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Keywords:

  • grand-offspring production;
  • maternal care;
  • mother–offspring conflict;
  • sibling rivalry;
  • sib mating

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Although inbreeding is commonly known to depress individual fitness, the severity of inbreeding depression varies considerably across species. Among the factors contributing to this variation, family interactions, life stage and sex of offspring have been proposed, but their joint influence on inbreeding depression remains poorly understood. Here, we demonstrate that these three factors jointly shape inbreeding depression in the European earwig, Forficula auricularia. Using a series of cross-breeding, split-clutch and brood size manipulation experiments conducted over two generations, we first showed that sib mating (leading to inbred offspring) did not influence the reproductive success of earwig parents. Second, the presence of tending mothers and the strength of sibling competition (i.e. brood size) did not influence the expression of inbreeding depression in the inbred offspring. By contrast, our results revealed that inbreeding dramatically depressed the reproductive success of inbred adult male offspring, but only had little effect on the reproductive success of inbred adult female offspring. Overall, this study demonstrates limited effects of family interactions on inbreeding depression in this species and emphasizes the importance of disentangling effects of sib mating early and late during development to better understand the evolution of mating systems and population dynamics.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Inbreeding is considered as one of the key parameters in the persistence of natural populations, as well as in the evolution of social systems, life history, morphology, physiology and behaviour (Keller & Waller, 2002; Frankham, 2008; Enders & Nunney, 2010). Inbreeding results from the mating of two related individuals such as brothers and sisters (i.e. sib mating). Sib mating is known to increase the degree of homozygosity in the offspring, which in turn can depress offspring fitness through the higher expression of deleterious recessive alleles or the induced reduction in heterozygote advantages (Charlesworth & Charlesworth, 1987; Roff, 2002). The negative impact of inbreeding on fitness-related traits, called inbreeding depression, has been shown to vary considerably across taxa, species and even populations (Crnokrak & Roff, 1999; Keller & Waller, 2002). For instance, some studies reported that sib mating dramatically reduces fitness-related traits such as the production and the survival of offspring (e.g. Keller, 1998; Slate et al., 2000; Saccheri et al., 2005; Vitikainen et al., 2011). Conversely, other studies found that inbreeding had limited effects on fitness-related traits (e.g. Kureck et al., 2012; Mehlis et al., 2012) or may even provide fitness benefits for the two mating partners (e.g. Waser et al., 1986; Lehmann & Perrin, 2003; Cohen & Dearborn, 2004; Neff, 2004; Kokko & Ots, 2006; Thünken et al., 2007; Tabadkani et al., 2012).

Across animal and plant species, multiple sources of environmental stress have been shown to exacerbate the negative effects of inbreeding, such as exposure to parasites or chemicals, food deprivation, competition and increase in the density of individuals in a population or a group (Armbruster & Reed, 2005; Konior et al., 2005; Fritzsche et al., 2006; Zajitschek et al., 2009; Fox & Reed, 2010; Michalczyk et al., 2010; Reed et al., 2012). The general importance of environmental stress on inbreeding depression recently received quantitative support from a meta-analysis based on 33 plant and animal species. In this study, Fox & Reed (2010) showed a positive and linear relationship between the severity of environmental stresses (listed above) and the lethal effects of inbreeding depression. In this analysis, environmental stresses explained 41% of the variance in the lethal effects of inbreeding depression measured across studies, which also indicates that other factors are (at least partly) responsible for the observed variation in the expression and magnitude of inbreeding depression (Keller & Waller, 2002; Armbruster & Reed, 2005; Waller et al., 2008).

