In insects, mating performance of each sex is associated with various factors such as mating/reproductive history (Uhia and Rivera 2005, Kant et al. 2012, Colares et al. 2015), age (Kawazu et al. 2014, Liu et al. 2014, Ekanayake et al. 2017), nutritional condition (Aluja et al. 2009), body size/mass (Aluja et al. 2009, De Luca 2015, Ekanayake et al. 2017), and population density (Shelly et al. 2013). When the relative density of mates changes, females or males may have different mate-encountering rates, searching costs, or mate-choosing opportunities, ultimately leading to changes in mating performance and reproductive outcomes (Kokko and Rankin 2006, Lehmann 2012). Therefore, mate density is a major factor in insects' reproductive biology and population dynamic mechanisms.

The effects of male density on female or male mating performances have been well documented for a number of insect species, e.g., water strider (Sih and Krupa 1995, Lauer et al. 1996), weevil (Flay et al. 2014), field cricket (Atwell and Wagner 2014), butterfly (Holveck et al. 2015), and damselfly (Gomez-Llano et al. 2018). However, little is known about the effects of female density, which have been studied only in a few insects. In the salt marsh-inhabiting planthopper Prokelisia dolus Wilson (Hemiptera: Delphacidae), female density was found to differentially influence males' ability to locate mates and male reproductive potential (Langellotto and Denno 2001). In the water strider Aquarius remigis (Say) (Hemiptera: Gerridae), however, no relationship between female density and male mating frequency was detected (Lauer et al. 1996). Da Silva et al. (2013) observed the mating frequency and mating duration of the citrus mealybug Planococcus citri Risso (Hemiptera: Pseudococcidae) and citrophilus mealybug Pseudococcus calceolariae Maskell (Hemiptera: Pseudococcidae) at different female densities (1, 2, 4, 8, and 16 females per male). Both mealybugs showed increased mating frequency as the female density increased. Moreover, P. calceolariae males mated significantly faster at the highest female density (i.e., 16 females) than at most of the other densities. The P. citri males also mated fastest at the highest female density, but the difference was not statistically significant. Because insects are highly diverse and different species may have evolved specific life history strategies (Bernays 2009), the responses to female density probably differ among species. Therefore, this topic needs to be studied in a range of insect species.

For females, their postmating performance, e.g., reproductive outputs and survivorship, as well as the traits of their offspring, are affected by several aspects of their mates or themselves. Female fecundity is partially determined by mating history and by the age of available males (Kant et al. 2012, Kawazu et al. 2014, Tobin et al. 2014, Colares et al. 2015, Wang et al. 2018). Specifically, females have a higher reproductive output if mated with virgin males (Torres-Vila and Jennions 2005) or experienced multiple matings (Kant et al. 2012, Wang et al. 2018). Mating can affect females' longevity positively (Villarreal et al. 2018), or negatively (Yu et al. 2014), through the delivery of male-derived substances, e.g., male accessory gland and seminal vesicle proteins, via ejaculates. The mating history of females can also affect several traits of their offspring, including the sex ratio (Kant et al. 2012), survival rate (Ivy and Sakaluk 2005), body size, and dispersal ability (Gottlieb et al. 2014). In short, life parameters of mated females and their progeny could be indicative of the nature of mating experienced by the females, and thus should be included in analyses of associations between male mating performance and female density.

The solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is native to North America, and is an invasive alien pest in Asia and beyond (Wang et al. 2010). It can rapidly establish and develop population in newly invaded regions, and is continually extending its geographic range because of its adaptive life features, including its broad host range, high fecundity, protective benefits from mutualistic ants, and its ability to adapt to diverse abiotic environmental conditions (reviewed by Tong et al. 2018). Both adults and crawlers damage plants by sucking cell sap from phloem tissue of leaves, stems, twigs, flower buds, and young bolls, causing premature leaf drop, dieback, and even the death of whole plant. Besides, honeydew excreted by these insects serves as a medium for the growth of sooty mold that hinders the photosynthetic ability of plants (Saeed et al. 2007). In some Asian countries, particularly India and Pakistan, cotton mealybug has caused serious economic losses to cotton production (Hodgson et al. 2008, Nagrare et al. 2009). In China, this mealybug was ranked as one of the 21 most important invasive insects to be managed with priority (Ministry of Agriculture 2013).

