Introduction
Multiple mating is a pervasive behaviour observed across the majority of polyandrous insect species, including ladybirds, that derive significant benefits (Arnqvist and Nilsson, Reference Arnqvist and Nilsson2000; Pervez and Maurice, Reference Pervez and Maurice2011). For instance, this behaviour typically enhances overall fitness, boosts reproductive success and increases the genetic diversity of offspring (Arnqvist and Nilsson, Reference Arnqvist and Nilsson2000). In females that receive nuptial gifts, multiple mating leads to higher average egg production, offspring yield and fertility (Wedell et al., Reference Wedell, Gage and Parker2002; Monalisa et al., Reference Monalisa, Pervez and Jahan2020). Conversely, in species lacking nuptial feeding, multiple mating generally results in no benefits for reproduction (Sadek, Reference Sadek2001; Osawa, Reference Osawa2005).
There are at least two causes of multiple matings involving sperm replacement. One is when females seek genetic benefits, such as replacing the sperm of a previous mate with that of another male to promote sperm competition, ensuring fertilisation by high-quality sperm, enhancing genetic diversity or avoiding genetic incompatibility (Thornhill and Alcock, Reference Thornhill and Alcock1983; Arnqvist and Nilsson, Reference Arnqvist and Nilsson2000; Simmons, Reference Simmons2001). The other cause is sperm depletion, where females may not obtain sufficient sperm to fertilise their full egg complement during their reproductive period from a single mating, or the sperm they receive may degrade during long-term storage (Thornhill and Alcock, Reference Thornhill and Alcock1983; Arnqvist and Nilsson, Reference Arnqvist and Nilsson2000). In the first case, sperm replacement occurs as a result of females seeking new mates to increase the genetic diversity of their offspring. Females deliberately discard previously acquired sperm to obtain a fresh supply from subsequent matings with other males, thereby continuing to fertilise their eggs (Sivinski, Reference Sivinski1980; Simmons, Reference Simmons2001, Reference Simmons2005). In the second scenario, all stored sperm in the spermatheca is depleted, necessitating replenishment for continued egg fertilisation. In both cases, the control over the use of stored sperm is exclusively managed by the female (Arnqvist and Nilsson, Reference Arnqvist and Nilsson2000; Xu and Wang, Reference Xu and Wang2011).
Studies on sperm depletion are primarily based on indirect observations, such as the proportion of fertilised eggs laid by females out of the total eggs produced. This methodology is widely used and broadly accepted across various studies (references in Thornhill and Alcock, Reference Thornhill and Alcock1983; Sakurai, Reference Sakurai1998; Awad et al., Reference Awad, Kalushkov, Nedvedovaá and Nedved2013). However, since females may choose to receive but not utilise sperm from males, changes and fluctuations in fertility do not necessarily indicate sperm depletion. Research on sperm depletion should specifically consider the initial quantity of sperm transferred during copulation, as well as its persistence in the spermatheca over time (Parker et al., Reference Parker, Simmons and Kirk1990), to ensure that the observed decrease in fertility is due to the reduction of sperm present in the spermatheca, thereby characterizing sperm depletion. On the other hand, if sperm are present in the spermatheca despite a decrease in fertility, it is reasonable to assume that the females chose not to utilise the received sperm. Thus, the hypothesis that the reduction in fertility results from sperm depletion can be dismissed.
The ladybird Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) exhibits a high, almost daily, frequency of copulation (Kaufmann, Reference Kaufmann1996; Jayanthi et al., Reference Jayanthi, Sangeetha and Verghese2013). In experiments with C. montrouzieri, it was observed that females achieved higher fecundity and fertility with continuous access to copulation, whereas females that copulated only once exhibited irregular fecundity over 20 days (De Lima et al., Reference De Lima, Nóbrega, Ferraz and Pontes2022; Felix et al., Reference Felix, De Lima, De Lima and Pontes2022). We hypothesise that this relationship between fertility and copulation frequency may be related to the need for sperm replenishment. To test this hypothesis, we conducted fertility assays in conjunction with sperm counting during copulation and sperm presence in the spermatheca, in three regimes of sperm replacement opportunities.
Material and methods
Individuals used in the experiments were obtained from an existing colony of C. montrouzieri at the Applied Entomology Laboratory (LEA) of the Federal University of Pernambuco, Recife, Brazil. The experimental conditions and insect rearing were conducted in an environment with controlled temperature and humidity (temperature of 25 ± 5°C, relative humidity of 70 ± 5%, with a photoperiod of 12:12).