Family life is a widespread phenomenon in nature that is often associated with competition between siblings over resources and parental care (Smiseth et al., 2012; Wong et al., 2013), two forms of social interactions that possibly shape the severity of inbreeding depression. Sibling rivalry is an important source of social stress that is known to negatively affect the development and/or the survival of offspring (reviewed in Roulin & Dreiss, 2012) and is typically intensified in large family groups and under limited food resources (reviewed in Mock & Parker, 1997). Conversely, the presence of parents is commonly associated with benefits to offspring, for instance through the provisioning of resources necessary for offspring development and survival (Smiseth et al., 2012). Hence, intense sibling rivalry is predicted to exacerbate inbreeding depression, and parental care expected to (at least partly) impede detrimental inbreeding depression (i.e. buffer against the poor quality of inbred offspring; Avilés & Bukowski, 2006). To date, the effects of sibling competition (during family life) on inbreeding depression remain largely untested, whereas mixed conclusion emerged from the few studies that examined the influence of maternal care on inbreeding depression. In the prairie voles, Microtus ochrogaster, inbreeding status of the tended offspring has been shown to not influence the level of parental care (Bixler & Tang-martinez, 2006). Conversely, the absence of inbreeding depression in offspring of the oldfield mice Peromyscus polionotus, the cichlid fish Pelvicachromis taeniatus and the subsocial spider Anelosimus jucundus has been suggested to result from the occurrence of parental care in these species (Margulis, 1997, 1998; Avilés & Bukowski, 2006; Thünken et al., 2007), although such a causal link remains to be tested.

Whether or not parents should provide more care towards inbred offspring (and thus counter inbreeding depression) may also depend on the cost of care. For instance under resource limitation, parents may weigh their own survival and/or future reproduction higher than the fitness of their current offspring and consequently compete with offspring for the limited resources, which may in turn exacerbates inbreeding depression. Mother–offspring competition over limited food resources has been recently described in the European earwigs, Forficula auricularia. When earwig families were reared under restricted food conditions, offspring suffered from maternal presence in terms of slower development and lower survival rates (Meunier & Kölliker, 2012b). Nevertheless, even under restricted food conditions, earwig offspring continue to actively aggregate with their mothers, possibly because other forms of care (e.g. protection against predators) continue to outweigh the cost of mother–offspring competition over food (Wong & Kölliker, 2012). Hence, under restricted food availability, the presence of mothers possibly becomes an environmental (i.e. nutritional) stressor that may exacerbate the effects of inbreeding depression in offspring.

The severity of inbreeding depression may also vary depending on the life stage of the inbred individuals (i.e. the progeny ensuing from sib mating). In populations with some inbreeding history, inbreeding depression is predicted to purge the highly deleterious recessive alleles, which typically affect early stages (such as embryonic development) and thus entail lower levels of inbreeding depression in early than late life stages (Husband & Schemske, 1996). Conversely, in populations without inbreeding history, the maintenance of these deleterious recessive alleles may dramatically affect the early life stages of individuals in case of inbreeding and thus result in higher levels of inbreeding depression in early than late life stages (Husband & Schemske, 1996). These predictions received support from a literature survey conducted in plants, where the predominantly outbreeding species showed greater inbreeding depression in early stages than the predominantly selfing species (Husband & Schemske, 1996). In a population of the subsocial spider, A. cf jucundus, where inbreeding is likely to occur, inbreeding depression was also detected in the late but not the early part of the life cycle (Avilés & Bukowski, 2006). Interestingly in this spider species, females provide care to their offspring only during early life stages, which could also explain the absence of inbreeding depression specifically at this stage (Avilés & Bukowski, 2006).

Finally, male and female offspring may exhibit different sensitivity to inbreeding depression. Inbreeding depression has been shown to specifically affect several fitness-related traits in males, such as fertilization success (Pray et al., 1994; Saccheri et al., 2005; Enders & Nunney, 2010), territory acquisition success (Potts et al., 1994; Meagher et al., 2000) or success in sperm competition (Konior et al., 2005; Fritzsche et al., 2006; Zajitschek et al., 2009; Michalczyk et al., 2010). Conversely, female-specific effects of inbreeding depression have been demonstrated in humans, where pedigree data from a Swiss mountain village population revealed that the most inbred mothers (but not fathers) had significantly fewer children (Postma et al., 2010). Although the sex-specific effect of inbreeding on individual reproductive success has been described in several species, it remains unclear to what extent mating with an unrelated but inbred partner alters the fitness of noninbred individuals. In particular, inbred males have been shown to either substantially reduce (Fox et al., 2011; Okada et al., 2011) or to not shape (Michalczyk et al., 2010) the fitness of the noninbred and unrelated females they mated with, whereas inbred females had limited effects on the fitness of the noninbred and unrelated males they mated with (Enders & Nunney, 2010).

