Daily rhythmicity in the sexual behaviour of Monema fl avescens ( Lepidoptera : Limacodidae )

Daily rhythmicity in the sexual behaviour of Monema fl avescens Walker (Lepidoptera: Limacodidae) was studied under laboratory conditions. There was a distinct diel periodicity in female calling, male responsiveness and mating behaviour of M. fl avescens. As females aged there was an advance in the onset and more time spent calling. One day old females started calling 4 h after the onset of the scotophase, and 5 to 6 day old females called during the fi rst hour of the scotophase. About 34.5% of females called on the night they emerged (1 day old) and the peak in calling of 97.8% was recorded for 2 day old females, after which the incidence of calling decreased rapidly with advancing age. Wind tunnel and copulation tests showed that males were sexually mature on the third night and female moths on the second night. The highest value for the percentage mating was recorded for 3 day old virgin females 4 to 6 h after the onset of the scotophase. In fi eld tests, traps baited with 2 day and 3 day old virgin females captured more males than any other trap and most males were captured 4 to 6 h (1 to 2 day old), 3 to 5 h (3 to 4 day old) and 2 to 4 h (5 to 6 day old) after the onset of the scotophase. These results indicate that there is a daily rhythm in the reproductive behaviour of M. fl avescens and provides a better understanding of its sexual behaviour. * Corresponding author; e-mail: zhangjintong@126.com. INTRODUCTION The Nettle caterpillar, Monema fl avescens Walker, 1855 (Lepidoptera: Limacodidae), is a serious defoliator of a wide variety of trees (Carrillo et al., 2012; Wang et al., 2013; Yang et al., 2016a) in many areas in eastern Asia including Japan, China, Korea and Russia (Siberia) etc. (Lammers, 2004; Yang et al., 2016b). Currently, M. fl avescens is increasing in abundance in China and overwintering cocoons are easily found in orchards and parks (Ju et al., 2008; Li et al., 2010, 2013; Han et al., 2013). Currently, the control of this insect pest relies mainly on spraying with chemical insecticides. However, as apples, pears and other fruit mature when the larvae of this pest are most active using pesticides to control them poses serious problems. Use of the female-produced sex pheromone may offer a promising alternative tool for controlling this pest. As recorded for other pests (Blackmer et al., 2008; Jing et al., 2010; Yang et al., 2012; Youm et al., 2012; Naka et al., 2018), sex pheromones could provide an environmentally friendly and effective technique for controlling these pests by selectively capturing the males and so reducing the likelihood of females fi nding a mate. Eur. J. Entomol. 116: 104–108, 2019 doi: 10.14411/eje.2019.012


INTRODUCTION
The Nettle caterpillar, Monema fl avescens Walker, 1855 (Lepidoptera: Limacodidae), is a serious defoliator of a wide variety of trees (Carrillo et al., 2012;Wang et al., 2013;Yang et al., 2016a) in many areas in eastern Asia including Japan, China, Korea and Russia (Siberia) etc. (Lammers, 2004;Yang et al., 2016b).Currently, M. fl avescens is increasing in abundance in China and overwintering cocoons are easily found in orchards and parks (Ju et al., 2008;Li et al., 2010Li et al., , 2013;;Han et al., 2013).Currently, the control of this insect pest relies mainly on spraying with chemical insecticides.However, as apples, pears and other fruit mature when the larvae of this pest are most active using pesticides to control them poses serious problems.Use of the female-produced sex pheromone may offer a promising alternative tool for controlling this pest.As recorded for other pests (Blackmer et al., 2008;Jing et al., 2010;Yang et al., 2012;Youm et al., 2012;Naka et al., 2018), sex pheromones could provide an environmentally friendly and effective technique for controlling these pests by selectively capturing the males and so reducing the likelihood of females fi nding a mate.the scotophase were calculated for all the groups.Three replicate groups were observed, with a total of 60 pairs in each group.

Field tests
Field tests were conducted in pear trees in Luanxian County, Hebei Province (39°66´N, 118°72´E), in June 2014.White delta sticky traps were suspended at a height of 1.5 m at intervals of 50 m.Virgin females aged 1 to 6 days old were placed in cages as bait beneath the roof of the traps.To compare the attractiveness of the virgin females, control traps without females were also used.The trap catches were counted every hour, and the moths on the sticky traps were recorded.The distribution of the treatments was randomized within six replicate series, using a total of 42 traps.