Pupae of C. montrouzieri were individually separated in Petri dishes (60 × 15 mm) with a perforated lid covered with fine mesh fabric to allow air circulation. After emergence, males and females were sexed based on the dimorphism of the first pair of legs (Pang and Gordon, Reference Pang and Gordon1986). Adults were used in the experiments only after reaching 5 days of age, to ensure that females had achieved ovarian maturation (Santos et al., Reference Santos, Rodrigues-Pedrosa and Pontes2023). Males and females were provided with females of Planococcus citri (Hemiptera: Pseudococcidae) and cotton soaked in water.
To investigate the relationship between mating frequency and fecundity and fertility, females were subjected to three mating scenarios: (i) no replenishment (n = 20); (ii) periodic replenishment (n = 20); and (iii) constant replenishment (n = 20). In the first treatment, each female, still virgin, was placed in a 10 ml acrylic tube with a virgin male for mating. Immediately after the end of copula, each female was isolated in a Petri dish (60 × 15 mm). Copulations lasting more than 16 min were considered successful (De Lima et al., Reference De Lima, Nóbrega, Ferraz and Pontes2022). Pairs with shorter copula duration were discarded and the process was repeated with new virgin individuals. In the second treatment, each female, still virgin, was mated as described above, but repeating this process every 10 days. In the third treatment, a male and a female, both virgins, were paired in a Petri dish. In that scenario, males were replaced with virgin ones in case of death or if paired females did not oviposit for at least 10 days. In all treatments, the food provided consisted of five P. citri females daily, per adult. Individuals from the three treatments were transferred to new Petri dishes daily, for egg counting and fertility monitoring. Eggs that did not hatch after 7 days were considered infertile. The experiment lasted for 30 days.
To test for sperm depletion over 30 days, it was first necessary to determine the average number of spermatozoa transferred by virgin males during a single copulation. For this purpose, females were dissected 1 h after copulation (n = 10) to count the spermatozoa present in the spermathecae. Subsequently, another group of females was mated (n = 30), isolated and fed as described above. Some of these females had their spermatheca dissected after 10 days (n = 10), another portion after 20 days (n = 10) and the last portion after 30 days (n = 10), and the number of spermatozoa was counted. The duration of all copulations was recorded.
The females were dissected in 0.9% saline solution using entomological pins and scissors. Spermathecae were individually macerated in 50 μl of saline solution directly onto microscope slides. After complete drying at room temperature, the slides were immersed in 10% formalin for 30 s. After drying, they were immersed in Giemsa stain for 30 s, rinsed and allowed to dry. The spermatozoa were counted using a light microscope (Leica DM500 with ICC50W camera) at 100× magnification (10× eyepiece and 10× objective). To minimise counting bias, the zigzag method was used, where the observer moved the slide in a zigzag pattern across the entire area of the slide, avoiding excessive or insufficient counting of spermatozoa.
Data normality was tested with the Shapiro–Wilk test and homogeneity with Levene's test. To test for differences between treatments (constant replenishment, periodic replenishment and no replenishment) in total fecundity values, a generalised linear model (GLM) was performed with R-E-G-W post hoc. For total fertility values, a Kruskal–Wallis test for independent k-samples was performed with Dunn's (all pairwise) post hoc. The daily oviposition pattern in each treatment was tested with the Friedman test (non-parametric equivalent to repeated measures ANOVA), and the difference between treatments over time was tested with GLM and R-E-G-W post hoc. The relationship between the number of spermatozoa and the variables copulation duration and post-copulation time was tested using Kendall's non-parametric bivariate correlation. Data were analysed using SPSS Statistics 20. Graphs were generated using Microsoft Excel and SPSS Statistics 20.
Results
There was high variability in the number of spermatozoa allocated in the spermatheca immediately after copulation, ranging from 34 to 6000, with an average of 876 ± 174.56 (figs 1 and 2). The duration of copulation varied from 20 to 29 min; however, there was no correlation between copulation duration and the number of spermatozoa transferred (fig. 2) (P = 0.782). The regression analysis resulted in low R 2 values (0.08–0.5) and showed no linear trends between these variables; actually, was best depicted by second-order polynomial equations. However, the P-values were not significant (fig. 2).
Figure 1. Comparison between fertility rate and sperm depletion in four moments after copula: 1 h, 10 days, 20 days and 30 days.
Figure 2. Correlation between copulation duration and number of sperm in the spermatheca, at four post-copulation moments.