The aim of this study was to disentangle whether inbreeding depression in the European earwig, Forficula auricularia, is shaped by mother–offspring and sibling competition over restricted food access, by life stages (early vs. late) and by the sex of adult offspring. In this promiscuous insect species, females tend their clutch of eggs and nymphs for several weeks and provide care in the forms of protection against natural enemies and food provisioning to nymphs (reviewed in Costa, 2006). Food restriction and large family groups (i.e. large clutches) are known to be major sources of stress that reduces the survival of earwig offspring and shape population density (Kölliker, 2007; Moerkens et al., 2009). The presence of mothers has been shown to enhance offspring survival under natural and good food conditions in the laboratory (Kölliker, 2007; Kölliker & Vancassel, 2007). Conversely, these benefits of maternal care are reduced under food restriction, possibly due to mother–offspring competition over the scarce food (Meunier & Kölliker, 2012b). Here, we used a series of experiments conducted under environmentally stressful conditions (limited food availability) to test whether (i) sib mating decreases the reproductive success of females due to the production of inbred offspring, (ii) sibling competition and/or mother–offspring competition over limited food resources exacerbate the effects of inbreeding depression in offspring, and finally whether (iii) inbreeding depression in the resulting inbred adult offspring is sex-specific. The results of these experiments will also reveal whether inbreeding depression is more (or less) severe in later than early life stages of individuals (i.e. juvenile development/survival and adult reproductive success) and to what extent mating with an inbred (but unrelated) male or female affects the fitness of the noninbred mating partner.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Origin of the tested individuals

The males and females used in the following experiments were the first laboratory-born generation from a field-sampled population of F. auricularia collected in May 2009 in Dolcedo, Italy. The sampling was made using 50 wood traps regularly spread over a 3000 m2 area to limit the risk of high relatedness among the collected individuals. The resulting field-sampled population was composed of 600 males and 600 females sampled as (virgin) fourth instar nymphs and maintained under standardized laboratory conditions for the rest of their life cycle. These standardized laboratory conditions are detailed in Meunier et al. (2012). Briefly, groups of 30 newly emerged males and 30 newly emerged females were maintained in large plastic containers for 5 months to allow mating (this species has a promiscuous mating system). They were kept at 60% humidity, at 14:10-h light–dark photoperiod and at a constant temperature of 20 °C (later called laboratory summer conditions) and provided ad libitum food [see food composition in Meunier et al. (2012)]. The resulting mated females were then isolated in Petri dishes (diameter 10 cm), in which they later produced their first clutch of eggs (approx. 50 eggs per female, see Meunier et al., 2012). From egg laying to egg hatching, females were maintained under complete darkness at 15 °C and 60% humidity (later called winter conditions), and no food was provided during this period (Kölliker, 2007). Eggs hatched approx. 24 days later (see Meunier et al., 2012). On day one after hatching of a clutch, the family (mothers and offspring) was transferred to a new Petri dish and received ad libitum food (see Meunier et al., 2012). The females were separated from their nymphs on day 14 after hatching, which corresponds to the approximate termination of food provisioning in F. auricularia (Wong & Kölliker, 2012). The mothers were isolated in new Petri dishes with ad libitum food until day 60 after hatching of their first clutch, during which 82% of them produced a second clutch (see Meunier et al., 2012). Simultaneously, 20 nymphs per clutch were setup in new large Petri dishes (diameter 14 cm) until they reached adulthood. The newly emerged adults were immediately separated by sex to avoid uncontrolled sib mating. These adult offspring were the individuals used in the present study.

