Identifying the Gut Virome of Diaphorina citri from Florida Groves

Abstract

Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

Keywords: ACP); Asian citrus psyllid (Diaphorina citri; CLas; D. citri densovirus (DcDV); D. citri flavi-like virus (DcFLV); D. citri reovirus (DcRV); D. citri-associated C virus (DcACV); gut virome; insect-specific viruses.

Figure 1

The percentage of viral sequence reads found in the HTS analysis of psyllid guts from each citrus production region in Florida. The composite sample represents the sequences that matched to known insect or plant virus species or unknown viruses.

Figure 2

Microscopic analysis of morphological abnormalities of nuclei in gut cells dissected from healthy, DcACV- and DcFLV-infected D. citri adults. (A) Light micrograph of dissected healthy gut showed normal morphology under color and black/white modes. Light micrographs of the dissected guts from DcACV- (B) and DcFLV-infected (C) guts showed dark or brown portions. The magnification photographs of dissected guts from healthy (D) and DcACV-infected (E) showed normal nuclei shapes and structures. The magnification image of the dissected gut from DcFLV-infected gut (F) showed that cell nuclei were irregular in shape and vacant in structure. Percentage of nuclei abnormalities (NA) of each three dissected guts (healthy, DcACV-, DcFLV-infected) is labeled at the bottom.