Browsing by Author "Coyne, Vernon E"

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    Detection and localisation of the abalone probiotic Vibrio midae SY9 and its extracellular protease, VmproA, within the digestive tract of the South African abalone, Haliotis midae
    (Public Library of Science, 2014) Huddy, Robert J; Coyne, Vernon E
    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.
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    Determining larval settlement, post-settlement and weaning substrates and regimes for the sea urchin Tripneustes gratilla in intensive aquaculture
    (2022) Bennett, Michael Andrew; Cyrus, Mark D; Bolton, John; Coyne, Vernon E; Macey, Brett M
    Sea urchins gonads are a highly valued and priced seafood. Demand is stable and unlikely to decline in the future. Successful echinoculture is limited by the ability to produce large enough quantities of viable larvae and/or juveniles economically and efficiently due to a bottleneck in production during the settlement and post-settlement phases. In this study, larval settlement, post-settlement growth, and weaning regimes were investigated in the collector urchin, Tripneustes gratilla. Two cohorts of urchins were spawned for two separate growth trials. Growth trials assessed settlement, post-settlement growth, and the timing of weaning onto macroalgae (Ulva lacinulata). Experimental substrates tested include: Ulvella lens, fresh Ulva, dried Ulva and alginate, dried Ulva and agar, Nitzschia sp. (undescribed diatom), dried Isochrysis galbana and alginate, probiotic Vibrio midae SY9 and alginate, V. midae SY9 and Ulva extract F9 and alginate, an ethanol-alcohol and alginate control, and a null-alginate control (replicates=4, n=35 individuals). The highest average settlement success was achieved on fresh Ulva (67.14% ± 8.45) followed by Ulvella lens (55.71% ± 12.26) and then Nitzschia sp. (40.71% ± 5.88). These treatments were significantly different from all the other treatments (p< 0.05) but not from each other (p< 0.05). U. lens facilitated the greatest significant change in test diameter in T. gratilla post-settlement (difference of 3013µm over 4 weeks) and maintained high survival over this time (61.43% ± 10.47). Weaning was successful at 4 weeks post-settlement but was accompanied by a lag-phase in observable growth that was not observed when urchins were subjected to delayed weaning (three weeks later). Survival of urchins with delayed weaning was significantly greater than that of juveniles subjected to early weaning (p< 0.05): 100.00% compared to 92.50%; and achieving a significantly greater size: ̴̴ 1.5 mm difference in test diameter over 6 weeks. Results suggest that U. lens can induce settlement while maintaining high survival. When inducing settlement using U. lens, fresh Ulva should be placed in the same tank to facilitate increased settlement, U. lens facilitating post-settlement growth thereafter. The timing of weaning is important in facilitating optimal growth.
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    Molecular characterization of Flavobacterium spp. isolated from Rainbow trout (Oncorhynchus mykiss) farmed in Southern Africa and development of a PCR-based tool for differentiating between isolates
    (2021) Komane, Godfrey M; Coyne, Vernon E; Macey, Brett M; Flemming, Leornard
    Bacterial fish diseases caused by yellow-pigmented, filamentous bacteria of the genus Flavobacterium are among those that lead to significant losses in the international aquaculture industry. An increasing number of Flavobacterium spp. have been isolated in association with diseased fish within the aquaculture industry. In salmonids, well known flavobacterial diseases include bacterial cold-water disease (F. psychrophilum), rainbow trout fry syndrome (F. psychrophilum), bacterial gill disease (F. branchiophilum, F. aquatile), and columnaris disease (F. columnare). Conventional diagnosis of Flavobacterium spp. is based on physico-chemical tests, but these tests are time-consuming and labor-intensive, and they are unable to distinguish between closely related species due to morphological similarities. Furthermore, little information exists on the diversity of fish associated Flavobacterium spp. from southern Africa. Recently, numerous, yellow-pigmented, filamentous bacteria were isolated from diseased rainbow trout farmed in South Africa and neighboring Lesotho. The aim of the present study was to elucidate the genotypic and phylogenetic diversity of the Flavobacterium spp. associated with these fish and to develop a new molecular system for rapid and accurate identification and differentiation of the isolates. In order to do this, the genotypic and phylogenetic diversity of ninety bacterial isolates, mostly yellow-pigmented, obtained from the gills, skin ulcers, liver, and kidneys of diseased Oncorhynchus mykiss was assessed following PCR and sequencing of the 16S rRNA gene. A BLAST search of the GenBank database revealed that 47 of the 16S rRNA gene sequences showed high similarity to several Flavobacterium spp.; 6 showed high similarity to Chryseobacterium spp., and 19 non-Flavobacterium isolates were identified, which included, amongst others, Hafnia spp. and Aeromonas spp. Fifteen isolates were excluded from further analysis due to poor DNA sequence data, whilst four isolates showed high similarity to uncultured bacteria and they were also excluded from further analysis. Isolate OM-46 was excluded from the phylogenetic analysis of Flavobacterium spp. since only the forward 16S rRNA sequence was available. Phylogenetic analysis based on the maximum likelihood method confirmed the allocation of 46 isolates as Flavobacterium spp., and 6 isolates as Chryseobacterium spp. Differential identification of the Flavobacterium spp. was achieved following PCR amplification of a hypervariable region of the 16S rRNA gene followed by high-resolution melt (HRM) analysis. Nine Flavobacterium isolates, presumed to be different species based on the phylogenetic analysis were identified and successfully differentiated using high resolution melt analysis. The present study has identified new Flavobacterium spp. (OM-13, OM-39, OM-51, OM-82, and OM84) from rainbow trout farmed in southern Africa and developed a tool that may be useful for the management of flavobacterial diseases worldwide.