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

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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.

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Notes

Credit
Bourne, David G, Dr (Principal Investigator)