Like other mealybug species, P. solenopsis exhibits typical sexual dimorphism. Females have three instars during the nymphal stage, and their adults are similar to nymphs morphologically but their body size increases by several times during feeding. Males proceed to the pupal stage after the second nymphal instar. Adult males have wings and a small body size, and do not feed (Zhu et al. 2011). Strikingly, female adults are sedentary, highly aggregative, and have a long lifespan (0.5–2 mo, depending on hosts and temperatures) (Prasad et al. 2012, Zhao et al. 2016), while male adults survive for only 2–4 d (Zhu et al. 2011). In the past decade, the mating and reproductive features of P. solenopsis have attracted much research interest, including its mating frequency (Wei et al. 2010), mating duration (Wei et al. 2010, Zhang et al. 2015), mating preferences (Xu et al. 2016), fecundity (Wang et al. 2014), and associations between mating and females' ovarian development and oviposition (Huang et al. 2013, Zhang et al. 2015, Arif et al. 2016). Wei et al. (2010) reported that males of cotton mealybug mated only once during their lifetime, but an average of 4.5 matings was reported by Wang et al. (2014). The mating history of males had no effect on the production of progeny (Huang et al. 2013).

Despite the studies mentioned earlier, little is known about the male mating responses to female density in P. solenopsis. In natural populations, female adults are expected to have a higher density than male adults during certain seasons, because females live much longer than males. Thus, each living male would have the chance to mate with several females, as observed by Wang et al. (2014). Considering that the short-living males might shift their mating performances to achieve the largest reproductive output, it is of great value to investigate their responses to female density, including mating frequency, mating interval, and mating duration.

Here, using USB digital microscopes, we observed the mating frequency, mating duration, and mating interval of P. solenopsis males that were supplied with different numbers of females (1, 5, 10, and 15 females per male). We also recorded the preoviposition period, merged ovipositional and postovipositional period, and fecundity of mated females in each group, and the sex ratio of their offsprings. The relationships between males' longevity and their mating frequency, and between females' longevity and their reproductivity, were analyzed. Our aims were to clarify the male mating responses to female density and their reproductive significance for this mealybug, and to improve our understanding of the importance of reproductive biology for population development in insects, particularly mealybugs.

Materials and Methods

Results

Discussion

Here, using USB microscopes, we recorded for the first time the detailed mating responses of P. solenopsis males to female density. As the female density increased, males increased their mating frequency and shortened their mating intervals; however, the mating duration did not change significantly. Surprisingly, female density had little effect on postmating reproductive performance. As we predicted, the longevity of males decreased as their mating frequency increased, but the longevity of females was not related to their fecundity.

Table 1.

Mating performance of P. solenopsis males in different female density treatments

Close relationships between female density and male mating performance have been observed in a few insect species, as described previously. In mealybugs, such as P. citri, P. calceolariae (Da Silva et al. 2013), and P. solenopsis (this study), each male is able to mate with several females. Some P. solenopsis males mated 6–9 times during their lifetime (Table 1, Fig. 1). This finding, together with the observed short mating intervals (Table 1), suggests that this mealybug has a high mating capacity. Such a feature may be a factor in its adaptive nature. First, because P. solenopsis females are highly aggregative (Huang et al. 2012), males will frequently find females at a high density and successfully mate with a number of females. Second, because males are short lived (Vennila et al. 2010, Huang et al. 2012) and lack a strong flight capacity (Wei et al. 2010), a high mating capacity would allow them to mate frequently and rapidly before death, thereby maximizing their reproductive output and promoting population development. Thus, these mating features can help this mealybug overcome the shortcomings of its life history, e.g., the short lifespan and weak flight capacity of males, the sedentary lifestyle, and the lack of wings in females.

