# Silencing the cyp314a1 and cyp315a1 Genes in the Aedes albopictus 20E Synthetic Pathway for Mosquito Control and Assessing Algal Blooms Induced by Recombinant RNAi Microalgae

## TL;DR

This study uses genetically modified algae to silence genes in Aedes albopictus mosquitoes, reducing their population and improving water quality.

## Contribution

A novel RNAi-based biocide using recombinant microalgae to control Aedes albopictus and alter aquatic ecosystems.

## Key findings

- RNAi recombinant algae caused high mortality and disrupted development in Aedes albopictus larvae.
- Recombinant Chlorella reduced mosquito populations and improved water quality in simulated field experiments.
- Algal blooms triggered biotic community changes, including proliferation of anaerobic bacteria.

## Abstract

This study explores a novel approach to controlling Aedes albopictus, a key vector for dengue fever, by using RNA interference (RNAi) mediated by genetically modified microalgae. We constructed double-stranded RNA (dsRNA) expression vectors that target the cyp314a1 and cyp315a1 genes of Ae. albopictus, and we transformed two species of algae, Chlamydomonas reinhardtii and Chlorella vulgaris, to express these dsRNAs. The recombinant RNAi algal strains were then fed to Ae. albopictus larvae, resulting in high mortality rates, disrupted pupation and reduced emergence. The algae triggered an increase in reactive oxygen species (ROS) and enzyme activity in the larvae, indicating redox stress. In simulated field experiments, RNAi recombinant Chlorella significantly reduced mosquito populations, and improved water quality by reducing nitrogen, phosphorus and others. Furthermore, with the release of RNAi recombinant Chlorella into the test water, the biotic community restructuring dominated by resource competition caused by algal bloom, as well as the proliferation of anaerobic bacteria and the decline of aerobic bacteria triggered by anaerobic conditions, are the main trends in the changes in the test water. This study is an important addition to the use of RNAi recombinant microalgae as a biocide.

As one of the key vectors for the transmission of Dengue fever, Aedes albopictus is highly ecologically adaptable. The development of environmentally compatible biological defence and control technologies has therefore become an urgent need for vector biological control worldwide. This study constructed and used double-stranded RNA (dsRNA) expression vectors targeting the cyp314a1 and cyp315a1 genes of Ae. albopictus to transform Chlamydomonas reinhardtii and Chlorella vulgaris, achieving RNA interference (RNAi)-mediated gene silencing. The efficacy of the RNAi recombinant algal strain biocide against Ae. albopictus was evaluated by administering it to Ae. albopictus larvae. The results showed that the oral administration of the cyp314a1 and cyp315a1 RNAi recombinant C. reinhardtii/C. vulgaris strains was lethal to Ae. albopictus larvae and severely affected their pupation and emergence. The recombinant algal strains triggered a burst of ROS (Reactive Oxygen Species) in the mosquitoes’ bodies, resulting in significant increases in the activities of the superoxide dismutase (SOD), peroxiredoxin (POD) and catalase (CAT), as well as significant upregulation of the mRNA levels of the CME pathway genes in larvae. In the simulated field experiment, the number of Ae. albopictus was reduced from 1000 to 0 in 16 weeks by the RNAi recombinant Chlorella, which effectively controlled the population of mosquitoes. Meanwhile, the levels of nitrogen (N), phosphorus (P), nitrate, nitrite, ammonia and COD (Chemical Oxygen Demand) in the test water decreased significantly. High-throughput sequencing analyses of 18S rDNA and 16S rDNA showed that, with the release of RNAi recombinant Chlorella into the test water, the biotic community restructuring dominated by resource competition caused by algal bloom, as well as the proliferation of anaerobic bacteria and the decline of aerobic bacteria triggered by anaerobic conditions, are the main trends in the changes in the test water. This study is an important addition to the use of RNAi recombinant microalgae as a biocide.