Extraction and Identification of the Black Cutworm’s Sex Pheromone and Correlative Studies on Its Biology
|Keywords||Agrotis ypsilon sex pheromone antenna sensilla standard chemicals EAG response wind-tunnel test field trap test|
The black cutworm [Agrotis ypsilon(Rottemberg)] belongs to Lepidoptera and Noctuidae, its common name is soil silkworm, land silkworm, black silkworm, black land silkworm, land scissors and roots-cutting worm etc. It took place and harmed the crops seriously in recent years in our country, has become an important countrywide agriculture pest, we have not effective methods to control it at present. We studied the sex pheromone communication system of the Chinese black cutworm by means of fight microscope, electron microscope(EM), gas chromatography (GC), gas chromatography/high-resolution mass spectrometry (GC-MS), electroantennogram(EAG), wind tunnel and field tests in this paper for the first time. By doing this, we aimed to prove up the black cutworm’s sex pheromone communication system and its variety character, and quest for a new method to control it with sex pheromone. The main study results were the follows.1. The study on the biology of the black cutworm. We continuously observed the biology and sex behavior of the black cutworm in the laboratory, it showed that the eclosion rate of the black Cutworm dropped with the rearing time extended, but its sex ration waved at the scope from 0.45 to 0.56. It could mate at the first day after eclosion, and the male and female black cutworm moth all could mate many times, but their mating ability was largely affected by sex ration. Its marry flying and mating behavior took place at the second half of night after the lights shut off(00: 30～04:00), and the mating behavior lasted for about 15 to 45 minutes. The longevity of female moths was longer than that of males, and varied with their mating conditions, the unmated adults’ longevity was a littler longer. The mating times could significantly affect the adult’s ovipositing quantities and the larva’s hatching rates, they would add with the mating times added.2. Observations of the location and ultrastructure of sex pheromone-producing gland in female black cutworm moth. We observed the location and ultrastructure of sex pheromone-producing gland in female black cutworm moth by means of fight microscope, scanning electron microscope(SEM) and transmission electron microscope(TEM). The results showed that the gland is an eversible ventral fold situated in the intersegmental membrane(ISM) between the eighth and ninth abdominal segments; it is covered in the seventh abdominal segment with the eighth and ninth abdominal segments; during courtship, the intersegmental membrane is extended into a white-yellow cyst. Many plump cones distribute on the surface of the gland. In the unmated female moth of 3 day old after eclosion, the glandular cells are arranged in one layer, among which the central cells are columnar and the nucleus is elliptical. There is evident conjugation between cells and basal involution is more in the basal membrane of cells. Microvilli are distributed on the cytoplasmic membrane and linked with endocuticle on which there are many layers of chitin. The outer horniness was dyed rather dark. The cells contain empty bubbles, mitochondria, lipid granule and smooth endoplasmic reticulum.3. The distilling and identification of the sex pheromone components from the female black cutworm moth. By means of GC and GC-MS, five sex pheromone components were identified in the female black cutworm moth’s sex pheromone producing-gland extracts, including Cis-7-dodecenyl acetate (Z7-12: Ac), Cis-9-tetradecenyl acetate(Z9-14: Ac), Cis-11-hexadecenyl acetate(Z11-16: Ac), Cis-5-decenyl acetate (Z5-10: Ac) and Cis-8-dodecenyl acetate (Z8-12: Ac). The content of each component per female is 0.245±0.098ng, 0.080±0.031ng, 0.089±0.033ng, 0.085±0.031ng and 0.105±0.065ng respectively. The percentages of Z7-12: Ac/Z9-14: Ac/Z11-16: Ac/Z5-10: Ac/Z8-12: Ac in pheromone gland extracts from individual females were(mean±SE) 40.451±13.66: 17.022±17.494: 14.943±5.142: 14.392±6.10: 17.225±9.792 respectively, and the percentages of Z7-12: Ac/Z9-14: Ac/Z11-16: Ac were 58.75±9.429: 18.91±7.539: 22.34±7.209.4. The study on the calling behavior and sex pheromone producing dynamic rhythms of the female black cutworm. The female black moth seldom called at the first day after eclosion, the calling rate reached the peak at the third day. The calling behavior all happened at scotophase, and reached the peak at the seventh to ninth hours at scotophase. The calling time would advance with the ages added. In general, both calling behavior and titers of five sex pheromone components showed distinct diel rhythms and time rhythms, and the two peals periods were synchronous at mid-later scotophase. The values of sex pheromone in female gland are very low at the first day after eclosion, it reached the peak at the third day and then declined. The values of sex pheromone in female gland did not change much from the starting of scotophase to 4.5 hours later, but from 4.5 to 7 hours the values of every component especially