Part 1: Effects of sib mating on parental reproductive success

Adult offspring produced by 46 females randomly chosen from the field-sampled population were used to test the effect of sib mating on offspring development and survival. Among them, 23 were experimentally mated to a genetically unrelated male (control), and 23 to one of their brothers (sib mating, Fig. 1a). These experimental matings were conducted by placing each virgin female with one virgin male (always different) in Petri dishes (diameter 10 cm) containing humid sand, a plastic shelter used as a nest and ad libitum food. After 4 weeks, males were removed from the Petri dishes and the females subsequently placed under laboratory winter conditions until egg hatching. At hatching, a random sample of 10 nymphs per clutch was weighed to the nearest 0.001 mg using a Mettler-Toledo MT5 Micro-balance (Mettler, Roche, Basel) to estimate the quality of newly produced nymphs. Then, the females and their clutches were transferred to new Petri dishes (according to the experimental design detailed in the Part 2; see also Fig. 1), in which they were kept under laboratory summer condition for 16 days. Females were then isolated in new Petri dishes to allow second clutch production.

image

Figure 1. Details of the experimental set-up allowing to (a) test the effects of sib mating of parental reproductive success, (b) disentangle the effects of offspring inbreeding status and social environment on nymphs development and survival rates and finally to (c) investigate the effects of adult inbreeding status on their reproductive success. *respectively.

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The influence of sib mating on the reproductive success of females was estimated in both first and second clutches using the four following measurements. First, we counted the number of eggs produced by each female 3 days after the first egg has been observed. In this species, all eggs are typically released within 1 day, but a very limited number of females may still produce a few eggs during the 2 days following the first egg release (J. Meunier & M. Kölliker, unpublished data). Second, we counted and weighed newly hatched nymphs the day after the first nymph has been observed. Egg hatching is particularly well synchronized in this species, where 97 ± 1% (mean ± SE, n = 46) of the newly produced nymphs emerge over the first day (Meunier & Kölliker, unpublished data). Third, we used the number of eggs produced and the number of hatched nymphs to calculate the hatching success of each clutch. Finally, we tested whether sib mating influenced the relative investment of females in their second clutches (compared with their first clutch), because this trait is known to vary naturally between females, to partly reflect the outcome of family conflicts and to be associated with other important life-history traits, such as the lifetime number of eggs produced or the level of food mothers provision to their offspring (Meunier & Kölliker, 2012a; Meunier et al., 2012). This relative investment was calculated by dividing the number of eggs produced in the second clutches by the lifetime number of produced eggs.

Part 2: Effects of inbreeding status on offspring development and survival

The effects of sib mating on the development and survival of offspring under high levels of environmental (social) stress were then tested using a split-clutch experimental design in which we manipulated both brood size and maternal presence (Fig. 1b). One day after hatching, 42 of the 46 above clutches (in the following referred to as ‘clutch of origin’) were divided into three experimental groups (notice that four clutches – three inbred and one noninbred – were excluded because they produced fewer than 40 nymphs): one group with a brood size of 10 nymphs (small group) and without mother, one group with a brood size of 20 nymphs (large group) without mother and one group with the mother and either 10 or 20 of its nymphs (Fig. 1b). This experimental design allowed us to generate a two-by-two complete block design with the presence/absence of mother and large/small brood size for both sib mating and control families. The 126 resulting experimental broods were maintained in small Petri dishes (diameter 6 cm) for the next 16 days. At that time, we counted the proportion of surviving nymphs that reached the second juvenile instar as a measure of developmental rate. All nymphs were subsequently transferred to new Petri dishes for the next 15 days, and then to large Petri dishes (diameter 14 cm) until they reach adulthood. The mothers were then isolated to allow second clutch production (see Part 1). Upon emergence as adults, we (i) determined the sex of all individuals, (ii) checked for the occurrence of developmental problems in terms of, for instance, anatomical ‘hermaphroditism’ (i.e. showing one female-like and one male-like forceps, a phenomenon sometimes also associated with mixed internal anatomy; Günther & Herter, 1974), wing cover abnormalities, mis-shaped forceps or antennae, (iii) counted the number of adults for each family for analyses of survival rates and (iv) separated brothers and sisters from each experimental group to prevent uncontrolled sib mating. The individual weight of adult male and female offspring was measured to the nearest 0.001 mg about 1 month after emergence and then averaged per family group. Because the weight of adults with developmental problems was generally much smaller than the one of adults from the same clutch with normal development, the former were excluded from the calculations of adult mean weights (note that their inclusion did not qualitatively change the results).

