Cell death localization in situ in laboratory reared honey bee (Apis mellifera L.) larvae treated with pesticides

In this study, cell death detected by DNA fragmentation labeling and phosphatidylserine (PS) localization was investigated in the honey bee (Apis mellifera L.) midgut, salivary glands and ovaries after treating larvae with different pesticides offered via an artificial diet. To do this, honey bee larvae reared in an incubator were exposed to one of nine pesticides: chlorpyrifos, imidacloprid, amitraz, fluvalinate, coumaphos, myclobutanil, chlorothalonil, glyphosate and simazine. Following this, larvae were fixed and prepared for immunohistologically detected cellular death using two TUNEL techniques for DNA fragmentation labeling and Annexin V to detect the localization of exposed PS specific in situ binding to apoptotic cells. Untreated larvae experienced ∼10% midgut apoptotic cell death under controlled conditions. All applied pesticides triggered an increase in apoptosis in treated compared to untreated larvae. The level of cell death in the midgut of simazine-treated larvae was highest at 77% mortality and statistically similar to the level of cell death for chlorpyrifos (65%), imidacloprid (61%), myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any pesticide-treated larvae. Indications of elevated apoptotic cell death in salivary glands and ovaries after pesticide application were detected. Annexin V localization, indicative of apoptotic cell deletion, had an extensive distribution in the midgut, salivary glands and ovaries of pesticide-treated larvae. The data suggest that the tested pesticides induced apoptosis in tissues of honey bee larvae at the tested concentrations. Cell death localization as a tool for a monitoring the subclinical and sub-lethal effects of external influences on honey bee larval tissues is discussed.

Graphical abstractDNA fragmentation and phosphatidylserine (PS) localization characteristic for cell death was investigated in the honey bee (Apis mellifera L.) larvae treated with different pesticides. Honey bee larvae were reared in an incubator and exposed to one of nine pesticides: chlorpyrifos, imidacloprid, amitraz, fluvalinate, coumaphos, myclobutanil, chlorothalonil, glyphosate and simazine. Two TUNEL techniques for DNA fragmentation labeling and Annexin V to detect the localization of exposed PS were employed to localize cellular death. All applied pesticides triggered an increase in apoptosis in treated compared to untreated larvae. The level of cell death in the midgut of simazine-treated larvae was highest at 77% mortality and statistically similar to the level of cell death for chlorpyrifos (65%), imidacloprid (61%), myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any pesticide-treated larvae. Indications of elevated apoptotic cell death in salivary glands and ovaries after pesticide application were detected. Annexin V localization, indicative of apoptotic cell deletion, had an extensive distribution in the midgut, salivary glands and ovaries of pesticide-treated larvae. Cell death localization as a tool for a monitoring the subclinical and sub-lethal effects of external influences on honey bee larval tissues is discussed.Figure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights► Different pesticides induced apoptotic cell death in the midgut, salivary glands and ovaries in honeybee larvae. ► In situ localization using TUNEL and Annexin V. ► Cell death as indicator of subclinical and sub-lethal effects on honey bee larvae.