Effects of the Monomeric Components of Poly-hydroxybutyrate-co-hydroxyhexanoate on the Growth of Vibrio penaeicida In Vitro and on the Survival of Infected Kuruma Shrimp (Marsupenaeus japonicus)

Abstract

Here, we investigated the inhibitory effects of the biodegradable, water-insoluble polymer poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH) and its two constituent monomers, the hydroxyalkanoic acids 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HH), on the growth of the shrimp-pathogenic bacterium Vibrio penaeicida. In vitro experiments revealed that 3HH showed greater growth inhibitory activity than 3HB against V. penaeicida. In addition, the activities of hydroxyalkanoic acids were pH dependent, being greater at pH 6.0 than at pH 7.0. Investigation of the pH of the shrimp gut revealed a pH range of 5.9-6.7 (mean 6.29 ± SD 0.20), indicating that the physiological environment was suitable for 3HB and 3HH to exert their inhibitory activities against V. penaeicida. In vivo bacterial challenge experiments revealed that survival rates in kuruma shrimp (Marsupenaeus japonicus) infected by V. penaeicida were significantly increased in shrimp reared on feed containing PHBH (0.1%-5% w/w PHBH) compared with that in shrimp reared on standard diet alone. Supplementation with PHBH had no significant effects on three shrimp growth parameters: increase in body weight, daily feeding rate, and feed conversion ratio. These results suggest that supplementation of standard diet with PHBH will increase shrimp resistance to infection by V. penaeicida, thereby increasing shrimp aquaculture productivity.

Keywords: Marsupenaeus japonicus; inhibitory effect; pathogenic Vibrio; poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH); short- and medium-chain hydroxyalkanoic acids; shrimp survival.

Figure 1

(A) Changes in pH of homogenized gut of Marsupenaeus japonicus versus the amount of seawater added to the homogenate. Shrimps were sacrificed after being reared for 6 weeks on standard diet (control) or experimental feed containing 5% (w/w) poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH). Different superscript letters show significant differences (p < 0.05). The pH of the gut was estimated by extrapolating back to the y-axis of the best fit line. (B) pH values of the gut of M. japonicus individuals with different body lengths and reared on standard diet. pH values were obtained by the method shown in (A).

Figure 2

Growth of the shrimp-pathogenic bacterium Vibrio penaeicida in nutrient medium containing different concentrations of the hydroxyalkanoic acids 3-hydroxybutyrate (3HB) (A,E) or 3-hydroxyhexanoate (3HH) (C,G), or the fatty acids butyrate (B,F) or hexanoate (D,H) at pH 6.0 (left half) or 7.0 (right half). CFU: colony-forming units; ND: not detected. Different superscript letters in each figure show significant differences (p < 0.05).

Figure 3

Increases in body weight (A) and changes in daily feeding rate (B) and feed conversion ratio (C) of Marsupenaeus japonicus during a 6-week feeding test with feed containing different amounts of poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH). Different superscript letters show significant differences (p < 0.05).

Figure 4

Survival rates of Marsupenaeus japonicus after infection with the shrimp-pathogenic bacterium Vibrio penaeicida. Nine weeks prior to bacterial infection and 1 week after infection, the shrimp were fed the feed containing different amounts of poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH). Different superscript letters show significant differences (p < 0.05).