Identification and Expression Profiling of the 5-HT Receptor Gene in Harmonia axyridis

SIMPLE SUMMARY: Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is a natural predatory enemy insect widely distributed in north China and has been widely used in agricultural practice. Owing to a low daily average temperature from December to February of the following year in greenhouses and the facultative diapause of adult H. axyridis, the application of this ladybird in greenhouses in north China is limited. It is of great importance to regulate its predatory ability to improve its biological control efficiency in greenhouses. The 5-hydroxytryptamine (5-HT) was found to play a regulatory role in the predation of H. axyridis in our previous study, but the 5-HT receptor of H. axyridis has not been clearly identified so far, and its expression characteristics are poorly understood. In this study, the 5-HT receptor gene of H. axyridis was cloned and identified by phylogenetic tree construction and multiple sequence alignments. The expression patterns of each receptor in different developmental stages and tissues were analyzed by quantitative real-time PCR (qRT-PCR). The results showed that H. axyridis expressed four 5-HT receptor subtypes, named 5-HT(1A)Har, 5-HT(1B)Har, 5-HT(2)Har, and 5-HT(7)Har. All 4 receptors were expressed at high levels in the adult stage, especially in 2-day-old adults, with expression levels of 18.72-fold (male) and 14.21-fold (female) of that in eggs for 5-HT(1A)Har, 32.27-fold (male) and 83.58-fold (female) of that in eggs for 5-HT(1B)Har, 36.82-fold (male) and 119.35-fold (female) of that in eggs for 5-HT(2)Har, and 165.47-fold (male) and 115.59-fold (female) of that in eggs for 5-HT(7)Har. The level of expression for each receptor gene decreased with the advance of day-age in adults. The levels of expression of 5-HT(1B)Har, 5-HT(2)Har, and 5-HT(7)Har were low at the egg, larval, and pupal stages, and 5-HT(1A)Har was not expressed in the larval stage. The four receptors were expressed in the nervous system, digestive tract, pectoral muscles, and male and female gonads of adults. The 5-HT(1A)Har was expressed at a high level in the pectoral muscle (6.75-fold of that in the nervous system), 5-HT(1B)Har in male gonads (1.02-fold of that in the nervous system) and the nervous system, 5-HT(2)Har in male gonads (5.74-fold of that in the nervous system), and 5-HT(7)Har in the digestive tract (1.81-fold of that in the nervous system). The results of this study will lay a foundation for research on the function of the 5-HT receptor by RNA interference in the regulation of predation by H. axyridis. ABSTRACT: It has been found that 5-hydroxytryptamine (5-HT) modulates the feeding of some insects, and this phenomenon was found in Harmonia axyridis (Pallas) by our previous study. An understanding of the 5-HT system in this beetle is helpful for utilizing 5-HT to modulate its predation to improve biological control efficiency, especially in greenhouses in winter in north China. This is because 5-HT influences diapause in insects by modulating the synthesis and release of prothoracic hormone (PTTH) and, therefore, influences feeding. To elucidate the molecular basis of the H. axyridis 5-HT system, reverse-transcription polymerase chain reaction (RT-PCR), multiple sequence alignment, and phylogenetic tree construction were used to identify the 5-HT receptor in H. axyridis, and quantitative real-time PCR (qRT-PCR) was used to analyze the expression pattern of these receptor genes in different developmental stages and in the nervous system (brain + ventral nerve cord), digestive tract, pectoral muscles, and gonads of the adult ladybird. The results showed that four 5-HT receptors were identified in H. axyridis, named 5-HT(1A)Har, 5-HT(1B)Har, 5-HT(2)Har, and 5-HT(7)Har. The four receptors were expressed at high levels in the adult stage, especially in 2-day-old adults, with expression levels of 18.72-fold (male) and 14.21-fold (female) of that in eggs for 5-HT(1A), 32.27-fold (male) and 83.58-fold (female) of that in eggs for 5-HT(1B), 36.82-fold (male) and 119.35-fold (female) of that in eggs for 5-HT(2), and 165.47-fold (male) and 115.59-fold (female) of that in eggs for 5-HT(7). The level of expression decreased with the advance of day-age in adults. The levels of expression of 5-HT(1B)Har, 5-HT(2)Har, and 5-HT(7)Har were low at the egg, larval, and pupal stages, and 5-HT(1A)Har was not expressed in the larval stage. The four receptors were expressed in the nervous system, digestive tract, pectoral muscles, and male and female gonads. The 5-HT(1A)Har was expressed at a high level in the pectoral muscle (6.75-fold of that in the nervous system), 5-HT(1B)Har in male gonads (1.02-fold of that in the nervous system) and the nervous system, 5-HT(2)Har in male gonads (5.74-fold of that in the nervous system), and 5-HT(7)Har in the digestive tract (1.81-fold of that in the nervous system). The results of this study will lay a foundation for research on the function of the 5-HT receptor by RNA interference in the regulation of predation by H. axyridis.