Data analyses
The statistical analyses were performed using the SPSS 19.0 statistical software package (SPSS Inc., Chicago, IL, USA).Analyses of variance (ANOVA) and Du ncan's multiple range test were used to assess the effect of age on the time spent calling.The percentages of moths calling, wind tunnel and copulation results were analyzed using Tukey's Honestly Signifi cantly Different (HSD) tests of arcsine-transformed data.The data on the number of moths captured in the fi eld were analyzed using nonparametric Friedman test analysis of variance followed by Bonferroni corrections.The level of signifi cance in all tests was set at 0.05.dividuals were considered to be 1 day old (1 d) between 0 and 24 h after emergence.

Calling behaviour
Observations of 30 M. fl avescens females were made every 15 min throughout the 10 h of the scotophase because preliminary observations indicated that adults were not sexually active during the photophase.Calling behaviour of female moths was accompanied by the full exposure of their ovipositors.If a female called during two consecutive observations, it was considered to have been calling for 30 min.If a female was recorded calling on only one of the two observations, it was considered to have been calling for 15 min.Calling behaviour was recorded on three occasions (three replicates) for a total of 90 females.

Wind tunnel tests
The wind tunnel experiments were performed in a glass wind tunnel (195 × 60 × 60 cm) maintained at 22 ± 2°C and 75% ± 3% RH, with an air speed of 0.5 m/s and a light intensity of approximately 0.5 lux.Extract of the pheromone glands of fi ve females was reduced to 10 μL under a gentle stream of N 2 and then dispensed onto a 2.0 cm 2 fi lter paper placed in the wind tunnel.The response of M. fl avescens males was tested daily using moths aged from 1 to 6 days old, at different times during the scotophase and when they were most sexually active (3 day old).The males were acclimated for at least 1 h to the light intensity and airfl ow in the tunnel before they were exposed to the pheromone.The males were released 150 cm downwind from the pheromone source.Two minutes were allotted for the male to respond and when there was no response the male was replaced.Once fl ight was initiated, the males were observed until they stopped responding.All treatments were tested in random order with three replicates, with each replicate consisting of approximately 15 males.The responses of the males was recorded in terms of the following behaviour: takeoff, directed fl ight, landing on the source and attempts at copulation.

Copulation
In order to record mating activity, six groups of 20 pairs of 1 to 6 day old virgin males and females were placed individually in small cages (60 × 60 × 60 cm) and mating recorded every 15 min during the test period.All these experiments were done in a test room at 25 ± 1°C, 75 ± 1% R.H and under a 14L : 10D photoperiod.Mating pairs were gently transferred into a small cage (30 × 30 × 30 cm) for measuring copulation duration.Mating pairs and the other moths were not disturbed during this process.When the scotophase ended at 5 a.m., females were dissected, and copulation was determined by the presence of a spermatophore.The percentages of pairs starting to copulate at different times during There were three replicates of each of the observations throughout the scotophase (N = 30).The different letters within columns indicate signifi cant differences at P ≤ 0.05 according to Tukey's HSD of arcsine-transformed data.

Calling behaviour
Calling behaviour of females of M. fl avescens of different ages was recorded at different times during the scotophase (Table 1).Females of M. fl avescens were only sexually active during the scotophase.Calling activity of 1 day old females (1 day old) started 3 h after the onset of the scotophase, and the mean time of the onset of calling occurred earlier as they increased in age.The 5 to 6 day old females called throughout the scotophase.Only 34.5% of the females called on the night (1 day old) after emergence, whereas almost all females (97.8%) called on the next night (2 day old) (Fig. 1).The percentage of females calling signifi cantly declined on the 5th night after emergence when about 66.7% of females called.The mean time spent calling increased signifi cantly with age (Fig. 2).

Wind tunnel experiments
The fi rst weak response of M. fl avescens males to the sex pheromone occurred on the night after emergence and the peak response on the third night (Table 2).Males responded to the pheromone throughout the scotophase, but the peak response occurred 4 to 6 h after the onset of the scotophase (Table 3).
Copulation M. fl avescens was only observed copulating during the scotophase.Copulations were fi rst recorded at the beginning of the second hour and reached a peak about 4 to 6 h into the scotophase (Fig. 3).The percentage mating was signifi cantly dependant on the age of the virgin females.The percentage was highest for 3 day old moths (41.7%), followed by 2 day old (40%), 4 day old (33.3%), 5 day old (23.3%), 1 day old (18.3%), and 6-d-old (16.7%).The average duration of copulation was 120.8 ± 12.6 min (mean ± SE; N = 20) and ranged from 60 to 240 min.

