# Methylene Blue Alleviates Thiamethoxam-Induced Toxicity in Honeybee Larvae by Activating Dihydrolipoyl Dehydrogenase

## TL;DR

Methylene blue reduces thiamethoxam pesticide toxicity in honeybee larvae by activating a key detoxifying enzyme.

## Contribution

Methylene blue is shown to mitigate neonicotinoid toxicity in bees via dihydrolipoyl dehydrogenase activation.

## Key findings

- Methylene blue reduced larval mortality from 47.2% to 25.0% when combined with thiamethoxam.
- AmDld gene expression and enzyme levels were restored by methylene blue in thiamethoxam-treated larvae.
- RNAi knockdown of AmDld increased mortality under thiamethoxam+methylene blue treatment.

## Abstract

The extensive use of thiamethoxam (TMX) pesticides has led to a significant decline in bee populations, causing irreversible damage to the sustainable development of bee colonies and attracting widespread global attention. TMX, a neonicotinoid insecticide that has gained significant popularity, has been observed to exert a deleterious effect on bee development. However, there is currently a lack of effective protective agents against TMX poisoning in bees. Preliminary laboratory experiments have identified methylene blue (MB) as a substance capable of protecting bee larvae against TMX poisoning. In this study, we utilized a multifaceted approach encompassing bioassays, transcriptome sequencing, RNA interference (RNAi), and enzyme content assays. This comprehensive strategy enabled us to demonstrate the detoxification efficacy of MB, identify target genes, and validate their functions. This provides a novel experimental approach for developing protective agents against pesticide poisoning in bees.

The extensive utilization of TMX, a substance characterized by its high toxicity towards honeybees, has exerted a deleterious influence on the employment of neonicotinoid insecticides and the proliferation of bee colonies. However, there is a lack of effective solutions to mitigate the toxicological impact of neonicotinoid insecticides on bees. The present study proposes a method of using MB to alleviate TMX poisoning in honeybee (Apis mellifera ligustica) larvae. The results demonstrated that when bee larvae ingested MB at a concentration of 0.32 mg·L−1, the mortality rate of larvae could be reduced from 47.2% to 25.0%. Transcriptome analysis identified the honeybee dihydrolipoyl dehydrogenase (AmDld) gene as one of the main genes involved in the function of MB. AmDld was highly expressed in larval hemolymph. Its expression levels and enzymatic content were suppressed by either TMX or MB alone but restored by the TMX+MB combination. RNAi-mediated knockdown of AmDld decreased AmDld content and increased larval mortality under the TMX+MB co-treatment from 25.0% to 40.6%. This indicated that the TMX+MB combination rescued AmDld levels, thereby alleviating TMX toxicity to bee larvae. The present study has demonstrated that the ingestion of MB by honeybee larvae has the capacity to reduce the toxicity of TMX, a toxic substance, through the action of the AmDld gene. This provides a novel approach to mitigating pesticide poisoning in bees.