Because restricted food conditions are known to create a stressful environment that exacerbates the negative effects of both sibling competition and mother–offspring competition over food resources in earwigs (Meunier & Kölliker, 2012b), the experimental earwig broods received a limited amount of the food. Specifically, food was provided every 6 days, and any leftover food was removed 3 days after supply (Meunier & Kölliker, 2012b). The quantity of food was adjusted according to the age of the nymphs, with 60, 120 and 240 mg of food provided in the small, normal and large Petri dishes, respectively. Females had access to ad libitum food once isolated for second clutch production. From hatching until adulthood, clutches were kept under laboratory summer conditions.

Part 3: Sex-specific effects of adult inbreeding status on reproductive success

We finally used a simple cross-breeding design involving adults produced in the 126 previously detailed experimental first clutches to disentangle the effects of inbreeding on adult males and females on their reproductive success (Fig. 1c). To this end, we haphazardly sampled one male and/or one female per clutch of origin across the three types of experimental clutches. These adults were then paired in new Petri dishes to obtain the four possible mating combinations: inbred female + inbred male, inbred female + outbred male, outbred female + inbred male and outbred female + outbred male. Each pair contained individuals from different families to avoid a second generation of inbreeding, and each combination had the same proportion of individuals previously reared in each type of experimental clutch. The sample size of each combination varied due to the number of males and females available in each experimental clutch. Three inbred families provided two males in the experiment.

The experimental mating and the rearing conditions until egg hatching were similar to the ones described above. At hatching, females were transferred with their nymphs in new Petri dishes for 16 days under laboratory summer conditions. At day 16, the number of nymphs was counted and the nymphs were discarded. The females were isolated in new Petri dishes to quantify second clutch production. When the first clutch eggs did not hatch, females were isolated in new Petri dishes 36 days after egg laying (a duration that approx. corresponds to the mean 23 days between oviposition and hatching of eggs, and 16 days of post-hatching family life, see above and Meunier et al., 2012). The sex-specific effects of adult inbreeding status were then tested by quantifying (in both first and second clutches) the number of eggs produced, the hatching success, the number of nymphs and the relative investment in second clutches (see above) of the mated females. The mean weight of newly hatched nymphs was also measured in the first clutches.

Statistical analyses

We first compared the reproductive success of females that were mated to their brother to the one of females that were mated to an unrelated male using a multivariate analyses of variance (manova). In this analysis, the measured traits (i.e. egg numbers in 1st and 2nd clutches, hatching success in 1st and 2nd clutches, nymph number and weight in 1st and 2nd clutches, and finally relative investment in second clutches) were entered as dependent variables and the mating type (sib mating vs. control) as fixed factor. Because the likelihood of second clutch production and the lifetime production of eggs and nymphs were tightly correlated with the number of eggs and nymphs produced in each or both clutches, these three measurements were not entered in the manova but analysed separately (see 'Results'). To confirm that traits of the second clutches were not shaped by the fact that females experimentally tended either 10 or 20 of their first clutch nymphs, we conducted another manova where the traits of the second clutch (i.e. egg numbers in 2nd clutches, hatching success in 2nd clutches, nymph number and weight in 2nd clutches and relative investment in second clutches) were entered as dependent variables, and the mating type (sib mating vs. control), the tended brood size (small or large) and their interaction as fixed factors.

