EUROPEAN JOURNAL OF ENTOMOLOGYENTOMOLOGY ISSN (online): 1802-8829 Eur. J. Entomol. 116: 104–108, 2019 http://www.eje.cz doi: 10.14411/eje.2019.012 ORIGINAL ARTICLE Daily rhythmicity in the sexual behaviour of Monema fl avescens (Lepidoptera: Limacodidae) SHUZHEN YANG 1, HONGXIA LIU 2 , MEIHONG YANG 2 and JINTONG ZHANG 2, * 1 Agronomy College, Shanxi Agricultural University, Taigu, Shanxi, 030801, China; e-mail: [email protected] 2 Institute of Chemical Ecology, Shanxi Agricultural University, Taigu, Shanxi, 030801, China; e-mails: [email protected], [email protected], [email protected] Key words. Lepidoptera, Limacodidae, Monema fl avescens, daily rhythmicity, sexual behaviour Abstract. 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 re- corded 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. INTRODUCTION In this study, daily rhythmicity and the effects of age on The Nettle caterpillar, Monema fl avescens Walker, both calling behaviour were investigated in M. fl avescens. 1855 (Lepidoptera: Limacodidae), is a serious defolia- These studies could be helpful in improving our under- tor of a wide variety of trees (Carrillo et al., 2012; Wang standing of chemical communication in the reproductive et al., 2013; Yang et al., 2016a) in many areas in eastern behaviour of Chinese populations of M. fl avescens and of Asia including Japan, China, Korea and Russia (Siberia) how to use pheromones to detect, monitor and mass-cap- etc. (Lammers, 2004; Yang et al., 2016b). Currently, M. ture this pest in the fi eld. fl avescens is increasing in abundance in China and over- wintering cocoons are easily found in orchards and parks MATERIALS AND METHODS (Ju et al., 2008; Li et al., 2010, 2013; Han et al., 2013). Insects Currently, the control of this insect pest relies mainly on Cocoons were collected from pear and apple trees in Luanx- spraying with chemical insecticides. However, as apples, ian County, Hebei Province, in late April 2014 and 2015. The pears and other fruit mature when the larvae of this pest are cocoons were kept at 25 ± 2°C, 75 ± 5% relative humidity (RH) most active using pesticides to control them poses serious and under a natural photoperiod until adult emergence. The adults problems. Use of the female-produced sex pheromone may of M. fl avescens started to emerge at the end of May. After emer- offer a promising alternative tool for controlling this pest. gence, the male and female moths were immediately segregated into different cages (40 × 40 × 60 cm; 15 moths per cage) con- As recorded for other pests (Blackmer et al., 2008; Jing et taining 10% (wt/vol) sugar solution in different rooms. One room al., 2010; Yang et al., 2012; Youm et al., 2012; Naka et al., was used for observing calling and copulation and the other for 2018), sex pheromones could provide an environmentally wind tunnel experiments under a 14L : 10D natural photoperiod friendly and effective technique for controlling these pests (sunrise about at 5:00 and sunset at 19:00). A single red incan- by selectively capturing the males and so reducing the like- descent light bulb providing a light intensity of approximately lihood of females fi nding a mate. 0.5 lux was used to observe sexual behaviour in both rooms. In- * Corresponding author; e-mail: [email protected]. Final formatted article © Institute of Entomology, Biology Centre, Czech Academy of Sciences, České Budějovice. An Open Access article distributed under the Creative Commons (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 104 Yang et al., Eur. J. Entomol. 116: 104–108, 2019 doi: 10.14411/eje.2019.012 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 dur- ing the photophase. Calling behaviour of female moths was ac- companied 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 occa- sions (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% ± Fig. 1. The percentage of females of Monema fl avescens of differ- 3% RH, with an air speed of 0.5 m/s and a light intensity of ap- ent ages calling during the scotophase. Percentages are means (± proximately 0.5 lux. Extract of the pheromone glands of fi ve fe- SE). The different letters indicate signifi cant differences at P ≤ 0.05 males was reduced to 10 μL under a gentle stream of N2 and then according to Tukey’s HSD of arcsine-transformed data. dispensed onto a 2.0 cm2 fi lter paper placed in the wind tunnel. The response of M. fl avescens males was tested daily using moths the scotophase were calculated for all the groups. Three replicate aged from 1 to 6 days old, at different times during the scotophase groups were observed, with a total of 60 pairs in each group. 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 Field tests the tunnel before they were exposed to the pheromone. The males Field tests were conducted in pear trees in Luanxian County, were released 150 cm downwind from the pheromone source. Hebei Province (39°66´N, 118°72´E), in June 2014. White delta Two minutes were allotted for the male to respond and when sticky traps were suspended at a height of 1.5 m at intervals of 50 there was no response the male was replaced. Once fl ight was m. Virgin females aged 1 to 6 days old were placed in cages as initiated, the males were observed until they stopped responding. bait beneath the roof of the traps. To compare the attractiveness of All treatments were tested in random order with three replicates, the virgin females, control traps without females were also used. with each replicate consisting of approximately 15 males. The The trap catches were counted every hour, and the moths on the responses of the males was recorded in terms of the following sticky traps were recorded. The distribution of the treatments was behaviour: takeoff, directed fl ight, landing on the source and at- randomized within six replicate series, using a total of 42 traps. tempts at copulation. Data analyses Copulation The statistical analyses were performed using the SPSS 19.0 In order to record mating activity, six groups of 20 pairs of 1 statistical software package (SPSS Inc., Chicago, IL, USA). to 6 day old virgin males and females were placed individually in Analyses of variance (ANOVA) and Du ncan’s multiple range test small cages (60 × 60 × 60 cm) and mating recorded every 15 min were used to assess the effect of age on the time spent calling. The during the test period. All these experiments were done in a test percentages of moths calling, wind tunnel and copulation results room at 25 ± 1°C, 75 ± 1% R.H and under a 14L : 10D photo- were analyzed using Tukey’s Honestly Signifi cantly Different period. Mating pairs were gently transferred into a small cage (30 (HSD) tests of arcsine-transformed data. The data on the number × 30 × 30 cm) for measuring copulation duration. Mating pairs of moths captured in the fi eld were analyzed using nonparametric and the other moths were not disturbed during this process. When Friedman test analysis of variance followed by Bonferroni cor- the scotophase ended at 5 a.m., females were dissected, and copu- rections. The level of signifi cance in all tests was set at 0.05. lation was determined by the presence of a spermatophore. The percentages of pairs starting to copulate at different times during Table 1. Percentage (%) of females of Monema fl avescens of different ages recorded calling at different times after the onset of the sco- tophase.