The number of spermatozoa found in the spermatheca 1 h after copulation did not differ significantly from the number of spermatozoa found in the spermatheca of females dissected after 10, 20 and 30 days (P = 0.07) (fig. 2). In 12.5% of the dissected females, the spermathecae showed no spermatozoa. In these cases, the females laid few eggs and all were infertile.
Females with no sperm replenishment had higher egg fertility than females with constant replenishment (χ2 (2) = 8.798; P < 0.05). However, there was no significant difference in fertility between the constant and periodic replenishment treatments (P = 0.232) and between periodic and no replenishment (P = 0.712) (figs 3 and 4). Nevertheless, females with access to constant sperm replenishment exhibited higher oviposition rates compared to those in other treatments (F (2) = 8.169, P < 0.005), with no difference in the number of eggs laid by females with periodic and no replenishment (P = 0.07) (figs 3 and 5). The only treatment in which female mortality was recorded was constant replenishment.
Figure 3. Fecundity and fertility daily rates (20 females mean) in the treatments of constant, periodic and no replenishment. In the periodic treatment, the points mean copula occurrence.
Figure 4. Fertility rate in the different sperm replacement regimes. Different letters indicate significant differences (P ≤ 0.05).
Figure 5. Fecundity rate in the different sperm replacement regimes. Different letters indicate significant differences (P ≤ 0.05).
The daily oviposition pattern shows that there were differences depending on the treatments (χ2 (29) = 187.46; P < 0.001). Females with constant replenishment oviposited more in the first few days (even though without significant difference), reaching an average of 97 ± 10.56 eggs. From days 11 to 20, oviposition increased by 50%, remaining constant until the end of the experiment. Females with periodic replenishment distributed oviposition over time, reaching an average of 74 ± 10.5 eggs in the first 10 days, followed by a 25% increase that remained stable until day 30. Without replenishment, the initial rate was similar to that of females with periodic replenishment, with 77 ± 7.87 eggs, but then there was a decline in the number of eggs until the end of the experiment (table 1). Additionally, there was an effect of days on the oviposition pattern in constant replenishment (χ2 (29) = 173.093; P < 0.001), periodic replenishment (χ2 (29) = 64.675; P < 0.001) and no replenishment (χ2 (2) = 88.586; P < 0.001), with significant differences between the first 5 days and the rest of the experiment.
Table 1. Effect of three sperm replenishment regimes on the fecundity of C. montrouzieri
Values are means ± SE. F-values are significant at P < 0.05. Different letters in the columns denote that means are significantly different in the post-hoc.
Discussion
No evidence was found to support the hypothesis that C. montrouzieri engages in multiple copulations due to sperm depletion. A single complete copulation is sufficient to maintain oviposition and fertility for at least 30 days in this specie, as also seen in Propylea dissecta (Mulsant) (Coleoptera: Coccinellidae) (Pervez et al., Reference Pervez, Omkar and Richmond2004). Although multiple copulations occur in several species of Coccinellidae (Yadav and Pervez, Reference Yadav and Pervez2022), it has been hypothesised that in C. montrouzieri, they occur due to sperm replenishment (Kaufmann, Reference Kaufmann1996). However, the present experiment clearly demonstrated that replenishment due to sperm depletion does not occur in this species.
Males of C. montrouzieri exhibit high variability in the number of spermatozoa transferred in the ejaculate, and the duration of a complete copulation is not related to the amount received and stored. Both characteristics have been observed in other ladybirds (Katakura, Reference Katakura1986; Yadav et al., Reference Yadav, Mishra and Omka2023). It is known that, in Drosophila Fallen (Diptera: Drosophilidae), the female can monitor the sperm supply received, possibly through receptors in the spermatheca (Thornhill and Alcock, Reference Thornhill and Alcock1983). Thus, the high individual variability in the number of spermatozoa transferred by each male may be a cryptic mechanism by which the female assesses the male's quality.
Females of C. montrouzieri can store viable sperm for at least a month after copulation. There are nutritive secretions produced by the female herself to maintain sperm viability in the spermatheca (Gillott, Reference Gillott, Adiyodi and Adiyodi1988). In Hippodamia undecimnotata (Schneider) and Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), sperm can be stored for more than 3 months after copulation (Awad et al., Reference Awad, Kalushkov, Nedvedovaá and Nedved2013; Susset et al., Reference Susset, Hemptinne, Danchin and Magro2018). This demonstrates that females do not use their sperm reserves at the same rate as copulations occur and, therefore, reinforces that sperm replenishment is not necessary.