Z7-12: Ac increased to the peak rapidly, then declined at 8 hours, and reached the level of 4.5 hours. Each component declined to zero when the scotophase ended. From this we can see that the peak of the sex pheromone is seventh hour at scotophase.5. The study of effects of mating behavior and temperature on the production of the female black cutworm moth’s sex pheromone. The study of effects of mating behavior and temperature on the production of sex pheromone indicated that the mating behavior did not significantly affect the production of sex pheromone. So mating behavior did not affect the calling and mating of the black cutworm again in the next scotophase. But the environment temperature could affect it significantly. The optimal temperature for the biosynthesis of sex pheromone was between 20～25℃, over lower and higher temperature all made against the natural producing and releasing of the sex pheromone.6. The observation of the morphologie characteristics of every sensillum on the male black cutworm moth’s antenna. Through the scanning electron microscope(SEM) technology, we observed the morphologic characteristics of every sensillum on the male black cutworm moth’s antenna. We found that there are seven types of sensilla on the male black cutworm moth’s antenna. They are Sensillum trichodea, Sensillum chaeticum, Sensilla squamiformia, Sensillum coeloconicum, Sensillnm coelonica, Sensillum auricillica and Bohm’s bristles. The Sensillum trichodea were the most numerous, they are the main chemical sensilla which can recept the sex pheromone.7. The EAG response of male black cutworm moth’s antenna to sex pheromone and standard chemicals. The EAG response of male black cutworm moth’ s antenna to sex pheromone and standard chemicals showed that the five components (Z7-12: Ac、Z9-14: Ac、Z11-16: Ac、Z5-10: Ac、Z8-12: Ac) all could arose EAG response, and their responding values were significant higher than that of blank control. This indicated that there are olfaction receptors of all five pheromone components on the male moth’s antenna. The responding values of male moth’s antenna to single components and compounds are very low at the first day after eclosion, and reached the peak at the third day, then descended with the adding of ages. The EAG response of male moth’s antenna to dosages of each components and compounds took on "S" type.8. The wind-tunnel test of the male black cutworm moth’s behavior to female’s sex pheromone. The wind-tunnel behavioral test indicated that any single component did not elicit complete behavioral responses of males to the sex pheromone source. Among the binary compounds, the attracting ability of AB to males was the largest, and was as effective as a 3-day old females. At the same dosage, the attracting ability of ternary compound ABC is the biggest, it is only inferior to live female and gland extracts. The ration of sex pheromone components cloud affect the male’s behaviors obviously, the test of different rations of binary compounds and ternary compounds indicated that the best ration of AB and ABC in rubbers were 3:1 and 3:1:1 separately. This result was consistent to the ration of sex pheromone components in female gland extracts. We use different dosages of ternary compound ABC at its best ration(3:1:1) to proceed wind-tunnel test and found that females all could respond to sex pheromone from 0.1-500μg, the response was the biggest at 100μg, 50μg was the second, there was not significant difference between them (P≥0.05). By using the percentages of males which reached the sex pheromone source and mated in advance as the target, we can see that the reaction percentages were significantly high than other dosages (P≤0.05).9. Field test of capturing male black cutworm moth by sex pheromone standard chemicals. By using the sex pheromone standard chemicals as lures to capture males in the field, we found that the results were consistent to the conditions in a wind-tunnel test. Single component of sex pheromone could not elicit the male’s complete behavior in the wind-tunnel, and captured no males in the field test. Choosing binary compound AB(3:1) and ternary compound ABC(3:1:1) to capture males in the field, we found the mean capture numbers of lure made with AB(3:1) was 2.6, the alluring effects of ABC(3:1:1) increased significantly, the mean capture numbers was 7.40, it was 2.8 times of AB(3:1). This showed that component C (Z11-16: AC) could add the effects of sex pheromone. In the dosage response, the capture numbers was the highest at 200μg in the field test.We distilled the sex pheromone from the China female black cutworm moth, identified and analyzed its precise contents and proportions for the first time in this paper. Through EAG response, wind-tunnel test and field trap test, we confirmed the active components of the sex pheromone and obtained the mixture of the best lure. This not only enriched the content of studying the insect sex pheromone, but also provide probably a new way and technique of high-effective, nonpoisonous, non-pollution, nonsocial effects of pollution for integrated control of the black cutworm by using the synthesized sex pheromone.