Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton

Abstract

Background More than half of the world’s population suffers from epidemic diseases that are spread by mosquitoes. The primary strategy used to stop the spread of mosquito-borne diseases is vector control. Interference RNA (RNAi) is a powerful tool for controlling insect populations and may be less susceptible to insect resistance than other strategies. However, public concerns have been raised because of the transfer of antibiotic resistance marker genes to environmental microorganisms after integration into the recipient genome, thus allowing the pathogen to acquire resistance. Therefore, in the present study, we modified the 3-hydroxykynurenine transaminase (3hkt) and hormone receptor 3 (hr3) RNAi vectors to remove antibiotic resistance marker genes and retain the expression cassette of the inverse repeat sequence of the 3hkt/hr3 target gene. This recombinant microalgal marker-free RNAi insecticide was subsequently added to the suburban water in a simulated-field trial to test its ability to control mosquito population. Methods The expression cassette of the 3hkt/hr3 inverted repeat sequence and a DNA fragment of the argininosuccinate lyase gene without the ampicillin resistance gene were obtained using restriction enzyme digestion and recovery. After the cotransformation of Chlamydomonas, the recombinant algae was then employed to feed Aedes albopictus larvae. Ten and 300 larvae were used in small- and large-scale laboratory Ae.albopictus feeding trials, respectively. Simulated field trials were conducted using Meishe River water that was complemented with recombinant Chlamydomonas. Moreover, the impact of recombinant microalgae on phytoplankton and zooplankton in the released water was explored via high-throughput sequencing. Results The marker-free RNAi-recombinant Chlamydomonas effectively silenced the 3hkt/hr3 target gene, resulting in the inhibition of Ae. albopictus development and also in the high rate of Ae. albopictus larvae mortality in the laboratory and simulated field trials. In addition, the results confirmed that the effect of recombinant Chlamydomonas on plankton in the released water was similar to that of the nontransgenic Chlamydomonas, which could reduce the abundance and species of plankton. Conclusions The marker-free RNAi-recombinant Chlamydomonas are highly lethal to the Ae. albopictus mosquito, and their effect on plankton in released water is similar to that of the nontransgenic algal strains, which reduces the abundance and species of plankton. Thus, marker-free recombinant Chlamydomonas can be used for mosquito biorational control and mosquito-borne disease prevention. Graphical Abstract

Use of micro and macroalgae extracts for the control of vector mosquitoes

“…on natural plankton was investigated in aquatic environments. As a result, it was reported that the reduction rates of plankton in the environment where transgenic algae were applied did not show a significant difference compared to the area where non-transgenic microalgae were applied ( Fei et al, 2023b ). Although transgenic microalgae have been shown to be highly effective in mosquito control, further studies are needed to investigate their toxic effects on the environment and non-target organisms.…”

Engineered Gut Symbiotic Bacterium-Mediated RNAi for Effective Control of Anopheles Mosquito Larvae

“…The use of dsRNA for RNAi-mediated degradation of targeted mRNA has shown promise as a novel tool for RNA-based mosquito management strategies ( 16 , 28 ). However, there are still several challenges that needs to be addressed, such as the high cost of dsRNA production, inconsistent RNAi efficiency, and the nonsustainable delivery methods used to administer dsRNA to mosquitoes.…”

“…However, these methods have limited feasibility for large-scale field applications due to the high cost of dsRNA production and its rapid degradation in the environment. Recent studies have explored alternative approaches for delivering dsRNA to mosquitoes, such as using transgenic nonsymbiotic bacteria, yeast, or algae ( 14 – 16 ). However, these studies used microorganisms that cannot colonize the mosquito gut, resulting in transient gene knockdown and limited sustainable effects of RNAi technology.…”