发现,人们进而发现了1个庞大的与性别决定有关的SRY盒-Sox(SRY-related HMG box gene)基因家族。锯缘青蟹是我国重要的海洋经济蟹类之一,其性染色体和性别决定机制仍处于进化的早期阶段,在分子水平上探讨其性别决定机制尚不多见。通过采用PCR技术,以特异扩增人SRY基因HMG盒保守区的1对兼并引物,扩增了锯缘青蟹基因组的Sox基因。结果表明锯缘青蟹雌雄个体与人一样,均能扩增出1条大小约为216bp左右的基因片段,显示出该基因在进化上的高度保守性,为探索锯缘青蟹的性别决定机制及Sox基因的进化提供了分子资料。