We then investigated the effects of maternal presence and increased sibling competition on inbreeding depression measured both in nymphs (nymph development rate, survival rate of nymphs until adulthood) and in adults (absolute number of adults per family, clutch sex ratio, mean weight of males and females, proportion of adults with developmental problems). To this end, we used generalized linear mixed models (GLMMs) with Gaussian or when required, binomial error distribution, where mating type (sib mating vs. control), maternal presence and experimental brood size were entered as fixed factors, and the clutch of origin as random factor. The GLMM on clutch sex ratio and mean weight of males and females also included the total number of adults per family group as covariate. All interactions were tested and removed from the GLMMs when nonsignificant (all > 0.12). Interactions between inbreeding and brood size and between inbreeding and maternal presence were kept for interpretation, but the results do not qualitatively change if they are removed from the models.

Finally, we analysed the influence of sib mating on the reproductive success of male and female offspring using a series of randomized general linear models (randomized GLMs; Manly, 1997). This method was used because the overall hatching success from the cross-breeding experiment was relatively low (mean ± SD: 16.3 ± 31.6%) so that the distribution of model residuals could not be properly tested. Randomized GLM consisted in using the type of father (inbred or outbred), the type of mother (inbred or outbred) and their interaction as fixed factors, although each of the 11 measures reflecting parental reproductive success (the 11 response variables are listed in Table 4) was randomly permuted 10 000 times within factors (R script available on demand, see Meunier et al., 2008). All statistical analyses were conducted using R v2.15.1.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Part 1: Effects of sib mating on parental reproductive success

Overall, the reproductive success of females mated to a brother was not significantly different from the one of females mated to an unrelated male (Table 1, manova conducted on the number of eggs produced in the 1st and 2nd clutches, their hatching success, the number and weight of nymphs produced in each of these clutches and the relative investment of females into their second clutch; Wilk's Lambda = 0.64, approx. F9,27 = 1.67, = 0.15). Sib mating had also no significant effect on the likelihood of 2nd clutch production (Table 1; Fisher's exact test, = 1.00), the lifetime production of eggs (t-test, t44 = -0.24, = 0.82) and the lifetime number of hatched nymphs (t-test, t44 = −1.41, = 0.16). Furthermore, measures of second clutches were not significantly influenced by our manipulation of first brood size (manova; Wilk's Lambda = 0.89, approx. F5,28 = 0.67, = 0.65), sib mating (Wilk's Lambda = 0.74, approx. F5,28 = 1.96, = 0.12) or an interaction between these two factors (Wilk's Lambda = 0.82, approx. F5,28 = 1.26, = 0.31).

Table 1. Short-term fitness correlates of females mated to an unrelated male (control) or to a brother (sib mating)
 ControlSib mating
Mean ± SE N Mean ± SE N
  1. a

    Hatching success was calculated using egg-laying females only.

  2. b

    Two clutches did not produced any nymph.

First clutch
Egg number66.52 ± 2.232362.70 ± 1.6923
Hatching success (%)a83.95 ± 3.972373.15 ± 5.7123
Newly hatched nymphs55.35 ± 3.012345.09 ± 3.4623
Mean nymph weight (mg)1.45 ± 0.03231.49 ± 0.0523
Second clutch
Egg number34.48 ± 4.052336.70 ± 3.8623
Hatching success (%)a79.35 ± 6.831974.99 ± 4.8420
Newly hatched nymphs28.09 ± 3.962327.30 ± 3.3023
Mean nymph weight (mg)1.46 ± 0.0317b1.46 ± 0.0520
General
Likelihood of 2nd clutch prod. (%)82.61 2386.96 23
Relative investment in 2nd clutch31.16 ± 3.432333.90 ± 3.2023
Lifetime egg number101.00 ± 4.872399.39 ± 4.8123
Lifetime newly hatched nymph83.43 ± 5.472372.39 ± 5.5823

