Social interactions throughout life impair longevity and age-specific mating success in male parasitoids

Abstract

Social interactions among individuals within a species profoundly influence behavioural and life history traits, impacting fitness. While extensively studied in cooperative and eusocial species, the effects of social environment on fitness in non-social species, particularly insects, remain less explored. Our study investigates the impact of social environment, specifically male density, on fitness-related traits in the hymenopteran parasitoid Venturia canescens. The research focuses on longevity, reproductive behaviours (latency before mating, mating probability and duration), and offspring production capacity. Through a lifespan study, males were exposed to either isolation or regular encounters with conspecific males, alternating with periods of female presence or absence. Results show a trend of reduced longevity in socially exposed males and a significant decline in mating success with age in the social context. However, reproductive behaviours and offspring produced by males remain unaffected. This study sheds light on the intricate interplay between social environment, ageing, and reproductive strategies in non-social insect species, emphasizing the need for further exploration of social effects on male behaviour and notably potential influences of interactions between male and females but also between females.

Significance Statement

Impacts of social interactions on individuals were largely explored in social species, but remain little studied in non-social ones. This study aims at testing the impact of male-male interactions before the encounter of a female (past social interactions) on male reproductive behaviours and longevity in a non-social parasitoid wasp. We show that longevity is negatively affected by the past social interactions in this wasp. Those interactions also impose a decline in male mating probability with age (senescence pattern) or mating experience (number of matings). Latency before mating, mating duration and offspring production are not influenced by social environment. On the other hand, offspring production increases with mating experience.

Appendices

Appendix 1

Global diagram of the experimental protocol

Fig. 6

Global diagram of the experimental protocol (A) and response variables measured (B). Focal males encountered one virgin female on days 1, 4, 6, 8, 11 and 13. Focal males from Social condition encountered 3 non-focal males on days 2, 3, 5, 7, 9, 10 and 12. After Day 13 (last possible male–female encounter), males were kept alone with food until they died: then, their longevity was calculated as the median between the last time they were seen alive and the first time they were seen dead. The eye represents the moments where individuals were observed and behaviours (mating characteristics) were measured for each male–female encounter. Offspring production from a mating event of a male was measured by counting the number of sons and daughters laid by the mated female and calculating the sex-ratio

Appendix 2

Model selection for the effect of male age and social environment on mating latency

In Table 2, we presented values of AICc for each possible model aiming to identify the effects of male age and social environment (and their interaction) on mating latency. In bold is indicated the best model (i.e. the one with the lower AICc).

Table 2 AICc of models describing the effect of male age, social environment and their interaction on mating latency

Appendix 3

Model selection for the effect of male age and social environment on mating duration

In Table 3, we presented values of AICc for each possible model aiming to identify the potential effect of male age and social environment (and their interaction) on mating duration. In bold is indicated the best model (i.e. the one with the lower AICc)

Table 3 AICc of models describing the effect of male age, social environment and their interaction on mating latency

Appendix 4

Male mating experience and ejaculate size

A complementary experiment was designed to test whether males' multiple mating experiences influenced the size of their ejaculates, estimated by the number of spermatozoa stored in the spermatheca, and therefore the number of spermatozoa that females could use to produce daughters. We predicted that the size of the ejaculate is smaller in males that have already experienced several matings (referred hereafter as “Experienced” males) than in males mating for the first time (“Inexperienced” males)

At emergence, males were randomly assigned to the “Experienced” or “Inexperienced” population cages, the former containing both males and females while the later containing only males (all individuals were randomly drawn from rearing boxes). Density within the cages varied slightly between treatments and replicates according to the number of males and females emerging each week. Individuals of both treatments were provided with food (honey) ad libitum.

At the end of the third day, females were removed from the “experienced” cages. On day 4 morning, food was removed from the cages, and 2 h later, as many one-day-old virgin females as males (already in the cages) were introduced in “Experienced” and “Inexperienced” cages, respectively. During 2 h, we observed and recorded matings. Each couple in copulation was gently withdrawn from the cage and kept in a tube. Then, mated females were dissected (from 2 to 24 h post mating) and spermatozoa transferred by males (i.e. stored in spermatheca) were counted with ImageJ software (Schneider et al. 2012) by experimenters who were blind to the experimental treatment (“Inexperienced” vs (“Experienced” groups). Thirty-seven and 32 matings occurred in the “Inexperienced” and “Experienced” treatments respectively, but due to technical problems during female dissections, 35 “Inexperienced” and 31 “Experienced” measurements only were included in the dataset. The size of mated males was measured. All details regarding the dissection procedure are provided below.

Detailed protocol for the dissection of spermatheca and counting of spermatozoa.

Females were dissected in one drop of PBS1X and spermatheca was isolated and transferred on another microscope glass slide with one drop of PBS1X/DAPI, DAPI diluted 1:1.000 (DAPI, Thermoscientific, reference 6224B). Spermatheca was crushed between the slide and a cover glass by thumb pressure on the cover glass. Cover glasses were sealed with varnish and stored at 4 °C. An image of each slide was taken with a confocal microscope (Fig. 7, Zeiss, LSM 800, magnification × 20).

Fig. 7

Spermatheca and spermatozoa coloured with DAPI between slide and cover glass, under confocal microscope. A—Image of the whole cover glass. Crushed spermatheca is indicated by the white circle. B—Cropped image corresponding to the red square in image A. Red arrows show examples of spermatozoa

Results—Effect of male mating experience on spermatozoa transfer

We first compared the number of spermatozoa counted by the two experimentalists with a Wilcoxon signed-rank test. As there was no difference (W = 458, p = 0.3), we retained the lowest value between the two experimentalists for further analysis (we draw qualitatively the same conclusions whatever the metric used: median sperm count or largest value). We also checked that there was no influence of the time since mating on spermatozoa number in the spermatheca (t = -0.06, df = 28, p = 0.95). The effect of male mating experience on the number of spermatozoa transferred was tested with a zero-inflated Poisson model, with Poisson distribution and the identity of the cage in which males were as random factor (glmmTMB packages Brooks et al. 2017). This allowed us to consider the differences in density between cage. As there were many zeros in the data which caused overdispersion, we used zero-inflated model in order to account for this.

We did not detect an effect of male mating experience, i.e. if the male could mate before the experiment or not, on the size of the ejaculate (Fig. 8). Indeed, male experience (“Experienced” and “Inexperienced”) did not influence the number of transferred spermatozoa in female spermatheca (χ² = 0.713, df = 1, p = 0.398).

Fig. 8

Effect of male mating experience on the number of transferred spermatozoa stored in the spermatheca. Each dot represents number of spermatozoa for one mating. Violin plots represent the probability density