Comparison of the sensitivity of Crustacean species against recombinant Photorhabdus insect-related (Pir) toxins

Abstract

Acute hepatopancreatic necrosis disease (AHPND), previously known as early mortality syndrome, was first identified in China in 2009; it is a serious emerging disease among shrimp in East/Southeast Asia and Latin America. The disease is caused by the Vibrio parahaemolyticus bacterium, which harbors a 70-kbp plasmid that encodes Photorhabdus insect-related (Pir) toxins designated PirAvp and PirBvp; these toxins are directly responsible for shrimp mortality. However, the molecular mechanism by which these two toxins cause pathogenesis has not been fully elucidated. In this study, we expressed recombinant PirAvp and PirBvp toxin proteins in Escherichia coli, designated rPirAvp and rPirBvp, respectively, then tested the sensitivities of 4 crustaceans via immersion tests. PirBvp toxin, analyzed as a representative toxin, was detected beginning at 2 h after incubation; it accumulated with bacterial growth. Culture filtrate diluted to a final concentration of 15 nM PirBvp toxin caused 100% mortality of Litopenaeus vannamei within 4 days after immersion. L. vannamei showed 100% mortality at 7 days after immersion in purified 4 nM rPirAvp, rPirBvp, or rPirAvp + rPirBvp mixture. The cumulative mortalities of Macrobrachium rosenbergii, Palaemon paucidens and Macrophthalmus japonicus in the rPirAvp, rPirBvp or rPirAvp + rPirBvp mixture were 50%, 83%, and 100% after 12 days of immersion; 11%, 77%, and 100% after 10 days of immersion; and 36%, 96% and 100% after 6 days of immersion, respectively. The observation of mortality caused by rPirAvp alone contradicts the findings in many previous reports; to our knowledge, toxicity testing with P. paucidens and M. japonicus has not been previously reported, although these species could be used as models for toxin susceptibility in crustaceans.