Mating Behavior And Cloning Of A Chemosensory Protein Gene Of Batocera Horsfieldi (Hope) (Coleoptera:Cerambycidae)

Batocera horsfieldi (Hope) (Coleoptera:Cerambycidae) is as a stem borer and a major destructive pest damaging various trees, which causing serious economic and ecology loss in China. It is difficult to control wood-boring pests because of the larvae cryptic feeding habit. With the development of chemecology, control strategies that are based on controlling or killing longhorned beetle adults which are exposed to wild for feeding and copulating. Although some species of cerambycid beetles have been studied, the mechanisms that are involved in behavior and chemical ecology of B. horsfieldi (which belongs to the lamiine) remain unknow. Research on the mating behavior and recognition mechanisms in this species will help in developing integrated pest management tools as well as increasing knowledge of this beetle. Moreover, a chemosensory protein (CSP) in maxillary palpi has also been researched to realize more about the chemosensory mechanism in this species.The main results are as follows:1.Mating behavior of B. horsfieldiWe observed the mating behavior of B. horsfieldi in the laboratory and confirmed that copulation of this longhorned beetle consisted of three phases:encountering and pair-bonding; mating attempt and ejaculation; post-copulatory guarding. Male showed a dash behavior (rapid approach toward mates) to the female without antennal contact on most occasions (48 of 60 pairs), and then the male mounted the female.Then, he attempted to mate, including licking the middle of the elytra of the female with his maxillary palpi repeatedly, and bending abdomen to contact the tip of the female genitalia. The average mating attempt time was 32.7 min. Only when the female was willing to copulate, ejaculation would be proceed and lasting 61.2 s average time. After ejaculation, 53 of 60 males (88.3%) remained astride the females showing behavior consistent with mate guarding (average time 25.5min).Five pairs of male and female has been observed for the mating behavior and oviposition behavior during their whole Adult stage. The results showed that the average mating behaviors are 26.2 times.The first peak appeared at 7d-8d after eclosion. The general trend of mating was decreased, but some secondary peaks appeared after then. The peak of mating occurred at 16:00-18:00 daily variation. The average oviposition period of female was about 30 min. During its whole life, the average oviposition rate of a female is 39.8.The average pre-oviposition period of female was 12d, the peak of oviposition occurred in 36d after eclosion. 2. Location and recognition for mate in B. horsfieldiIn olfactometer bioassays, males were significantly attracted to odors from females' volatiles compared with the control air, but were not attracted to volatiles from females with Rosa multiflora Thunb.It is indicating that there are volatiles of females (non-contact sex pheromone) for males to locate but the function of location is weaker than the feeding-host. All of the beetles tested showed no significant different responding in other groups,suggesting that the female was not attracted by the male. Wind tunnel tests showed that sex pheromone was of great importance for orientation of female by male, but the vision effect was limited. Male showed few attractions for female.The males showed behaviors of mating attempt (licking and abdominal bending) with living females and frozen females.Frozen females, with cuticular hydrocarbons stripped by hexane extraction showed no attraction for tested males, suggesting that males did not recognize females.Reapplying the solvent extract of frozen females to both washed dead males and females caused mating attempts by the tested males, confirming that cuticular hydrocarbons (contact pheromones) played an important role in recognition of females by male B. horsfieldi. The male presented with a glass tube coated with female extract showed the same bending behavior, providing further evidence that contact pheromones play an essential role in mate recognition, and female hexane extract containing female contact pheromones.Gas chromatography-mass spectrometry analyses of whole-body hexane extract of male and female revealed substantial differences in hydrocarbon profiles, with eight of the compounds in the extracts from females (total 28 compounds) being absent from those of males (total 24 compounds).For limitation of beetle resource and time, we need to do further researches on these compounds to obtain the active components.3.Cloning of a chemosensory protein (CSP) gene of B. horsfieldiIt was essential of male licking behavior with his maxillary palpi in mate recognition. A full-length BhorCSP2 gene was cloned from maxillary palpi of B. horsfieldi. The open reading-fragment of BhorCSP2 was 384bp, encoding 127 amino acid residues.This deduced amino acid sequence shared some common structural features with chemosensory proteins in insects with four conserved cysteines and a signal peptide with 19 aa in the N-terminal sequence. The amino acids sequence analysis of BhorCSP2 indicated that it was the highest identitiy with that of Tribolium castaneum CSP11 among 30 insect CSP tested.