# Recombinant cystatin ingestion by Diaphorina citri reduces insect survival: insights into the HLB host–bacteria interaction (D. citri–CLas) focused on DcCathL studies

## TL;DR

Researchers found that feeding a protein inhibitor to citrus psyllids reduces their survival, offering a potential strategy to control the spread of citrus greening disease.

## Contribution

The study demonstrates that recombinant cystatin ingestion disrupts DcCathL activity in Diaphorina citri, increasing mortality and suggesting a novel control method for HLB.

## Key findings

- DcCathL expression and protein levels are significantly higher in CLas-infected D. citri insects.
- Recombinant CsinCPI-2 significantly increases mortality in both healthy and infected D. citri.
- GFP detection confirms the successful delivery of recombinant protein via artificial diet to D. citri.

## Abstract

Huanglongbing (HLB) is considered the most serious Citrus disease for which there are currently no effective control methods. The putative agents of HLB, Candidatus Liberibacter asiaticus (CLas), and the vector Diaphorina citri, are widespread in citrus regions, causing losses in citrus cultivation worldwide. Studying the interactions between D. citri and CLas can elucidate disease epidemiology and determine specific targets for HLB control. This work aimed to improve our understanding of the host–bacteria (D. citri–CLas) relationship, focusing on cysteine peptidase (DcCathL) and its inhibition by citrus cystatin (CsinCPI-2). In this study, a combination of qPCR, FISH, and immunolocalization techniques was employed to detect DcCathL genes or proteins in CLas-free or -infected D. citri samples. To verify the effect of DcCathL inhibition on insect survival, an artificial diet assay containing recombinant CsinCPI-2 was performed with infected and healthy D. citri nymphs and adults. Accordingly, a relative expression of DcCathL 1.23 times higher and an approximately 3.3 times greater DcCathL transcripts in gut tissue were demonstrated in CLas-infected compared to CLas-free insects. Furthermore, the presence of DcCathL protein was detected in the gut, ovary, and salivary glands of D. citri, concentrated at the peripheral regions of the cells. The fluorescence signal associated with DcCathL indicates that the gut of infected D. citri contains 4.81 times more protein than the gut of a healthy insect. Similarly, the protein levels in the salivary glands and ovaries of infected insects were 1.38 and 1.1 times higher, respectively, compared to those of CLas-free insects. The efficacy of the artificial diet in delivering the recombinant protein to the insect was demonstrated by the detection of green fluorescent protein (GFP) in the intestinal tract of nymphs and adults. Finally, the CsinCPI-2 demonstrated a substantial increase in mortality among CLas-free nymphs (df = 1, p < 0.0001) and CLas-free or CLas-infected adults (df = 1, p = 0.0001). Thus, the development of inhibitors that can disrupt the interactions between bacteria and vectors by blocking DcCathL activity may represent a promising strategy to prevent the spread of HLB disease.