A marine probiotic treatment against the bacterial pathogen Vibrio coralliilyticus to improve the performance of Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oyster larvae
Introduction
In the USA, oyster farms are largely dependent on high-quality seed (“eyed” larvae) from oyster hatcheries. Oyster hatcheries, however, have periodically experienced severe larval losses during the past two decades, leading to seed shortages and supply disruptions (Elston et al., 2008; Richards et al., 2015). Initially, these losses were mainly attributed to ocean acidification (Barton et al., 2012; Gray et al., 2022), and currently many hatcheries employ sophisticated systems that measure and correct acidified incoming water. Unfortunately, these treatments have not entirely resolved the problem, suggesting other factors, such as pathogens, likely play a significant role in these losses and contribute to various sub-lethal effects on oyster health (Marques et al., 2006).
Bacteria from the genus Vibrio are omnipresent in marine and brackish waters as commensals, mutualists, or pathogens (Takemura et al., 2014). They are highly adaptable to changing ocean conditions, including increasing temperature, lower pH, and salinity, and can make up >50% of all detectable microbes during favorable conditions (Gilbert et al., 2012; Oh et al., 2009; Vezzulli et al., 2010). Vibrio coralliilyticus has been linked to massive die-offs of Pacific oyster (Crassostrea gigas) larvae in U.S. West coast hatcheries (Elston et al., 2008; Estes et al., 2004; Richards et al., 2015) and, occasionally, mortalities in Eastern oysters cultured in U.S. East coast hatcheries (Kehlet-Delgado et al., 2017). In addition to Pacific and Eastern oysters, this pathogen affects commercially important Kumamoto oysters (C. sikamea), greenshell mussels (Perna canaliculus), and geoduck clams (Panopea generosa) (Elston et al., 2008; Estes et al., 2004; Kesarcodi-Watson et al., 2009; Richards et al., 2015). Furthermore, pathogenic V. coralliilyticus has been identified globally as a deadly pathogen in various marine species, including finfish, corals, and several bivalve species (Alves Jr et al., 2009; Austin et al., 2005; Jeffries, 1982; Kim et al., 2020).
Antibiotic interventions against bacterial infections in aquaculture are either not available or restricted due to the risks of promoting widespread anti-microbial resistance in bacterial populations (Cabello et al., 2013; Kesarcodi-Watson et al., 2008); furthermore, prophylactic treatments, such as vaccines and probiotics, are either not feasible or not yet commercially available for molluscan aquaculture (Pérez-Sánchez et al., 2018). This is in contrast to the many probiotic products used in crustacean and finfish aquaculture (reviewed by El-Saadony et al., 2021).
The definition of probiotics has been expanded in aquatic environments to reflect that they are not solely antagonists of pathogens, but also can interact positively with both the environment and their hosts. Verschuere et al. (2000), for example, state”[…] a probiotic is defined as a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment”.
Included in this definition are probiotic-induced modifications of the microbial community that affect host life stages known to be influenced by microbial cues. Bivalves undergo metamorphosis and settlement, where they permanently transition from planktonic larvae to sessile juveniles referred to as spat. Metamorphosis often results in high mortality (Durland et al., 2019). Some biofilms (Campbell et al., 2011; Devakie and Ali, 2002; Rodriguez-Perez et al., 2019; Tritar et al., 1992; Wieczorek and Todd, 1998; Zhao et al., 2003), individually added bacterial isolates (Freckelton et al., 2017), and bacterial supernatants (W. K. Fitt et al., 1990; William K. Fitt et al., 1989; Walch et al., 1999) improve larval oyster metamorphosis, while others result in inhibitory effects (Devakie and Ali, 2002; Dobretsov et al., 2006). Therefore, studies on the use of probiotics in bivalve aquaculture should include their effects on larval settlement and metamorphosis.
This study aimed to develop a combination of beneficial probiotic bacterial isolates that reduced acute mortalities of early larvae of Pacific oysters (Crassostrea gigas) resulting from exposure to pathogenic V. coralliilyticus strain RE22. We further evaluated whether single or repeated probiotic additions resulted in longer-term benefits to larval growth and metamorphosis of Pacific oysters. Lastly, we determined the effect of a single dose of the combined probiotics on metamorphosis of the Miyagi and Midori strains of the Pacific oyster as well as the Kumamoto oyster (Crassostrea sikamea).
