Abstract
Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline. To study changes in gene expression in insects upon exposure to pesticides or other external stimuli, appropriate reference genes are required for data normalization. Since there is no such gene that is absolutely invariable under all experimental conditions, the aim of this study was to identify the most stable targets suitable for subsequent normalization in quantitative experiments based on real-time polymerase chain reaction in honeybee research. Here, we evaluated the expression of fifteen candidate housekeeping genes from three breeding lines of honeybees treated with pyrethroids to identify the most stable genes. The tested insects were exposed to deltamethrin or lambda-cyhalothrin, and then, changes in the accumulation of selected transcripts were assessed, followed by statistical analyses. We concluded that AmRPL32, AmACT and AmRPL13a were the commonly recorded most stable genes in honeybees treated with the selected pyrethroids.
Highlights
Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline
We analysed the stability of the expression of fifteen genes, used previously in selection of reference genes in insects[12], involved in basic metabolism of the honeybees, namely, actin (AmACT), α-tubulin (AmTUB), glutathione-S-transferase (AmGST1), glyceraldehyde-3-phosphate dehydrogenase (AmGAPDH), porphobilinogen deaminase (AmHMBS), ribosomal protein L32 (AmRPL32), 60S ribosomal protein L13a (AmRPL13a), 40S ribosomal protein S18 (AmRP18S), succinate dehydrogenase (AmSDHA), TATA-box-binding protein (AmTBP), elongation factor 1-alpha (AmEF1α), arginine kinase (AmARGK), chitin synthase 6 (AmCHS6), dorsal (AmDORS), and 18S ribosomal RNA (Am18S), in A. mellifera L. exposed to two types of pyrethroids: deltamethrin and lambda-cyhalothrin
We validated the results and used the selected reference genes to measure the expression of two cytochrome P450 monooxygenase (AmCYP450) genes described previously to be influenced in honeybees treated with insecticides[13,14], and the expression of AmCYP6AQ115 and AmCYP305a113 assayed in the research
Summary
Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline. The experimental design is based on, among other factors, the selection of a good internal control, that is, a gene that exhibits stable expression under the experimental conditions being tested By using this approach is it possible to accurately estimate the accumulation of target mRNA molecules. We analysed the stability of the expression of fifteen genes, used previously in selection of reference genes in insects[12], involved in basic metabolism of the honeybees, namely, actin (AmACT), α-tubulin (AmTUB), glutathione-S-transferase (AmGST1), glyceraldehyde-3-phosphate dehydrogenase (AmGAPDH), porphobilinogen deaminase (AmHMBS), ribosomal protein L32 (AmRPL32), 60S ribosomal protein L13a (AmRPL13a), 40S ribosomal protein S18 (AmRP18S), succinate dehydrogenase (AmSDHA), TATA-box-binding protein (AmTBP), elongation factor 1-alpha (AmEF1α), arginine kinase (AmARGK), chitin synthase 6 (AmCHS6), dorsal (AmDORS), and 18S ribosomal RNA (Am18S), in A. mellifera L. exposed to two types of pyrethroids: deltamethrin and lambda-cyhalothrin. We validated the results and used the selected reference genes to measure the expression of two cytochrome P450 monooxygenase (AmCYP450) genes described previously to be influenced in honeybees treated with insecticides[13,14], and the expression of AmCYP6AQ115 and AmCYP305a113 assayed in the research
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