Cryptolaemus montrouzieri appears to fit different daily oviposition models depending on the frequency of copulations. When females have constant copulation opportunities, fecundity is higher than in females with a single copulation. Interestingly, the fertility of females with a single copulation is higher than that of females with constant access to males. Males may invest nutrients and substances through their ejaculates to increase female oviposition, expecting that females will use their sperm to fertilise them, thereby increasing their paternity in the offspring (Smith, Reference Smith1979; Wedell, Reference Wedell2006). This is observed in various species of ladybirds, where the number of copulations tends to be related to increased fecundity and fertility (Haddrill et al., Reference Haddrill, Shuker, Amos, Majerus and Mayes2008; Hodek et al., Reference Hodek, van Emden and Honek2012; Yadav and Pervez, Reference Yadav and Pervez2022). However, this does not seem to occur in C. montrouzieri, as it does not in Leptiontarsa decemlineata (Say) (Coleoptera: Chrysomelidae) (Orsetti and Rutowski, Reference Orsetti and Rutowski2003).
The increase in fecundity of C. montrouzieri females paired with males may be explained by the mechanical stimulation of copulation (Lange and Loughton, Reference Lange and Loughton1985) or by the transfer of stimulating seminal fluids that trigger egg production (Shahid et al., Reference Shahid, Siddiqui, Omkar and Mishra2016). The stimulants passed during copulation do not have a long-lasting effect, as C. montrouzieri females that copulated only once showed a reduction in fecundity after 20 days. Thus, it is likely that multiple copulations induce higher fecundity through the transfer of oviposition-stimulating substances rather than spermatozoa themselves (Xu and Wang, Reference Xu and Wang2011; Worthington and Kelly, Reference Worthington and Kelly2016).
The reduction in fertility of C. montrouzieri females paired with males, without a corresponding decrease in fecundity, suggests that the presence of the male stimulates females to produce more eggs but not necessarily to increase fertility. In insects in general, females often control the fertilisation of their eggs (Simmons, Reference Simmons2001). A plausible hypothesis would be that paired females choose to fertilise fewer eggs from a single male but are unable to control the effects of the oviposition stimulus induced by the male's presence. It has been suggested that C. montrouzieri engages in multiple copulations to increase the genetic diversity of the offspring (Kaufmann, Reference Kaufmann1996; Kairo et al., Reference Kairo, Paraiso, Gautam and Peterkin2013). Following this reasoning, females confined with a single male would have less incentive to fertilise more eggs from the same partner, as observed in Gryllus bimaculatus De Geer (Orthoptera: Gryllidae) (Tregenza and Wedell, Reference Tregenza and Wedell1998). However, this hypothesis has not been experimentally tested.
In some way, the constant presence of the male seems to affect female fertility. In C. montrouzieri, when pairs remained together, the average fertility was 51%, close to the value observed by other authors (De Lima et al., Reference De Lima, Nóbrega, Ferraz and Pontes2022). When females were kept isolated and only met with males at the time of copulation, fertility rose to over 80% (Xie et al., Reference Xie, Zhang, Wu, Liu, Deng and Pang2014), which was also observed in the present study and in other Coccinellidae species (Omkar and Mishra, Reference Omkar and Mishra2005; Awad et al., Reference Awad, Piálek, Krejčí, Laugier and Nedved2017). Several studies suggest a reproductive cost due to the presence of the male, where females that mate frequently show a decrease in fertility and longevity (Nielson and Toles, Reference Nielson and Toles1968; Pyle and Gromko, Reference Pyle and Gromko1981; Mishra and Omkar, Reference Mishra and Omkar2006; Omkar and Sahu, Reference Omkar and Sahu2012).
Cryptolaemus montrouzieri females constantly accompanied by a male may suffer significant energetic wear, as this was the only treatment that presented female mortality. Even if the female does not need material or is not receptive due to copulation itself, the male continues to attempt copulation, leading to a cycle of harassment and females being overcome by exhaustion. In Gryllus texensis Cade and Otte (Orthoptera: Gryllidae), females paired with males show a significant increase in injury and death (Worthington and Kelly, Reference Worthington and Kelly2016). In the ladybird Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae), female mortality was only observed in paired couples (Bayoumy and Michaud, Reference Bayoumy and Michaud2014). It is likely that this scenario results from two energetically costly activities: constant escape and/or elevated oviposition. The male has little to gain by giving up on an uncooperative female, as his only option would be to seek another partner who would be equally reluctant. Thus, it may be advantageous for him to persist for a long time with any female he can find (Thornhill and Alcock, Reference Thornhill and Alcock1983). This behaviour also alludes to the hypothesis of copulation for convenience (Parker, Reference Parker and Smith1984).