Section snippets
Isolation and initial screening of probiotic candidates
All experiments were conducted at Oregon State University's facilities, including the research hatchery at the Hatfield Marine Science Center (HMSC) in Newport, Oregon, USA. Over 311 bacterial isolates were collected from water samples, microalgae tanks, oyster feces, mantle, gills, the gastrointestinal tracts of healthy adult bloodstock and cultures of juvenile spat and larvae of Pacific oysters (C. gigas) from the HMSC research hatchery. Additional samples also originated from a commercial
Initial screening of probiotic candidates
From all microbial strains collected for probiotic screening, approximately 28.3% were not revivable after being frozen with glycerol or failed to sufficiently grow in LBSw within 48 h, 36.7% grew on TCBS agar and were consequently excluded, leaving 35% screened on agar plates against V. coralliilyticus strain RE22. Ultimately 13 strains were suggestive of contact inhibition or zones of clearing on the V. coralliilyticus lawn. These strains proceeded to pathogenicity testing with oyster larvae
Discussion
This study describes the development of a promising novel probiotic treatment for both disease prevention and enhancement of larval development. The best treatment consisted of a combination of three individual beneficial bacterial isolates added to the larvae culture once at 24 hpf. This treatment significantly improved the survival of Pacific oyster larvae exposed to a lethal dose of a highly virulent V. coralliilyticus strain RE22 added 24 h after the probiotic addition. In addition, a
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank the MBP staff for their support in rearing larvae, Jennifer Hesser for assisting in some of the experiments, MK English for the 16S rRNA gene sequencing of the probiotic candidates. Whiskey Creek hatchery and Oregon Oyster Farms, Oregon, kindly provided biological materials for isolation of candidate probiotics. This work was supported by a NOAA National Sea Grant award [NA18OAR4170346] awarded to CS, CL, and RM, and a NOAA Saltonstall-Kennedy grant [NA18NMF4270220] awarded to
References (62)
- et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Chemical production of cultchless oyster spat using epinephrine and norepinephrine
Aquaculture
(1986) - et al.
Effective use of plastic sheet as substrate in enhancing tropical oyster (Crassostrea iredalei Faustino) larvae settlement in the hatchery
Aquaculture
(2002) - et al.
The functionality of probiotics in aquaculture: an overview
Fish Shellfish Immunol.
(2021) The use of probiotics in aquaculture
Aquaculture
(1999)- et al.
The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms
Aquaculture
(2000) - et al.
Hatchery crashes among shellfish research hatcheries along the Atlantic coast of the United States: a case study of production analysis at horn point laboratory
Aquaculture
(2022) Three Vibrio strains pathogenic to larvae of Crassostrea gigas and Ostrea edulis
Aquaculture
(1982)- et al.
Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes
Aquaculture
(2008) - et al.
Protective effect of four potential probiotics against pathogen-challenge of the larvae of three bivalves: Pacific oyster (Crassostrea gigas), flat oyster (Ostrea edulis) and scallop (Pecten maximus)
Aquaculture
(2012)
Yields of cultured Pacific oysters Crassostrea gigas Thunberg improved after one generation of selection
Aquaculture
Bacteriophages improve survival and metamorphosis of larval Pacific oysters (Crassostrea gigas) exposed to Vibrio coralliilyticus strain RE98
Aquaculture
Introduction and evaluation on the US west coast of a new strain (Midori) of Pacific oyster (Crassostrea gigas) collected from the Ariake Sea, southern Japan
Aquaculture
Biological approaches for disease control in aquaculture: advantages, limitations and challenges
Trends Microbiol.
Review of probiotics for use in bivalve hatcheries
Vet. Microbiol.
Conservation and restoration of a keystone species: understanding the settlement preferences of the European oyster (Ostrea edulis)
Mar. Pollut. Bull.
Bacterial diversity in a marine hatchery: balance between pathogenic and potentially probiotic bacterial strains
Aquaculture
Larval settlement of the silver- or goldlip pearl oyster Pinctada maxima (Jameson) in response to natural biofilms and chemical cues
Aquaculture
Diversity and pathogenic potential of vibrios isolated from Abrolhos Bank corals
Environ. Microbiol. Rep.
Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii
Environ. Microbiol.
The Pacific oyster, Crassostrea gigas, shows negative correlation to naturally elevated carbon dioxide levels: implications for near-term ocean acidification effects
Limnol. Oceanogr.
Recent advances in probiotic application in animal health and nutrition: a review
Agriculture
The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer
Science
Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition
ISME J.
Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health
Environ. Microbiol.
Effects of age and composition of field-produced biofilms on oyster larval setting
Biofouling
Bacterioplankton community shifts in an Arctic lake correlate with seasonal changes in organic matter source
Appl. Environ. Microbiol.
Inhibition of biofouling by marine microorganisms and their metabolites
Biofouling
Effects of marine bacteria on the culture of axenic oyster Crassostrea gigas (Thunberg) larvae
Biol. Bull.
Comparison of larval development in domesticated and naturalized stocks of the Pacific oyster Crassostrea gigas exposed to high pCO2 conditions
Mar. Ecol. Prog. Ser.
Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management
Dis. Aquat. Org.
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Present address: JL. Trans Balauring, Desa Merdeka, Kecamatan Lebatukan, Kab. Lembata, 86681 NTT, Indonesia.