Field tests
Unba ited traps set during the scotophase and traps baited with virgin female set during the photophase failed to attract males of M. fl avescens in the fi eld.Traps baited with 2 day old and 3 day old virgin females captured more males than the other traps (Table 4).The number of males captured signifi cantly depended on the age of the virgin females and the time from the onset of the scotophase.Most males were captured in traps 4 to 6 h (1 to 2 day old fe-   The different letters within columns indicate signifi cant differences at P ≤ 0.05 according to Tukey's HSD of arcsine-transformed data.73.3 ± 7.7b 62.2 ± 5.9b 37.8 ± 2.3b 28.9 ± 2.2b 8~10 33.3 ± 5.9c 28.9 ± 3.8c 13.3 ± 2.2c 6.7 ± 5.9b The Monema fl avescens males tested were 3 days old (N = 45).
The different letters within columns indicate signifi cant differences at P ≤ 0.05 according to Tukey's HSD of arcsine-transformed data.
males), 3 to 5 h (3 to 4 day old) and 2 to 4 h (5 to 6 day old) after the onset of the scotophase (Table 4).

DISCUSSION
Female calling, male responsiveness and mating behaviour of M. fl avescens had a distinct diel periodicity.Under natural conditions, 34.5% of the females that called during the fi rst night were fertilized that night, the number of females calling on the second night was 97.8%, and the number of females older than 2 days recorded calling was almost zero.These results indicate that most female moths were sexually mature on the second night.One day old females started calling 3 h after the onset of the scotophase, but 5 to 6 day old females called during the fi rst hour of the scotophase.The result that females began calling earlier during the scotophase and for a longer time as they aged is in agreement with that recorded for several other species of moths (Seol et al., 1986;Delisle, 1992;Spurgeon et al., 1995;Mazor & Dunkelblum, 2005).Older females advance their calling time to increase their mating success relative to younger females, otherwise they would be at a disadvantage compared to the younger females if they did not spend more time calling (Delise, 1995;Xiang et al., 2010;Liu, 2013).
Wind tunnel and copulations tests indicate that the males were sexually mature on the third night, one day later than female moths.This accounts for why most 2 day old females called but were less likely to be mated than 3 day old females.Similar results are reported for the insect tomato looper, Chrysodeixis chalcites (Snir et al., 1986).
Field tests indicate that none of the traps caught male moths during the day, but were most attractive 4 to 6 h (1 to 2 day old), 3 to 5 h (3 to 4 day old) and 2 to 4 h (5 to 6 day old) after the onset of darkness, respectively.These results also indicate that the diel periodicity in calling conform with the result of the fi eld tests, and similar results are reported for Maruca vitrata (Lu et al., 2007) and Isoceras sibirica (Lu et al., 2013).
The calling and mating in Lepidoptera have a certain rhythm.Studying the rhythm of their reproductive behaviour can provide an important theoretical basis for the ef-fective control of insects and reducing the economic losses they cause.However, the effects of some other factors such as temperature and photoperiod should also be studied in the future.Means in a column followed by different letters are signifi cantly different after Bonferroni correction (1 day old: H = 30.5,d.f.= 8, P = 0.000; 2 days old: H = 38.9,d.f.= 8, P = 0.000; 3 days old: H = 38.0,d.f.= 8, P = 0.000; 4 days old: H = 29.7,d.f.= 8, P = 0.000; 5 days old: H = 27.7,d.f.= 8, P = 0.001; 6 days old: H = 21.5, d.f.= 8, P = 0.006).

Fig. 1 .
Fig. 1.The percentage of females of Monema fl avescens of different ages calling during the scotophase.Percentages are means (± SE).The different letters indicate signifi cant differences at P ≤ 0.05 according to Tukey's HSD of arcsine-transformed data.

Fig. 2 .
Fig. 2. The mean time females of Monema fl avescens of different ages spent calling in the scotophase.Values are means (± SE).Values followed by different letters are signifi cantly different (P ≤ 0.05) based on Duncan's multiple range test.

Fig. 3 .
Fig. 3.The percentage of Monema fl avescens of different ages recorded mating at different times after the onset of the scotophase.Values are means (± SE).Values followed by different letters are signifi cantly different (P ≤ 0.05) based on Tukey's HSD of arcsinetransformed data.

Table 1 .
Percentage (%) of females of Monema fl avescens of different ages recorded calling at different times after the onset of the scotophase.

Table 2 .
Effect of age on response (%) of Monema fl avescens males to pheromone extracts in a wind tunnel.

Table 3 .
Effect of time after the onset of the scotophase on the response (%) of Monema fl avescens males to pheromone extracts in a wind tunnel.

Table 4 .
Effect of the photophase and the time after the onset of the scotophase on catches of male M. fl avescens in traps baited with 1 to 6 day old virgin females.