Protogyny after hibernation and aestivation in Cheilomenes sexmaculata (Coleoptera: Coccinellidae) in central Japan

Differences in the time of occurrence of both sexes of adult ladybirds, which refl ect differences in the longevity and reproductive strategies of the sexes, is important from the perspective of applied entomology, including biological control, as well as basic entomology. Nevertheless, there is little fi eld census data on this topic. We investigated the seasonal occurrence of both sexes of Cheilomenes sexmaculata (Fabricius) in Osaka City, central Japan, from 2003 to 2011. Female adults tended to appear earlier than males after hibernation or aestivation. That males never appeared earlier than females during the nine year period of this study indicate that protogyny occurs after hibernation and aestivation in this species.


INTRODUCTION
Despite the fact that male and female insects should occur simultaneously so that they are more likely to encounter each other for mating, the sexes of many insects differ in their seasonal occurrence.For example, in butterfl ies in temperate zones, there is a general tendency for males to emerge before females, i.e. protandry (Wiklund & Fagerström, 1977).In addition, male butterfl ies, which overwinter as adults, appear earlier than females after hibernation (Wiklund et al., 1996).The occurrence of this pattern could be because monogamous (single-mated) male butterfl ies that emerge before females are more likely to mate with virgin females than their counterparts that emerge later (Wiklund & Fagerström, 1977;Iwasa et al., 1983).
In addition, reproductive physiology may constrain the timing of occurrence of both sexes, for example, females maximize their body mass by prolonging development, because generally female fecundity is directly correlated with body size; in contrast, males with a lower body mass may suffer negligible disadvantages during reproduction (Kleckner et al., 1995).Differences in the time of occurrence of both sexes may arise because of differences in the mortality and/or demography of the sexes (Carey et al., 1995).In many species of insects, males emerge concurrently with or before females; however protogyny, in which females appear earlier than males, is recorded in caddisfl ies and bagworm moths (Degen et al., 2015;Nowinszky et al., 2016).In bagworm moths, protogyny is associated with a reduction in female encounter rates with males; however, as females live longer than males this may compensate for this disadvantage (Degen et al., 2015).Taken together, sexual dimorphism in the timing of emergence is affected by differences in reproductive strategies, reproductive physiology and demographics (e.g.temporal pattern in abundance and mortality) of the sexes.(Kawakami et al., 2016).On the basis of these fi ndings, the generation periods of the adults that overwintered and oversummered from 2003 to 2011 are shown in Table 1.Autumn adults hibernate and emerge as overwintered adults, and summer adults aestivate and emerge as oversummered adults (Kawakami et al., 2016).Differences in the time of occurrence of both sexes may lead to a decline in the mating rate.Therefore, to investigate the time of mating with regard to the abundance of both sexes, the relationship between the number of mating pairs and the number of adults of both sexes was analyzed using Pearson's correlation coeffi cient.The difference in the sex ratios of the generations that overwintered and oversummered, indicated by a slight protogyny in the line diagram, was examined because a more abundant sex may appear earlier and remain longer than a less abundant sex.Thus, any difference in the sex ratio (male/ male + female) from 0.5 was analyzed using a test for independence for the two generations.We used JMP 11 Discovery (SAS maculata were counted on shrubs and weeds where prey aphids occurred.Using these data, population changes in the abundance of larvae, teneral and post-teneral adults from 2003 to 2011 are described in Kawakami et al. (2016).
To determine the difference in the occurrence and periods of mating activity of the sexes, the average numbers of males, females and mating adults per 30 min are described using a line diagram.Sex ratio and mating pairs were analyzed using Pearson's correlation coeffi cients for each year.As C. sexmaculata adults hibernate and aestivate (Kawakami et al., 2016) like other ladybird beetles (Nedvěd & Honěk, 2012), the demography of both sexes and the sex ratio of the generations that overwinter and oversummer were determined.

RESULTS AND DISCUSSION
The results of fi eld surveys of the changes in the number of larvae, teneral and post-teneral adults in the Osaka population revealed that C. sexmaculata completed three generations a year Institute Inc., Cary, NC, USA) to carry out all the statistical analyses.
Like other ladybird beetles, which hibernate as adults (Nedvěd & Honěk, 2012), C. sexmaculata adults also hibernate and aestivate (Kawakami et al., 2016).In Osaka female adults tended to appear earlier than males after hibernation and aestivation during the period 2003 to 2011 (Fig. 1).Differences in the day of appearance of both sexes are small; however, the fact that males never appeared earlier than females over the nine year period of this study indicates that protogyny occurs after both hibernation and aestivation in this species.Multiple mating may occur in the Osaka population of C. sexmaculata because mating pairs were recorded throughout each year except in summer (Fig. 1) and tended to be recorded on days when there were many adult individuals of both sexes (Fig. 1).Ladybirds often mate several times and the sperm of the last male a female copulates with is used to fertilize the eggs (Katakura, 1981;De Jong et al., 1993, 1998;Ueno, 1994; for the Indian population of this species, see Chaudhary et al., 2016).Therefore, for males there would be few benefi ts from protandry, which may be one of the factors favouring protogyny in C. sexmaculata.Indeed, for Adalia bipunctata at emergence from pupae reared under laboratory conditions, slight protogyny is documented (Hemptinne et al., 2001).Alternatively, for the females to be sexually mature it may be necessary for them to begin feeding on temporary food resources earlier than males.
The sex ratio remained the same throughout this study (Kawakami et al., 2016); however, females that overwintered were significantly more abundant than overwintered males and females that oversummered tended to be more abundant than oversummered males (Table 2).This indicates that the demographic differences between sexes (more females appeared than males in early spring or early autumn) may also contribute to protogyny.
In conclusion, taken together, our fi ndings indicate that protogyny in the Osaka population of C. sexmaculata may be due to the rare occurrence of competition between males for females and the differences in the demographics of the sexes.

Fig. 1 .
Fig. 1.Seasonal changes in the numbers of male and female adults of Cheilomenes sexmaculata recorded per 30 min in Osaka City from 2003-2011.

Table 2 .
Sex ratios (male/male + female) of the generations of Cheilomenes sexmaculata that overwintered and oversummered in Osaka City from 2003 to 2011.The bias in the sex ratios were analyzed using χ 2 -test for independence for the adult numbers of both sexes.df = 1; n -sample size.