Another important finding was that P. solenopsis female density did not significantly affect male mating duration or female reproductive performance (Tables 1 and 2). Moreover, males' mating experience appeared to have little effect on mating duration (otherwise the value would vary more widely than that observed among the density treatments where males mated multiple times). This result differs from those reported for other insects (Liu et al. 2010, Omkar and Singh 2010, Omkar et al. 2013, Wang et al. 2016, Wu et al. 2018). Thus, we argue that P. solenopsis males might have evolved a sperm investment strategy that meets females' reproductive requirements: once meeting a suitable female, they would transfer a ‘definite’ amount of sperms to the female, and the females, once mated, would produce a ‘definite’ amount of offspring, irrespective of their density in the population. This strategy may decrease the risk of sperm loss caused by the death of mated female, and contribute to the population development during their invasion. Further work is required to learn whether the sperm investment strategy of P. solenopsis males will be affected by their age.

Fig. 1.

Linear regression (fitting line) between P. solenopsis males' longevity and their mating frequency. Each circle represents one male.

We found a medium level of mate choice in P. solenopsis, as observed in the one-female treatment where approximately half (52.8%) of tested males mated. This mating rate was similar to that reported for P. citri, where 50% of males mated with single paired females (Da Silva et al. 2013). In P. calceolariae, however, a higher mating rate, 88%, was detected for males in pair settings (Da Silva et al. 2013). Overall, mealybugs do not have a high mate choice, that is, P. solenopsis males tend to mate with females once they meet rather than waiting for the most suitable ones during their short lifespan. In terms of biological invasion, this is much favorable for establishment in newly invaded regions, where both female and male mates are normally scarce.

Most females mated only once in all treatments; however, there were one and two females that mated twice in the 5- and 15-fe-male treatments, respectively. Thus, females could mate multiple times in their lifetime, especially when sufficient males are available. The same result has been reported previously for cotton mealybug (Wei et al. 2010), and for a number of other mealybug species, such as the long-tailed mealybug Pseudococcus longispinus (Targioni Tozzetti) (Hemiptera: Pseudococcidae), obscure mealybug Pseudococcus viburni (Signoret) (Hemiptera: Pseudococcidae), vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) (Waterworth et al. 2011), and the grape mealybug Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae) (Waterworth and Millar 2012). Multiply mating (with same and/or different males), by contrast to single mating, would favor P. solenopsis females to enhance their reproductive success. Further research is needed to investigate the mating and reproductive outputs of P. solenopsis females maintained at different densities while supplied continuously with male(s).

In insects, there may be a trade-off between mating and survival, as their longevity tends to decrease as the mating frequency increases (Oliver and Cordero 2009, Metzler et al. 2016). This trade-off may not exist for some insects (Camacho-Garcia et al. 2018). It exists in P. solenopsis males, as revealed by the significantly negative relationship between males' mating frequency and their longevity (Fig. 1). Notably, the males that mated 0–3 times did not differ significantly in their lifespan (Fig. 1), suggesting that such a trade-off is not strong. This is consistent with our speculation that males of cotton mealybug have a high mating capacity. To gain such a capacity, males (which do not feed during their lifetime, and have a low flight capacity) probably allocate a large proportion of energy to reproduction and little energy to flight or other behaviors.

Table 2.

Reproductive performance of mated P. solenopsis females and sex ratios of their offspring in different female density treatments

Fig. 2.

Linear regression (fitting line) between P. solenopsis females' longevity and their fecundity (number of offspring). Each circle represents one reproductive female.

Insect lifespan and reproduction are strongly associated with nutrition, in that the ratio and amount of nutrients consumed can affect life expectancy and reproductive investment (Malod et al. 2017). However, such a nutrient-related effect does not exist in P. solenopsis females (Fig. 2). This could be partially explained by the fact that P. solenopsis females do not need to spend too much energy on flight, movement, defense against natural enemies, or other nonreproductive performances, because they are wingless, sedentary, covered with wax (Zhu et al. 2011), and have a mutualistic relationship with ants (Zhou et al. 2014). Therefore, most energy could be allocated to reproduction and the maintenance of the somatic body.

In conclusion, we discovered a number of mating and postmating reproductive performances in P. solenopsis with potential adaptative significance during its life history. This is one of the few studies on cotton mealybugs that has addressed mating capacity, reproductive outcome, trade-offs, and their associations with female density. Our findings help to explain why P. solenopsis can establish and increase its population size so rapidly after arriving at new regions (Huang et al. 2011). The results may also be useful for further research on reproductive biology in other short-lived insect species.