Probiotic bacteria for hatchery production of Greenshell mussels, Perna canaliculus
- Publication Type:
- Thesis
- Issue Date:
- 2009
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The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is
the largest aquaculture sector in the country. In 2006, the export earnings were valued at
US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at
present, GSM production involves the capture of wild mussels on ropes followed by on-growing
of these animals to market size (approximately 14 months). However, hatchery
production of GSM has been developed in recent years. Hatchery production will
alleviate the seasonal uncertainties of current techniques and allow the benefits of
selective breeding programs. To date, efforts to produce commercial quantities of GSM
in hatcheries have been hampered by unreliable larval rearing. These problems were often
alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the
ongoing use of antibiotics is not sustainable because of increasing legislative restrictions
on their use and the possible emergence of antibiotic resistant bacteria. Hence, the
identification and use of novel probiotics was investigated as an alternative.
Because of a lack of previous work, it was necessary to investigate the bacterial
pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of
disease against which the probiotics would be active. Twenty-two bacterial strains,
isolated from compromised larvae, were screened for larval toxicity using a larval
bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S
rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio
coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the-
ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration
102 CFU ml-1. Histopathology indicated the route of infection was via the digestive
system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the
two isolates.
Although two bacterial pathogens were identified, the successful design and
implementation of protective measures in the hatchery still required an understanding of
the dynamics of the infection process. Developing an in situ experimental model for
infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of
V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for
GSM larvae during hatchery production. In a flow-through water hatchery system, larvae
given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough
processes resumed, averaged 58% and 69% cumulative mortality, respectively,
on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of
magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V.
splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were
significantly greater than those of the non-exposed control larvae, averaging 23% in the
experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were
repeated four times to establish reproducibility. The infection models were reproducible
and provided a tool to assess measures for the protection of GSM larvae against infection
in the hatchery environment.
A bioassay was developed to screen and select bacterial strains as potential probiotics for
GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were
tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue
culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty
of the tested isolates afforded larval survival significantly greater than pathogen controls
(p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative
probiotics and highlighted the benefit of including the host animal in the first stage of the
screening procedure. The time of inoculation of putative probiotic strains prior to
pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20
hours revealed a greater number of potential probiotics than a two-hour pre-exposure
period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness
of the TCD screening method in recognising effective probiotics.
Following hatchery pilot trials, two probiotic strains were chosen for further study,
namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic
analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp.
0536. Both probiotics were evaluated separately in a GSM hatchery facility during
routine larval rearing and when the larvae were challenged with a high and low
pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic
application significantly improved larval survival, if administered prior to pathogen
exposure. Across all experiments, larvae that were exposed to the high and low dosages
of pathogens averaged 14% and 36% survival respectively on the fourth day following
pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval
survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae
administered the probiotic alone demonstrated 67% and 79% survival respectively. In a
repeat experiment, these benefits were reproduced, with the exception of A. macleodii
0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress
naturally occurring vibrios in the culture environment of healthy GSM larvae. This was
the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic
bacteria.
Many studies document probiotic application in aquaculture under conditions of pathogen
attack, yet few describe the use of probiotics during routine production. The effects of
administering the probiotic, A. macleodii 0444, during routine GSM larvae production,
were compared against larvae from the same cohort that were not treated with the
probiotic. The probiotic was administered daily for the first 11 days of the larval period
and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of
larval swimming activity, gut colouration, lipid levels, larval survival, larval size and
settlement success were recorded. There were minimal differences in all parameters
between larvae provided the probiotic and control larvae. Probiotic treated larvae
consumed more food and had higher lipid levels at the end of the larval period, but these
were not statistically significant. All treatments completed the larval phase and settled
successfully after metamorphosis. Survival at the end of the larval period was 37.2%,
38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively.
The probiotic was still detected in larvae seven days after the final addition to the tanks.
Animals were further grown in the field at a commercial farm. The probiotic was not
detected in mussels at four months after leaving the hatchery.
Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536,
was investigated to determine whether additive protection against pathogen attack with
Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of
combination administration were compared with larvae administered each probiotic as
single strains and non-inoculated larvae. Additionally, two concentrations were tested for
each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were
administered probiotics daily for the first six days, challenged with pathogens on the third
day and then reared until settlement (day 19). Although protection against pathogen
attack was observed in combination treatments, when compared with single-strain
administration, additive protection was not apparent. Administration of 108 CFU ml-1
levels of probiotics, both singly and in combination, afforded larval survival slightly
better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the
other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the
majority of the 19-day trial. At the end of the study, larval sizes were smaller in the
treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other
treatments. Additionally, towards the end of the larval period, feed consumption in the
combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic
treatments one day previously. This suggested that either the larvae were compromised or
they were growing slower. Despite a lack of additive protection against a single strain
pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as
prophylactic measures against every-day microbial encounters in larviculture, would
remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack
slightly better, they were also potentially detrimental to normal larval rearing when
administered in combination. Following the successful completion of the larval period
and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this
level was recommended as the best concentration of each probiotic where combination
administration would be applied.
The work presented in this thesis supports the use of A. macleodii 0444 and
Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce
settled juvenile mussels, equal in numbers to those produced in normal healthy
conditions, plus the benefits against pathogen attack led to the recommendation of their
use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is
intended in the near future. A provisional patent has been prepared and will be submitted
shortly. It is anticipated that future work will continue with these probiotic strains to
determine their potential benefit for other aquaculture species.
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