DAC20Y01 - Investigation of black lip oyster larvae crash and potential role of Vibrio spp.

Background from Sam Nowland

In the past, mass mortality of black lip oyster larvae have occurred on day 7 coinciding with the appearance of green colonies cultured on TCBS plates. Research has optimised larval rearing conditions resulting in significant improvements in the survival and performance of black-lip larvae.

In February 2020 the DAC had a failed hatchery run (Run 0220) on day 12. Larvae dropped out of the water column and by day 14 there was 100% mortality. The cause of this mortality was suspected to be the green vibrio, as larvae were healthy and performing well up to this point. When testing for presence/ absence of vibrios by culturing on TCBS plates, small green colonies grew from the incoming 1 m filtered seawater sample and the larval tank sample. The only recent change made to the larvae was the addition of Muelleri (Chaetoceros muelleri ) algae species on day 10. Therefore, we suspect that the origin of the green vibrio colonies is from either the incoming seawater (perhaps a bloom in the ocean or our piping system) or from the Muelleri algae.

Further evidence that the algae may be contaminated with vibrio is:
(1) A subset of larvae from run 0320 fed Muelleri algae species on day 10 with subsequent drop out of larvae on day 13, with the unfed main batch remaining healthy; TCBS plates showed yellow colonies in wash tank, new tank, from the current larval tank and the subset larval tank, with the green colonies only appearing on day 12 algae (with Muelleri), just before the larval tank dropout.
(2) Main batch of larvae run 0320 dropped out 29 March; green colonies from TCBS plates containing samples from 3 algal species Pavlova spp.(Pav), Isochrysis lutea (Iso) and Chaetoceros muelleri (Muel) with a different Chaetoceros muelleri (Muel) batch with no green colonies

Which Vibrio species are causing these mortality events in black-lip oyster hatchery production and what are the potential sources of these species?

Suggested plan (time frame):

1. Identify which Vibrio species formed the the green (and yellow) colonies from algal cultures growing on TCBS plates
• Isolate and grown 20 green colonies + 3 yellow colonies in TSB/NaCl
• Extract DNA from 1 ml overnight culture
• Perform hsp60 PCR and Sanger sequencing
• Identify closest sequence match against NCBI database (~ 2-3 weeks)

2. To ensure we find the species that caused the larval mortality and in case it is not cultivable (and therefore not the green colony forming species), identify all Vibrio spp. in water, algae and oyster larvae samples (~6-8 weeks)
• Extract DNA from all samples including water, larvae, algae and green/yellow colonies from (1) above (approx. 55 samples)
• Send to UTS for hsp60 whole Vibrio community PCR and Ramaciotti Centre for Ecogenomics for high throughput sequencing
• Analyse data and identify Vibrio species and relative abundance in each sample

We can then establish a quick low-cost presence/absence specific vibrio PCR test for future surveillance once the Vibrio identity is known.