We observed that 12.5% of females with a single copulation exhibited low oviposition and no fertile eggs. When dissected, 20 and 30 days post-copulation, they had no sperm stored in their spermathecae. However, all females dissected 1 h after copulation had some amount of sperm stored, indicating a higher probability that sperm discard by the female may have occurred, as observed in other beetles (Rodriguez, Reference Rodriguez1995, Reference Rodriguez1999; Fedina, Reference Fedina2007). Most failures in copulation result from the non-transfer of a spermatophore (Tyler and Tregenza, Reference Tyler and Tregenza2012) or the transfer of only an empty sperm sac, which is not related to copulation duration (Bloch Qazi et al., Reference Bloch Qazi, Herbeck and Lewis1996). Beyond the inability to transfer, the absence of sperm in dissected females may also indicate that the male passed few spermatozoa, as one of the females, despite showing good fertility for 10 days, no longer had spermatozoa in the spermatheca.
In summary, the hypothesis of sperm depletion does not apply to C. montrouzieri, as a single copulation maintains fertility for at least 30 days. The constant presence of the male reduces female fertility, indicating a reproductive cost of frequent mating. Although multiple copulations increase fecundity, this appears to be due to the transfer of stimulating substances and does not reflect greater fertility. Our results suggest that periodic copulations in C. montrouzieri represent an optimal female mating rate (average fecundity and average fertility) and that a regime of constant copulations, with males and females housed together, may be higher than optimal and therefore result in lower fertility and higher female mortality.
These data may be useful in the management and rearing of C. montrouzieri, aiming to optimise mass production and application of this species in pest control. In laboratory and mass-rearing setups of C. montrouzieri, adult individuals are generally kept in cages containing ample quantities of mealybugs for feeding and oviposition, where males and females are housed together (Sanches and Carvalho, Reference Sanches and Carvalho2010; Júnior et al., Reference Junior, Silva, Santos, Santos and de Francisco2019; Gunawardana and Hemachandra, Reference Gunawardana and Hemachandra2020). Under these conditions, an average of 337 eggs per female per month has been recorded, with a viability rate of 62%, resulting in approximately 208 adults (Sanches and Carvalho, Reference Sanches and Carvalho2010).
In our experiments, evaluating individually paired adults, we observed an average of 363 eggs per female per month, with a viability rate of 52%. However, there was a 40% mortality rate among females, necessitating replacements. On the other hand, our data showed that in females exposed to males for periodic copulation only, the viability rate increased to 59%, with no recorded mortality. In our laboratory rearing setup, males and females are reared as described in the literature, and we also observed high female mortality, similar to our observations in the constant replacement treatment. After a few weeks, the initial 1:1 sex ratio became skewed towards males, with ratios reaching as high as 5:1 (Personal communication, unpublished data).
Thus, while continuous copulations might appear to be the best option at first glance, the absence of early female mortality until at least the end of the second generation (in the periodic copulation scenario) would allow all initial females to contribute their maximum reproductive potential. Cages with high male density can almost completely suppress oviposition (Bayoumy and Michaud, Reference Bayoumy and Michaud2014). In a broader context, such as the mass-rearing of C. montrouzieri, we suggest a simple strategy to reduce the cost associated with the constant presence of males, which consists in sexing the colony periodically to discard the excess males and restore the 1:1 sex ratio. Subsequently, the females would remain isolated for a period to ensure optimal oviposition, before being reintroduced to the males. This minor adjustment could result in an improvement in the productivity of a rearing programme.
Acknowledgements
We thank the members of the Laboratório de Entomologia Aplicada (LEA-UFPE), especially Andrea Sena for help in developing the methodology and experiment. Our gratitude extends to our collaborators from Laboratório de Porifera (LabPor-UFPE) (Dr Ulisses Pinheiro, Thales Almeida and Alan Dias) and from Aggeu Magalhães Institute – FIOCRUZ-PE (Dr Rosângela Barbosa and Tainá Santos) for the partnership in the microscopic analysis. We also thank Programa de Pós-Graduação em Biologia Animal of the Universidade Federal de Pernambuco (PPGBA/UFPE). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 and under grant 88887.702876/2022-00.
Competing interests
None.
