Microbial diversity of mid-stage Palinurid phyllosoma from the Great Barrrier Reef

Samples of Phyllosoma were collected from trawls conducted on the Great Barrier Reef at depths of 1-100 m in May 2005. Phyllosoma were identified to genus (and to species level when possible) immediately upon collection using a dichotomous key.The total bacterial DNA associated with phyllosoma was extracted from two separately homogenized P. ornatus phyllosoma and one homogenized Panulirus penicillatus phyllosoma using the Qiagen DNeasy Tissue Kit (Qiagen, Hilden, Germany). Bacterial 16S rRNA gene was amplified from DNA extracted from phyllosoma using the primers 27f and 1492r.Partial and complete 16S rRNA gene sequences of bacterial clones were obtained using the primers 27f, 339f, 732f and 1492r; bacterial representatives were found from the following: a-Proteobacteria, Firmicutes, y-Proteobacteria, e-Proteobacteria, Bacteroidetes. Various indices and models were used to analyse the variation of microbial diversity within clone libraries. The genus affiliation of dominant bacterial sequences (Sulfitobacter, uncultured e-proteobacterium, Erythrobacter, Roseobacter, Exiguobacterium, Psychrobacter, Clostridium, Pseudomonas, Alteromonas, Paracoccus) retrieved from the clone libraries was recorded for wild Panulirus ornatus (PO1 and PO2) and Panulirus penicillatus (PP) phyllosoma samples.Phyllosoma samples were analysed by histology (examined using light microscopy to determine any internal lesions or disease); scanning electron microscopy (SEM) to determine bacterial colonization (external or internal); and fluorescence in situ hybridization (FISH) to examine bacteria in tissue sections (without detectable bacterial cells, with low bacterial density, occasional cell accumulations).The GenBank accession numbers assigned for nucleotide sequence data of all phyllosoma clones submitted are:DQ985883, DQ985884, DQ985885, DQ985886, DQ985887, DQ985888, DQ985889, DQ985890, DQ985891, DQ985892, DQ985893, DQ985894, DQ985895, DQ985896, DQ985897, DQ985898, DQ985899, DQ985900, DQ985901, DQ985902, DQ985903, DQ985904, DQ985905, DQ985906, DQ985907, DQ985908, DQ985909, DQ985910, DQ985911, DQ985912, DQ985913, DQ985914, DQ985915, DQ985916, DQ985917, DQ985918, DQ985919, DQ985920, DQ985921, DQ985922, DQ985923, DQ985924, DQ985925, DQ985926, DQ985927, DQ985928, DQ985929, DQ985930, DQ985931, DQ985932, DQ985933, DQ985934, DQ985935, DQ985936, DQ985937. To determine the bacterial community associated with wild-caught, mid-stage larvae of spiny lobsters (Palinuridae) in their native oligotrophic marine environment, and to compare their diversity and composition with communities associated with aquaculture-reared larvae of the tropical rock lobster Panulirus ornatus, the current identified candidate for aquaculture in Australia. The phylogenetic tree included closest relativeves (and accession numbers): Acidovorax sp. (AJ534865), Alteromonas sp. (AF427478), Citromicrobium bathoceanense (Y16267), Clostridium quinii (X76745), Cytophaga marinoflava (M58770), Erythrobacter citreus (AF118020), Exiguobacterium sp. (AY205564), hodothermus marinus (X77140), Pseudoalteromonas sp. (AF239705), Pseudoalteromonas sp. (AJ704790), Pseudoalteromonas sp. (AY258115), Pseudoalteromonas sp. (U80834), Pseudomonas saccharophila (AF368755), Psychrobacter arenosus (AJ609273), Psychrobacter marincola (AJ309941), Psychrobacter pacificensis (AB016054), Psychrobacter pacificensis (AB016055), Psychrobacter sp. (AJ551125), Roseobacter sp. (AJ536669), Staphylococcus saccharolyticus (L37602), uncultured a-Proteobacterium (AY033306), Uncultured e-Proteobacterium (AF367487), Vibrio alginolyticus (AF513447).

