Abstract
Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone.
Highlights
South Africa has a rapidly developing abalone aquaculture industry, based on the cultivation of Haliotis midae [1]
V. midae SY9 was cultured in marine broth (MB) [(wt/vol) 3% NaCl, 0.23% MgCl2.6H2O, 0.03% KCl, 0.2% glucose, 0.5% casamino acids, 0.1% yeast extract] or peptone marine basal medium (P-MBM) [(wt/vol) 3% NaCl, 0.23% MgCl2.6H2O, 0.03% KCl, 1% peptone, 0.1% yeast extract] with shaking at 100 rpm at 22uC and maintained on marine agar (MA) [MB supplemented with 2% bacteriological agar, Unilab] at 22uC
Transposon Mutagenesis of V. midae SY9 Fifty eight V. midae SY9Smr cells growing on VNSS agar supplemented with Sm and Kan were screened by PCR amplification in order to confirm chromosomal integration of the mini-Tn10-gfp-kan cassette
Summary
South Africa has a rapidly developing abalone aquaculture industry, based on the cultivation of Haliotis midae [1]. The relatively slow growth rate of abalone represents a major constraint on the aquaculture industry. The use of probiotic microorganisms is becoming increasingly accepted as a means of improving the health and growth of aquacultured species [2]. Several possible modes of action have been proposed for probiotic effects observed within aquaculture environments [5,6], including the production and secretion of extracellular hydrolytic enzymes that contribute to, and improve, the digestion efficiency of the host. Several studies have demonstrated the effect of probiotic supplementation on abalone digestive enzyme activity levels and/or growth, and have suggested a possible role for ‘nutritional probiotics’ in abalone aquaculture [7,8]
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