Part 2: Effects of inbreeding status on offspring development and survival

The effect of sib mating on nymph developmental rate depended on maternal presence, as revealed by the significant interaction term between sib-/nonsib mating and presence/absence of mothers (Table 2). In the presence of mother, no significant inbreeding depression was observed (Welch's t-test, t38 = 0.71, = 0.484), whereas in the absence of mothers, the developmental rate of inbred nymphs was reduced by 51% relative to outbred nymphs (Fig. 2, Welch's t-test, t39 = −2.16, = 0.037). Conversely, sib mating had no significant effect (neither as main effect, nor in an interaction with maternal presence and experimental brood size) on the survival rate of nymphs until adulthood, the absolute number of adult produced, the sex ratio of adults produced or the mean body weight of newly produced males and females (Tables 2 and 3).

Table 2. Influence of inbreeding status (inbred vs. noninbred) and two sources of stress (maternal presence and brood size) on nymph development and adult production. Statistics obtained using GLMM.
 Nymph developmental rateNymph survival rate until adulthoodAbsolute number of adults produced
LR inline imageP-valueLR inline imageP-valueLR inline imageP-value
  1. *Significant P-values are in bold. LR, likelihood ratio.

Inbreeding status3.340.0681.440.2301.750.186
Maternal presence46.89 <0.0001 24.77 <0.0001 33.46 <0.0001
Experimental brood size63.04 <0.0001 71.00 <0.0001 0.120.728
Inbreed. stat.: Maternal presence6.38 0.012 0.010.916<0.010.974
Inbreed. stat.: Exp. brood size0.840.3570.130.5770.130.723
Table 3. Influence of adult inbreeding status, maternal presence, brood size and total number of adult produced on clutch sex ratio and on the mean weight of males and females. Statistics obtained using GLMMs
 Clutch sex ratioMean weight of malesMean weight of females
LR inline imageP-valueLR inline imageP-valueLR inline imageP-value
  1. *The significant P-value is in bold. LR, likelihood ratio.

Total adult produced0.630.4281.350.2456.97 0.008
Inbreeding status1.250.2641.890.1701.650.199
Maternal presence0.790.376<0.010.9253.300.069
Experimental brood size2.530.1120.150.695<0.010.926
Inbreed. stat.: Maternal presence1.560.2121.660.1980.850.356
Inbreed. stat.: Exp. brood size0.250.6210.980.322<0.010.933
image

Figure 2. Interacting effect of inbreeding and maternal presence on nymph developmental rate.

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The proportion of offspring turning into adults with developmental problems was significantly smaller in the presence of a tending mother (1.7% of 116 adults) than in the absence of a mother (5.7% of 418 adults, Binomial GLMM, LR inline image = 6.27, = 0.012). This proportion was slightly smaller in outbred (3.4% of 295 adults) than inbred groups (6.7% of 239 adults), albeit this difference was not significant (Binomial GLMM, LR inline image = 3.41, = 0.065). Neither brood size (Binomial GLMM, LR inline image = 1.62, = 0.20), interactions between maternal presence and inbreeding (Binomial GLMM, LR inline image = 1.44, = 0.23) or between brood size and inbreeding (Binomial GLMM, LR inline image = 0.69, = 0.41) significantly influenced the proportion of adults with developmental problems.

Independently from mating type, nymph developmental rate and survival until adulthood were significantly higher in small as compared with large experimental clutches (Table 2, Developmental rate in small clutches: mean ± SE = 0.31 ± 0.04 and in large clutches: 0.15 ± 0.04; survival rate in small clutches: 0.42 ± 0.03 and large clutches: 0.20 ± 0.01). As predicted under restricted food availability where females may compete with offspring over the scarce food (see 'Introduction'), the clutches reared in the absence of mothers overall exhibited a higher nymph survival rate until adulthood and produced a larger absolute number of adults than the clutches reared in the presence of a mother (Table 2; survival rate: absence = 0.36 ± 0.02, presence = 0.22 ± 0.03; absolute number of adults: absence = 4.69 ± 0.21, presence = 2.71 ± 0.38). Our results also show that brood size and maternal presence had no significant effect on the adult sex ratio of clutches and the mean weight of newly produced males and females (Table 3). Finally, the total number of adult offspring produced in a clutch significantly decreased the weight of newly produced females, but had no significant effect on the weight of newly produced males (Table 3, Fig. S1).