2005年5月，研究人员于大堡礁1~100米水深的拖网作业中采集了叶状幼体（Phyllosoma）样本。采集完成后，立即使用二歧分类检索表对叶状幼体开展属级鉴定，条件允许时可鉴定至种级。
分别选取两份均质化的锦绣龙虾（Panulirus ornatus）叶状幼体样本与一份均质化的杂色龙虾（Panulirus penicillatus）叶状幼体样本，使用德国希尔德市Qiagen公司的DNeasy组织试剂盒（Qiagen DNeasy Tissue Kit, Qiagen, Hilden, Germany）提取与叶状幼体共生的细菌总DNA。以引物27f和1492r，从提取的共生细菌DNA中扩增细菌16S rRNA基因。
使用引物27f、339f、732f与1492r获取细菌克隆的部分及完整16S rRNA基因序列，所获细菌类群涵盖α-变形菌门（α-Proteobacteria）、厚壁菌门（Firmicutes）、γ-变形菌门（γ-Proteobacteria）、ε-变形菌门（ε-Proteobacteria）及拟杆菌门（Bacteroidetes）。采用多种指数与模型分析克隆文库内微生物多样性的变化情况，并记录了从野生锦绣龙虾（PO1、PO2）与杂色龙虾（PP）叶状幼体样本的克隆文库中获取的优势细菌序列的属级分类地位，包括嗜硫杆菌属（Sulfitobacter）、未培养ε-变形菌、赤杆菌属（Erythrobacter）、玫瑰杆菌属（Roseobacter）、微小杆菌属（Exiguobacterium）、嗜冷杆菌属（Psychrobacter）、梭菌属（Clostridium）、假单胞菌属（Pseudomonas）、交替单胞菌属（Alteromonas）、副球菌属（Paracoccus）。
对叶状幼体样本开展三类检测：组织学检测（通过光学显微镜观察是否存在内部病变或病害）、扫描电子显微镜（SEM）分析以确定细菌定殖部位（体表或体内），以及荧光原位杂交（FISH）技术检测组织切片中的细菌，分为未检出细菌细胞、细菌密度较低、偶见细胞聚集三种情况。
本次提交的所有叶状幼体克隆的核苷酸序列数据所获得的GenBank登录号为：DQ985883、DQ985884、DQ985885、DQ985886、DQ985887、DQ985888、DQ985889、DQ985890、DQ985891、DQ985892、DQ985893、DQ985894、DQ985895、DQ985896、DQ985897、DQ985898、DQ985899、DQ985900、DQ985901、DQ985902、DQ985903、DQ985904、DQ985905、DQ985906、DQ985907、DQ985908、DQ985909、DQ985910、DQ985911、DQ985912、DQ985913、DQ985914、DQ985915、DQ985916、DQ985917、DQ985918、DQ985919、DQ985920、DQ985921、DQ985922、DQ985923、DQ985924、DQ985925、DQ985926、DQ985927、DQ985928、DQ985929、DQ985930、DQ985931、DQ985932、DQ985933、DQ985934、DQ985935、DQ985936、DQ985937。
本研究旨在明确原生贫营养海洋环境中野生捕获的龙虾科（Palinuridae）中期幼体的共生细菌群落组成，并将其与澳大利亚当前拟开展养殖的热带岩龙虾——锦绣龙虾（Panulirus ornatus）的养殖幼体共生群落的多样性与组成进行对比。本研究构建的系统发育树包含如下近缘物种（及对应登录号）：食酸菌属（Acidovorax）sp.（AJ534865）、交替单胞菌属（Alteromonas）sp.（AF427478）、深海柠檬杆菌（Citromicrobium bathoceanense, Y16267）、奎氏梭菌（Clostridium quinii, X76745）、海洋噬纤维菌（Cytophaga marinoflava, M58770）、柠檬色赤杆菌（Erythrobacter citreus, AF118020）、微小杆菌属（Exiguobacterium）sp.（AY205564）、hodothermus marinus（X77140）、假交替单胞菌属（Pseudoalteromonas）sp.（AF239705、AJ704790、AY258115、U80834）、嗜糖假单胞菌（Pseudomonas saccharophila, AF368755）、沙栖嗜冷杆菌（Psychrobacter arenosus, AJ609273）、海洋嗜冷杆菌（Psychrobacter marincola, AJ309941）、太平洋嗜冷杆菌（Psychrobacter pacificensis, AB016054、AB016055）、嗜冷杆菌属（Psychrobacter）sp.（AJ551125）、玫瑰杆菌属（Roseobacter）sp.（AJ536669）、解糖葡萄球菌（Staphylococcus saccharolyticus, L37602）、未培养α-变形菌（uncultured α-Proteobacterium, AY033306）、未培养ε-变形菌（Uncultured ε-Proteobacterium, AF367487）、溶藻弧菌（Vibrio alginolyticus, AF513447）。