2.1 The relationship between calling and mating
The ovaries of 0-day-old female moths was immature and developed at grade 1 when they emerged, but quickly developed into grade 2 on the same day. The ovaries of female moths are fully developed to grade 3 at 1 day of age (Fig.1).
The age of moths had significant effects on mating rate (F=11.08,df=29, P<0.001). The mating rate of 0-day-old male and female moth was only 2.5%, and the 1day old mating rate was the highest, 43.2±10.1%. The mating rate of 2 day old moths was 38.2±7.7%, and that of 3 day old was 13.9±4.5%. The mating rate of 1 and 2 day old moths was significantly higher than that of other ages, accounting for 81.4% of the total (Fig.2A).
When the female moth releases sex pheromone during calling, the ovipositor at the end of the abdomen will extrude outward in the dark period (Fig. 3a). The percentage and time of the ovipositor extrusion are related to the age of the female moths. The average extrusion time of day 1 moths was 1.8 ± 0.2 h, The average extrusion time to day 4 or 5 moths was the highest, 5.2 ± 0.2 or 4.9 ± 0.4 h (Fig. 2B). The 1day old female moth had a low extrusion rate of ovipositor and extruded late, starting after 0:00 and ending before 5:00, and the time lasted short. As age increased, the extrusion time was gradually earlier and longer, and the percentage increased. The highest number was observed at 3:00 in 2 day old female moths. 3 day old female moths began to extrude the ovipositor at 19:00, and the percentage of extruded ovipositor gradually increased, reaching the highest at 2:00. The ovipositor was extruded 100% after 23:00 for the 4-day-old and 5-day-old moths(Fig.2D).
The mating of male and female moths of H. armigera has a remarkable circadian rhythm (F=22.094,df=71,P<0.001 ). The mating percentages at 23:00 and 01:00 were 26.9%±4.2% and 31.0%±2.1%, was significantly higher than other time periods. The mating percentages at 00:00 and 02:00 were 15.4±3.4% and 17.1%±2.1%, respectively, and the total mating rate between 23:00 and 01:00 accounted for 90.4% (Fig. 2C). Here, it indicated that the age of ovipositor extension was later than that of mating age, and in the circadian rhythm, the time of ovipositor extension was also later than the mating time. The percentage and time of ovipositor extrusion reached the maximum at 4 or 5 days of age.
When the ovipositor was not extruded, Z11-16: Ald was not detected or very minor in the headspace extraction by SPME(Fig.3d). After the ovipositor was extruded, the release of the major component Z11-16: Ald increased from 0.0009 ± 0.0005 ng to 0.0568 ± 0.013 ng at 25 °C, an elevation of 63 times compared to no extrusion (F = 26.2, df = 17, P = 0.006). When the temperature increased from 15 °C to 35 °C, the release amount was greatly elevated from 0.0138 ± 0.012 to 0.59 ± 0.263ng, which was 42.7 times higher than that at 15 °C (F = 6.965, df = 45, P = 0.002) (Fig. 3e).
2.2 Age and circadian rhythm in the titer of sex pheromone
Z11-16:OH was not detected in the headspace extraction and in the outer cuticle surface of pheromone glands by SPME adsorption, but only in the fluid of pheromone glands. Z11-16:Ald, Z9-16:Ald and 16:Ald were not detected in the fluid of pheromone glands. All components were detected in the solvent extract of pheromone glands(Suppl Fig.1). Female H. armigera moths at 0-day-old do not have Z11-16:Ald, and the release gradually increases after 1 day of age. At 3 days old, the maximum is 0.12±0.06 ng before the release amount starts to gradually decrease. The titer of Z11-16:Ald in the cuticle surface gradually increased with the increase of day age, reaching a maximum of 5.36±3.14 ng at 3 days of age, which was significantly higher than other days of age. Z11-16:OH in the solvent extracts of pheromone glands has the highest titer at the age of 3 days, which is 5.96±2.60 ng. The minimum titer at 0 days of age was 0.03 ± 0.02 ng，There was significant difference in the titer of Z11-16:OH between ages（F=4.168, df=23, P=0.011）. Z11-16: Ald titer is the largest when the female moth is 2 days old, 8.06±4.99 ng, but no significant difference in solvent extracts of different ages was analyzed(F=2.22, df=23, P=0.097). Z11-16:OH exist in the extracts of gland fluid of different female moths, there was significant difference between moth ages (F=3.219, df=23, P=0.03), the minimum titer at 0 days of age was 0.02±0.01 ng (Fig.4A).
In the headspace extract, there was no significant difference in the titer of Z11-16: Ald from 19:00 to 07:00 (F=1.60, df=33，P=0.185), and the titer gradually increased into scotophase. The highest peak was reached at 03:00, and the titer was 0.20±0.10 ng before gradually decreasing. When extracting the pheromone in the cuticle surface by the SPME adsorption, the highest peak was reached at 03:00, and the titer was 4.55±2.47 ng. However, in the solvent extraction of pheromone gland, the highest peak of Z11-16: Ald titer was 23:00, and the highest peak of Z11-16: OH was 01:00. In the inner fluid of gland, the highest titer of Z11-16:OH was at 03:00 (Fig.4B).
2.3 The effect of mating on the release of sex pheromone
After mating, the synthesis and release of Z11-16: Ald were suppressed significantly, and recovered slowly. At 24 hours after mating, the total pheromone titers extracted by solvent remained low, and Z11-16:Ald could not be detected by headspace and SPME adsorption. At 48 hours after mating, the total pheromone titer gradually recovered, but was relatively lower than just after mating. Z11-16:Ald could be detected in some of females by the headspace SPME (F=2.163, df=31, P=0.10). Z11-16:OH in the inner fluid of gland did not change significantly before and after mating(F=1.345, df=28, P=0.282 ). At 72 hours after mating, the total pheromone titer significantly recovered, and reached the level just after mating (Fig. 5A).
However, for those female moths that mated earlier in the dark period, its sex pheromone was extracted by four methods at the normal release peak time (10 h into scotophase) in the same scotophase. The results showed that the titer of female moths decreased rapidly on the night after mating, and only solvent extraction had trace amounts of Z11-16: Ald and Z11 -16:OH(Fig. 5B). It was not detected in the sample with headspace SPME, and very trace amount in the sample with SPME adsorption. Z11-16:OH was not even detected in the gland fluid.
2.4 Circadian rhythm of male moth trapping in the field
The circadian rhythm of H. armigera male moth catches in 4 locations was recorded by pheromone trapping (Fig.6). In Xinxiang, Henan, on August 8-13, 2021, male moths were caught at the beginning of the dark period, reaching the peak of moth trapping 8 h into scotophase, and ending 10 h into scotophase. In Tianjin City on June 17-23, 2021, the circadian rhythm was the same. The moth trapping in Aksu, Xinjiang on July 9-16, 2021 started 2 h into scotophase, and the peak period was 7 h into scotophase. Male H. armigera moths in Changji, Xinjiang, on June 2-9, 2021 were trapped 1 h into scotophase, but there was no obvious peak catch of moths between 2 and 7 h into scotophase. Thus, the activity time of male moths to sex pheromone was 8 -10 h, which was more wider than that of female moths.