Cloning, Expression And RNAI Of Pheromone Biosynthesis Activating Neuropeptide Receptor, A Sex Pheromone Biosynthesis Gene In The Black Cutworm, Agrotis Ipsilon

The black cutworm moth, Agrotis ipsilon is one of the most caustic migratory and disastrous controversial turf grass insect pest of the order Lepidoptera attacking upon diverse field crops around the world. Pheromone biosynthesis activating neuropeptide receptor(PBANR) is a neuropeptide receptor that regulates the sex pheromone production in a variety of moth species. One of the GPCRs plays a very important role to regulate sex pheromone biosynthesis in ligand to PBAN. Mating interrupts permanent in female‘s receptivity and ceases transient loss sex pheromone production in adult Lepidoptera. In this present study, first time the full-length 3539 bp nucleotide clone of pheromone biosynthesis activating neuropeptide receptor was subsequently cloned by using the rapid amplification of cDNA ends PCR(RACE-PCR) method from the female pheromone gland of the adult A.ipsilon and named as(Aips-PBANR). The multiple protein sequence alignments revealed that proteins codified by A. ipsilon PBANR gene shared high identity with homologs from other Lepidoptera organisms. The RT-PCR screening amplified preferably Aips-PBANR from pheromone gland of female and male aedeagus of male tissues. The real-time quantitative PCR(qRT-PCR) was used for investigating the expression profile of Aips-PBANR gene in different tissues, days and times. The qRT-PCR results for tissue distribution showed maximum expression in Aips-PBANR in pheromone gland; however a little was expressed in male aedeagus tissue of male adult. For different days and times, the Aips-PBANR gene expression was found significantly highest at the 4th day after emergence, and 2:00 am in its scotophase period. In addition, qRT-PCR experiment revealed that mating significantly inhibited the Aips-PBANR gene expressions, which shows that mating suppresses insect pheromone production.RNA interference(RNAi) holds great potential as a new measure for pest management. However, RNAi effectiveness varies greatly in insects from species to species. Insects in the order Lepidoptera are among the most difficult species for utilization of RNAi-based methods. In this study, we chose the black cutworm, A. ipsilon as a target for optimizing conditions for RNAi-based gene silencing. We used siRNA and dsRNA to knockdown the PBANR involved in the production of sex pheromone in adult A. ipsilon females. We injected 20μl of siRNA, dsRNA, dsGFP, and water into the 8th and 9th abdomen segment of an adult A.ipsilon female and evaluated its expression 72 hrs after injection. The results of qRT-PCR demonstrated that the mRNA levels were significantly reduced more than 90% in PBANR siRNA-treated A. ipsilon, however, 56% knockeddowned by dsRNA, respectively than in dsGFP, water and non-injected controls.Transversely with astonishing pliability in A.ipsilon in its growth, population, survivor and reproduction with relation to timing and spacing, the outbreaks of larvae are closely related to temperatures encountered by the immigrant adults. But mechanisms linking population outbreaks and migration are not well understood. We investigated the effect of exposing A. ipsilon adults to different temperatures ranging from 160 C to 360 C on its mating, oviposition, and longevity. Our results showed that both mating percentage and frequency were highest at 240C–280C and decreased at temperatures outside this optimal range. Time of night when mating began was delayed at higher temperatures, while mating duration progressively decreased with increasing temperature. Both pre-oviposition period(POP) and oviposition period(OP) decreased linearly with increasing temperature. Peak daily and lifetime fecundity were highest at 240C–280C and declined gradually with decrease in temperatures, suggesting that 240C–280C is the optimal thermal range for A.ipsilon oviposition. Adult longevity was negatively correlated with temperature. Males lived longer than females at lower temperatures, but females lived longer than males in the range between 320C–360C treatments. Together, our findings suggest that reproduction occurs when the prevailing temperature is around 240C–280C. We predict that larval damage or outbreaks of A.ipsilon will occur only in areas where the prevailing temperature is around 240C–280C, which provides a key basis for the prediction of A.ipsilon population outbreaks in areas of immigration.As a whole, PBANR, a sex pheromone gene was cloned first time from adult female pheromone glands and named as Aips-PBANR after the organism. Quantitative studies on the expression of Aips-PBANR gene on different tissues, days, times and pre-post mating were done. Silencing of Aips-PBANR was also carried out by injection of siRNA and dsRNA in the abdomen of adult female.These results provide solid framework for essential targets in A. ipsilon that may help in disruption of reproductive functions as pest management tool and diversified functions of this diverse neuropeptide receptor. In addtion, the results provided knowledge about different temperature levels for survival of black cutworm their effects on mating factors, which could help us to mornitor and forcaste accurately the occurrence of A. ipsilon.