Microbiological aspects of larval rearing of the ornate rock lobster, Panulirus ornatus

A polyphasic approach was used to study the microbial community, incorporating direct microscopic analysis, culture-based and molecular microbiological methods, as well as analyses of quorum sensing (QS) molecules in phyllosoma and biofilm samples.The microbial community from the water column, the biofilm, live feeds and phyllosomas in the larval-rearing tank were investigated by standard microbial plating techniques. Biofilm samples were sampled in two ways: by random swabbing of the side of the tank (10 cm below surface), and fromspecifically constructed slides (similar to those used in FISH and SEM analysis) from the larval-rearing tank. Larval samples were prepared by collecting 10 moribund phyllosomas. Artemia samples were prepared by collecting 50 animals.For the detection of quorum sensing signal molecules (N-acyl-homoserine lactones (AHLs)) the bacterial AHL monitor strain JB357 was applied which harbours the reporter plasmid pJBA89.Genomic DNA was extracted from pure cultures using a QIAGEN DNeasy® Tissue Kit. Partial and complete 16S rDNA sequences of bacterial isolates were obtained using the primers 27F, 63F, 339R, 1387R, 907R and 1492R. Sequence data were analysed with the ARB software package. Phylogenic associations were evaluated by using evolutionary distance, maximum parsimony and maximum likelihood analyses.Histopathological preparations from early stage (day 1 post-hatch before feeding) phyllosomas were compared to 20-day-old phyllosomas: the gut, hepatopancreas and mid-gut were examined. Cell necrosis was also noted in the 20-day-old phyllosomas.Fluorescence in situ hybridisation (FISH) analysis using a universal bacterial probe on early stage (day 1 post-hatch) phyllosoma demonstrated bacteria only on external surfaces of the animals whereas later stage phyllosoma had bacteria within and through the hepatopancreas tubule lumen and intestine. A Vibrio specific probe showed these species formed the majority of the bacterial community.Scanning electron microscopy (SEM) was employed to follow external fouling over 21 days on the animals, and was used to investigate 3 areas on the larval animal: the mouth parts (high nutrient input), the anus (high nutrient output) and the eye (control area) over time following a moult. Filamentous bacteria adhesion to the carapace was observed within 2-3 days post-moult. Targeted FISH Eubacterial (Eub+) probes and Thiothrix specific rRNA targeted probe found that the majority of filamentous bacteria associated with phyllosoma fouling were Thiothrix species.
To obtain an understanding of the microbial community and dynamics within the larval-rearing system, the larval-rearing environment was examined as four microbiological compartments: 1) the water column; 2) the biofilm; 3) the live phyllosoma feed (Artemia); 4) the phyllosomas.Dividing the system into compartments was useful in obtaining an understanding of the microbial community as well as interactions and dynamicsof the entire larval-rearing system.
The major cause of phyllosoma mortality is believed to be due to bacteria, especially opportunistic pathogens.

本研究采用多相研究方法对微生物群落展开探究，涵盖直接显微分析、基于培养的微生物学方法与分子微生物学方法，以及对叶状幼体（phyllosoma）和生物膜样品中的群体感应（quorum sensing, QS）分子的分析。本研究对育苗水槽内水柱、生物膜、活饵料以及叶状幼体的微生物群落，采用标准微生物涂布平板技术开展了调查。生物膜样品通过两种方式采集：一是随机擦拭水槽内壁（水面下10 cm处），二是从育苗水槽内特制的载玻片（与荧光原位杂交（fluorescence in situ hybridisation, FISH）和扫描电子显微镜（scanning electron microscopy, SEM）分析所用载玻片类似）采集。幼虫样品采集10只濒死叶状幼体制备获得，卤虫（Artemia）样品则采集50只个体制备。
为检测群体感应信号分子N-酰基高丝氨酸内酯（N-acyl-homoserine lactones, AHLs），本研究使用了携带报告质粒pJBA89的细菌AHL监测菌株JB357。使用QIAGEN DNeasy®组织试剂盒从纯培养物中提取基因组DNA。利用引物27F、63F、339R、1387R、907R及1492R，获取了细菌分离株的部分及完整16S rDNA序列。使用ARB软件包对序列数据进行分析。通过进化距离、最大简约法以及最大似然法分析，对菌株的系统发育关联进行了评估。
将孵化后第1天（投喂前）早期叶状幼体的组织病理切片，与20日龄叶状幼体进行对比，重点观察其肠道、肝胰腺及中肠。研究同时观察到20日龄叶状幼体存在细胞坏死现象。对孵化后第1天的早期叶状幼体使用通用细菌探针开展荧光原位杂交分析，结果显示细菌仅定殖于动物体表；而后期叶状幼体的细菌则遍布肝胰腺小管腔及肠道内部。弧菌属特异性探针检测结果表明，该类菌群构成了微生物群落的主体。采用扫描电子显微镜对叶状幼体体表21天内的附着情况进行追踪，并在蜕皮后不同时间点，对幼虫体表3个区域展开研究：口器（高营养输入区）、肛门（高营养输出区）以及眼（对照组区域）。研究发现，蜕皮后2-3天内即可观察到丝状细菌附着于甲壳表面。通过靶向细菌（Eub+）探针以及硫丝菌属（Thiothrix）特异性rRNA探针的FISH分析，证实叶状幼体体表附着的丝状细菌多数为硫丝菌属物种。
为阐明育苗系统内的微生物群落及其动态变化，本研究将育苗环境划分为4个微生物学研究分区：1）水柱；2）生物膜；3）活饵料卤虫；4）叶状幼体。将系统划分为分区的研究方式，有助于解析整个育苗系统的微生物群落、物种互作及动态变化过程。
目前认为，叶状幼体死亡的主要诱因为细菌感染，尤其是机会性致病菌。