Study on the attractivity of female adults to the males and the peak of mating activity in Diaphorina citri Kuwayama

Abstract:【Objective】Citrus is an important economic crop in China, and Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a serious pest of citrus, which is also an important medium for spreading the most devastating diseases in citrus production huanglongbing (HLB). Generally, the areas where the HLB occurs are also the distribution areas of D. citri. The control of D. citri relies largely on chemical insecticides currently, but has led to insect resistance and serious environmental problems. One of the al- ternatives is to use insect pheromones that have the advantages of trace, high efficiency, non-toxicity and no harm to environment and natural enemy, and play an important role in the integrated pest man- agement. The purpose of the present work is to study the attractivity of adult D. citri between two sexes and the peak of mating activity of D. citri to provide scientific basis for accurate extraction of the sex pheromone of D. citri.【Methods】The Y-tube olfactometer was used to test the attractivity of adult D. citri between two sexes. The Y-tube olfactometer consisted of a 30-cm-long, 3-cm-diameter central tube and two 15-cm-long, 2-cm-diameter lateral arms, which were individually connected to the odor stimuli and the control through a Teflon connection. The olfactory bioassays were conducted at room tempera- ture (27±2 °C) from 10:00 to 20:00. Ten virgin females or males were used as lures, while the empty bottle was as the control. For each bioassay, a single insect was introduced into the central arm of Y- tube. The insect walked into one of the lateral arms when they came over 1/3 and remained for at least 60 s in that lateral arm can be recorded. If an insect did not make a choice in 5 min after being released into the olfactometer, it was considered as a non-responder. The mated and single older nymphalD. citri samples were fed on Murraya exotica seedlings at (27±2) °C, 60%-70% and 14L: 10D photope- riod. M. exotica seedlings were pruned 8-10 cm in height 15 days before the beginning of the experi- ment to allow emission of flushes. Seedlings containing flushes of 1 cm in length were allocated to cov- er with the bigger plastic cup, which was 5.5-cm-diameter bottom, 8.5-cm-diameter top opening, and 13.5-cm-height with air hole. The bigger plastic cup (top one) and the small plastic cup (bottom one) that was 4.5-cm-diameter bottom, 6.5 cm-diameter top opening, and 8.5-cm-height with drain water hole in the bottom for planting the seedlings formed an experimental unit (cage). After that, one virgin male and one virgin female (couple) with 1, 2, 3, 4, 5, 6, 7, and 8 days after emergence (treatments) were released to inside the cage. For each treatment (couple age), 30 replicates were performed. The mating activity of each couple were assessed every 30 min (24 h a day) for 7 days.【Results】Using our laboratory colony, we studied the mating behavior of D. citri when individual couples were kept in the laboratory on a common host, M. exotica, and under conditions of photoperiod, relative humidity, and temperature mimicking those found under natural conditions in most citrus fields in south China. The re- sults showed that there was a sex pheromone association between the D. citri adults. The virgin males were significantly attracted to the virgin females (x2=6.533, p<0.05) over control. Neither the virgin females nor the mated adults had significant preference for the virgin adults of the opposite sex. There was no mating activity of D. citri observed on 1- and 2-days after emergence. The mating behavior ofD. citri occurred from 3- to 14-days after emergence, and reached a clear peak with 6-day old insects with the highest frequency of copulation (24.4%) and recopulation (79%). During the 3-6 days after emergence, the mating rate gradually increased with the increase of the emergence days, and then gradu- ally decreased at 6- 10 days after emergence. The mating rate at 11- days after emergence increased again. Subsequently, the mating rate decreased again at 12-14 days after emergence, but the decline was small. Next, we studied the diel rhythm of mating activity by 6- day- old couples. Mating occurred throughout light period (6:00—20:00) and dark period (20:00—6:00), but mainly in the light period, es- pecially at 14:30—18:30. Within the light period, the mating rate of D. citri was low without a regular distribution from 6:00 to 14:00. During the period from 6:00 to 15:30, the mating rate generally increased, and then slightly decreased at 16:00. The mating rate decreased from 16:30 to 20:00. It is obvious that the mating rate in the dark period was lower than 10%. Moreover, no mating activity was ob- served at 00:30, 01:00, 03:00 and 04:30【. Conclusion】The virgin males were significantly attracted to the virgin females. The mating behavior of D. citri occurred until 3 days after emergence. The peak of mating activity occurred at 14:30—18:30 in the afternoon of the 6th day after emergence. It was sug- gested to extract the sex pheromone of D. citri using the 6-day-old couples during this period. To study the mating behavior and pattern of D. citri can provide a reliable basis for the extraction, isolation and identification of its sex pheromone components.