Probiotic and pathogenic bacteria in larval rearing of spiny lobsters, Jasus edwardsii and Jasus (Sagmariasus) verreauxi

Rock lobster hatchery rearing for the supply of juvenile seedstock to aquaculture has yet to be commercialized due to the complex and prolonged larval phase, which is further confounded by bacteria-associated infections and mortalities. However, the adoption of improved larval rearing technology and the practice of hygienic husbandry at the Tasmanian Aquaculture and Fisheries Institute Marine Research Laboratories (TAFI MRL) have been responsible for closing the life cycle of the southern rock lobster Jasus edwardsii and more recently of the eastern rock lobster Jasus (Sagmariasus) verreauxi. The aim of this research was to control the microorganisms that colonize the phyllosoma during their early stages of development, especially by application of bacteria with probiotic characteristics. Bacteria were isolated and identified from the microbial community of phyllosoma cultured at TAFI MRL and were examined for their probiotic or pathogenic effects. Since pathogenic, opportunistic, beneficial and innocuous Vibrio spp. are ubiquitous in crustacean hatcheries and due to the prevalence of Vibrio spp. in clear water larval rearing techniques, the probability of obtaining autochthonous Vibrio spp. probionts that have better colonizing efficacy than probionts that are used in terrestrial animals was considered to be high. Bacterial isolates from live and dead J. edwardsii phyllosoma (instar I, n = 100; instars III and VI, n = 50) were randomly selected from bacteria enumerated on Johnson's marine agar and TCBS agar plates, and subculturing produced 239 viable isolates from 25 different species. The isolates were identified using PIBWIN based on the biochemical reactions conducted with the MicroSys® Vibrio identification kit. Different species that constituted the microbial community of the phyllosoma were designated as putative probionts or likely pathogens based on their association with live and dead phyllosoma, respectively. High recovery of V. chagasii from dead instar I phyllosoma and its complete absence from live instar I phyllosoma indicated that it was likely to be pathogenic. Similarly, high occurrence of V. splendidus from dead instar III and VI phyllosoma indicated it to be a virulent isolate. Following this hypothesis, V. mediterranei, V. alginolyticus, Phenon 36, Phenon 52, V. cyclitrophicus, and V. calviensis were also presumed to be pathogenic. Phenon 8, V. orientalis, V. anguillarum and V. penaecida were associated with healthy, live phyllosoma. Isolates that were identified as Phenon' were V. alginolyticus-like isolates. The development of a probiotic model to protect phyllosoma was an important aspect of this research. Since hatchery-reared phyllosoma derive their gut microflora from Artemia, the delivery of isolates via Artemia during the present study mimicked the normal route through which the bacterially-free phyllosoma become colonized. The Artemia were subjected to a short disinfective purge (with formaldehyde and concentrated microalgae) and immediately suspended in microalgae inoculated with the axenic bacterial isolates. The bacterially colonized Artemia were dispensed into the phyllosoma rearing containers and the gut evacuation of the isolates by the Artemia was determined. During enrichment, Artemia accumulated 1.3-4.9 ± 0.02-0.1 x 107 and 1.2-2.5 ± 0.01-0.07 x 107 heterotrophic and Vibrio cells Artemia-1 , respectively, and 24 h later, the numbers had diminished 100-fold. Experiments determining the effect of monoxenic isolates on the survival of newly hatched phyllosoma were conducted with isolates that were often or exclusively obtained from either dead or live phyllosoma. During these experiments, which lasted for 14 days, V. penaecida (Sr. No. 232) and V. chagasii (Sr. No. 64) were pathogenic towards phyllosoma with 22.5% and 58% survival, respectively, compared to 68% survival in the Control. The survival in phyllosoma exposed to monoxenic cultures of Phenon 8 (Sr. No.148), Phenon 52 (Sr. No. 14), V. cychtrophicus (Sr. No. 152) and V. orientalis (Sr. No. 229) was 67-87%. V. mediterranei (Sr. No. 93), V. calviensis (Sr. No. 30), V. alginolyticus (Sr. No. 25), V. splendidus (Sr. No. 166), V. anguillarum (Sr. No. 221) and Phenon 36 (Sr. No. 215) were mildly virulent towards phyllosoma (61-65% survival) compared to the Control. The histological sections of dead phyllosoma revealed proliferation of bacterial cells in the lumen. Probiont-pathogen challenge experiments were conducted on phyllosoma, wherein V.\ud cyclitrophicus (Sr. No. 152), V. orientalis (Sr. No. 229), Phenon 8 (Sr. No.148) and Phenon 52 (Sr. No. 14) were used as putative probionts while V. penaecida (Sr. No. 232) and V. chagasii (Sr. No. 64) were used as pathogens. Probiont-pathogen treatments received putative probionts exclusively until Day 4 and thereafter also received the pathogens until the experiments were terminated. Survivals in the probiont-pathogen treatments were significantly higher than in the treatments receiving pathogen-only, and were not statistically different from survivals in the Control or the probiont-only treatments. Highest survival (88%) was in the Phenon 8 (Sr. No.148) - V. chagasii (Sr. No. 64) treatment. Other probiont-pathogen treatments had 86-82% survival with the exception of V. orientalis (Sr. No. 229) — V. penaecida (Sr. No. 232), which had 75% survival. Probiotic effects are exerted through mechanisms such as in vivo antagonism, competitive exclusion of the pathogens by the probionts, activation of the innate crustacean immune responses and production of siderophores. During the present study, the observed probiotic effect may have involved either a single mechanism or a combination of mechanisms of probiosis. High phyllosoma survival was associated with high abundance of sucrose fermentors while sucrose non-fermentors typically accompanied poor survival. The therapeutic effect of the probionts was determined by administering the probionts to phyllosoma that had been previously colonized by V. chagasii (Sr. No. 64) and V. penaecida (Sr. No. 232). The administration of probionts was able to halt the phyllosoma mortality and gradually displaced the pathogen. The administration of the probionts and pathogen simultaneously to phyllosoma infected with the pathogens improved the survival compared to the treatments that received the pathogen exclusively throughout the experiment. There was no benefit of mixed-culture probionts over axenic probionts on the survival of phyllosoma. Further, the application of probionts and intermittent ozonation of the culture water did not improve larval survival, although the effect of ozonation resulted in a reduction in the bacterial abundances. The application of autochthonous probionts as prophylactic and therapeutic measures ensured high survival of phyllosoma during their critical early larval phase. The results of this study may assist in manipulating and controlling the bacterial colonization of early stage phyllosoma during hatchery rearing