Vibrio parahaemolyticus
becomes lethal to post-larvae shrimp via acquiring novel virulence factors
Authors: Liu Shuang , Wang Wei , Jia Tianchang , Xin Lusheng , Xu Ting-ting , Wang Chong , Xie Guosi , Luo Kun , Li Jun , Kong Jie , Zhang Qingli 
Published: October 2023

Translucent post-larvae disease (TPD), caused by
Vibrio parahaemolyticus
(
Vp
TPD
), has become an emerging shrimp disease, affecting more than 70%–80% of coastal shrimp nurseries in China in spring 2020. Here, we investigated the key virulence factors of
Vp
TPD
by analyzing protein fragments, related genomic information, as well as experimental challenge tests. After investigating the toxic effects of purified protein fragments with different molecular weights (MWs) from
Vp
TPD,
we found that only the protein fragments with MW >100 kDa showed similar lethality to live
Vp
TPD
in the experimental challenge test using post-larvae shrimp. Meanwhile, similar histopathological changes exhibiting in the hepatopancreas and midgut of the diseased individuals were observed in the post-larvae shrimp challenged with either bacterial protein fragments (MW >100 kDa) or live
Vp
TPD
. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, two novel proteins,
Vibrio
high virulent protein (VHVP)-1 and VHVP-2, were identified as the candidates of key virulence factors to cause TPD. Moreover, VHVP-1 and VHVP-2 were found to be encoded by two genes (
vhvp-1
and
vhvp-2
) tandemly located on a 187,791-bp plasmid and were predicted to depend on the same promoter following a comparative genomic analysis. Further epidemiological investigation and challenge test indicated that the
V. parahaemolyticus
isolate carrying only the
vhvp-1
gene and lacking
vhvp-2
gene could not cause mortality of experimental
Penaeus vannamei
post-larvae. The mutant (Δvhvp-2) by deleting
vhvp-2
gene could only cause 4.92% of accumulative mortality of post-larvae that is similar to the non-
Vp
TPD
Vibrio
strain. Additionally, the complemented strains, Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, showed similar virulence to the wild-type
Vp
TPD
. The results demonstrated that
V. parahaemolyticus
becomes lethal to post-larval shrimp by acquiring the VHVP-2 virulence factor. This study sheds light on further investigations of the pathogenic mechanism of
Vp
TPD
and the development of strategies for early diagnosis of TPD in shrimp hatcheries.

IMPORTANCE

As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the
Vp
TPD
, for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein,
Vibrio
high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of
V. parahaemolyticus
to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like
Vp
TPD
, in aquaculture shrimps and would be beneficial to the fisheries department in early warning of
Vp
TPD
emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of
Vp
TPD
could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.