And You Thought That Was It For Cucumbers!

Following on from my previous post; the aquaculture of the Japanese Sea Cucumber Apostichopus japonicus is a rapidly developing and important area of industry for China. The cucumbers are plagued with a number of diseases, especially skin ulceration syndrome. It has been shown that the main pathogens which cause this disease include Vibrio splendidus, Vibrio alginolyticus and Vibrio cyclitrophicus.  Probiotics are a constantly increasing market in the health industry. They are also gaining much attention in the aquaculture industry due to their environmental friendliness. Recently over 30 genera of bacteria have been suggested for use in aquaculture, some of which have been isolated from the marine environment. In sediments and water columns but also in gastrointestinal tract of marine animals.

In this study Liu et al identify a bacterial strain from an aquaculture cuttlefish which could inhibit the growth of V.splendidus. The authors investigate the mechanism of the ability to aquire iron and the effects of this bactria on the survuval rate of the Japanese Cucumber when infected with V.splendidus. Bacterial antagonists were isolated from the cuttlefish Sepia pharaonis and V.splendidus Vs were isolated from A.japonicus sufffering from ulceration syndrome. Both bacterial strains were incubated overnight. V.splendidus Vs was then spread onto 2216E marine agar.The antagonistic bacteria being screened were then applied to sterile filter paper and applied to the agar using the disk diffusion method. After 24 hours incubation, the zones of no growth were observed similar to determine the antagonistic effect. The same method was then applied to V. alginolyticus, V. cyclitrophicus and V.parahaemolyticus.

16S rRNA analysis was performed on the anatgonistic bacteria and it was identified as Vibrio sp. V33. Growth inhibition was tested in two methods. Equal amounts of V33 and Vs were mixed into sterile seawater and incubated for 24 hours. After which the culture was diluted and spread onto 2216E media. The media was incubated for 24 hours and colonies were counted. The second method tested only the supernatant to determine if it had an inhibitory effect. V33 was grown for 12 hours before being centrifuged and filtered through a poly-carbonate membrane to remove bacteria. The filtered supernatant was added to Vs suspensions and after 12 hours incubation the cell density was measured at 600nm in a spectrophotometer. The thermo-stability of the inhibitory substance was tested. The same steps as described above were followed but the supernatant was heated to 100 °C for 5 minutes before being introduced to the Vs suspension. The results suggest that V33 might secrete the inhibitory substance into the supernatant as 37.5% of the growth of Vs was inhibited. The inhibitory substance was also shown to be thermo-stable.

Siderophores are high affininty iron binding compounds secreted by bacteria, known to be some of the strongest iron binding agents. The siderophore production of V33 and Vs were determined using disk diffusion on Chrome Azurol S (CAS) agar. Both strains were grown on iron limited 2216E media before being transferred to the sterile filter paper. The size of the orange haloes formed around the colonies was then measured. V33 showed a 1.75 fold larger halo than that of Vs, suggesting V33 produced more or higher affinity siderophores than Vs.  Both V33 and Vs were then incubated in 2216E separately. 1 ml of cell free suspension was then added to CAS bright blue dye, allowed to stand for 15 minutes and then measure at OD630 in a spectrophotometer. The quantity of siderophore was determined using an equation: siderophore % = (Ar − As) / Ar × 100, where Ar = reference absorbance and As = sample absorbance. The results from both the qualitative and quantitative methods suggest that V33 produces more siderophores than Vs.

40 sea cucumbers were acclimated for 3 days before being artificially immerse infected. They were divided into 4 treatment groups randomly; V33 only, Vs only, V33 and Vs and a control. After 5 days the percentage of survival (ROS) was calculated using RPS = [1 − (% mortality co-infected with Vibrio sp. V33 and V. splendidus Vs / % mortality infected with V. splendidus Vs)] × 100. The treatment which contained only Vs showed 70% mortality after the 5 days. The treatment containing both Vs and V33 only showed 40% mortality. V33 gave an RPS of 43% and the protective effect of V33 suggest that it has the potential to treat ulceration syndrome. Interestingly the treatment containing only V33 showed no difference to the control, suggesting it is avirulent to Japanese Sea Cucumbers.

Overall a very positive study using robust, known and proven methods. The authors not only discover a new strain of Vibrio sp. but provide evidence for its antagonistic effect on ulceration syndrome causing pathogens, looking into the mechanisms behind this. This is strong evidence of alternative methods to reduce disease in aquaculture other than the widely abused use of antibiotics. From previous work by another author Vibrio sp. V33 could be combined with bacteriophages that target V. splendidus to further combat disease in aquaculture. This would require further study, however, as discovery of a phage that targets V.splendidus and not Vibrio sp. V33 would not be an easy task. A cocktail of mutliple phages would also be required for it to be effective, making the task even more monumental. What is interesting about this study is Vibrio sp. V33 was isolated from the gastrointestinal tract of a cuttlefish species. So rather than large scale commercial production of this strain, cuttlefish could possibly be introduced to the aquaculture system. This could provide much cheaper, long term protection for the cucumbers. Further study would be required.

Liu, N., Zhang, S., Zhang, W. and Li, C. (2017). Vibrio sp. 33 a potential bacterial antagonist of Vibrio splendidus pathogenic to sea cucumber (Apostichopus japonicus). Aquaculture, 470, pp.68-73.