# RNAi-mediated knockdown of the poultry red mite cathepsin D-1 impacts haemoglobin digestion

## TL;DR

Scientists used RNA interference to reduce a specific enzyme in poultry red mites, finding it impacts their ability to digest blood meals.

## Contribution

The study demonstrates that RNAi-mediated knockdown of Dg-CatD-1 affects haemoglobin digestion in poultry red mites.

## Key findings

- RNAi knockdown of Dg-CatD-1 reduced gene expression by up to 91% after a single feeding.
- Repeated dsRNA feeding reduced the mites' ability to process blood meals compared to controls.
- Effective knockdown required specific dsRNA lengths and concentrations.

## Abstract

The poultry red mite, Dermanyssus gallinae, is a haematophagous ectoparasite causing significant economic losses in the commercial egg-laying sector. Blood meal digestion by D. gallinae is required for nutrient acquisition, with acidic lysosomal proteinases such as cathepsin L and cathepsin D playing a critical role in haemoglobin digestion. This study investigated the role of a cathepsin D-like aspartyl proteinase, Dg-CatD-1, in the haemoglobin digestion cascade.

Haemoglobin processing was investigated by RNA interference (RNAi)-mediated silencing of Dg-CatD-1 and assessing the impact on haemoglobin digestion. RNAi-mediated knockdown of Dg-CatD-1 was achieved by feeding a target-specific double-stranded RNA (dsRNA) to D. gallinae in a blood meal. The minimum length and concentration of Dg-CatD-1 dsRNA for effective knockdown was determined. In addition, the effect of Dg-CatD-1 knockdown on mite digestive physiology, haemoglobin digestion, and egg-laying by adult female mites was assessed.

Feeding Dg-CatD-1 dsRNAs via a blood meal to adult female D. gallinae mites resulted in a substantial knockdown of target gene expression. The minimum length and concentration of dsRNA required for effective Dg-CatD-1 knockdown were 25 base pairs (bp, at 200 ng/μl) (61% knockdown) and 25 ng/μl (at 485  bp) (42% knockdown), respectively. When Dg-CatD-1 dsRNA was delivered as a single feed it resulted in up to 91% reduction in Dg-CatD-1 expression, although no observable effect on blood digestion was observed. The phenotypic impact of Dg-CatD-1 knockdown was demonstrated following two consecutive rounds of Dg-CatD-1 dsRNA feeding where knockdown reduced the ability of mites to process and clear their blood meal relative to control non-specific dsRNA-fed mites.

This work highlights the importance of Dg-CatD-1 as an essential enzyme in the haemoglobin digestion pathway of D. gallinae. These findings open avenues for the development of targeted control strategies aimed at disrupting the digestive processes of D. gallinae. Furthermore, this research suggests that reductions in gene expression via RNAi do not always lead to corresponding decreases in protein levels or observable phenotypes. Repeated exposure to dsRNA may be necessary to reveal phenotypic effects of gene knockdown.

The online version contains supplementary material available at 10.1186/s13071-026-07254-y.