Part 3: Sex-specific effects of adult inbreeding status on reproductive success

Sib mating of parents significantly reduced the reproductive success of the (correspondingly inbred) male offspring, but had only limited negative effects on the reproductive success of the female offspring. Of the 10 measures taken to characterize the reproductive success of inbred males and females, four were significantly influenced by the inbreeding status of the father, one by the inbreeding status of the mother and none by an interaction between the inbreeding status of the two parents (Tables 4 and S1). In particular, comparing our measures of reproductive success between inbred and outbred males, the hatching success of first clutch eggs was 75% lower, the number of first clutch nymphs 70% lower, the number of eggs in the second clutches 35% lower and the lifetime number of nymphs 64% lower.(Fig. 3, Table 4). By contrast, inbred and noninbred females showed only marginally significant differences in measures of reproductive success, with the inbred females having first clutch nymphs 12% heavier than outbred females, and inbred females producing 28% fewer second clutch eggs than outbred females (Fig. 3; Tables 4 and S1).

Table 4. Sex-specific effect of inbreeding. P-values were obtained using randomized GLMs
 Female offspringMale offspringInteraction
P-valueP-valueP-value
  1. *Significant P-values are in bold.

First clutch
Egg number0.4500.5840.785
Hatching success0.353 0.007 0.330
Newly hatched nymphs0.407 0.023 0.460
Mean nymph weight 0.049 0.4340.676
Second clutch
Egg number 0.043 0.028 0.459
Hatching success0.7410.1140.521
Newly hatched nymphs0.2150.1220.523
General
Likelihood 2nd clutch production0.1040.2220.933
Relative investment in 2nd clutch0.0810.0780.622
Total egg number0.1170.1140.566
Total nymph number0.323 0.030 0.453
image

Figure 3. Sex-specific effects of inbreeding. Adults were originating from sib mating (inbred) or control (outbred) clutches. Values are presented ± SE. *P < 0.05, **P < 0.01

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

This study demonstrates that offspring life stage and sex had strong influence on the expression of inbreeding depression, but that the stress from competition over restricted food among family members (sibling competition and parent–offspring competition over restricted food resources) only had limited effects in the European earwig, F. auricularia. In particular, we found that the presence of the mother did not influence the developmental rate of inbred nymphs, whereas it significantly reduced the one of noninbred nymphs. Conversely, brood size did not influence the expression of inbreeding depression in offspring both in terms of developmental or survival rates. These limited effects of inbreeding depression measured in the young offspring contrast to the ones we found when the offspring became adults. In particular, we showed that inbreeding dramatically reduced the reproductive success of male adult offspring (but had only marginal effects on the reproductive success of inbred female adult offspring). Hence, inbreeding depression is sex-specific and more expressed in the late than the early life stages in earwigs.

The benefits associated with maternal presence during family life are commonly thought to be strong enough to buffer against severe forms of inbreeding depression in offspring (e.g. Avilés & Bukowski, 2006; Thünken et al., 2007). Although maternal presence has been shown to provide benefits to earwig offspring reared under natural conditions and under ad libitum food in the laboratory (Kölliker, 2007; Kölliker & Vancassel, 2007), maternal presence is also known to reduce offspring developmental and survival rates under limited food condition, possibly due to a mother–offspring competition over the scarce food (Meunier & Kölliker, 2012b). Hence, we hypothesized that under the latter conditions, mother–offspring competition should exacerbate inbreeding depression in earwig offspring. Our results first confirmed that maternal presence reduced developmental and survival rate of outbred nymphs under restricted food conditions and, more surprisingly, revealed that such presence decreased the rate of morphological malformation in (both inbred and outbred) newly produced adults. This finding demonstrates that maternal presence under limited food conditions is also associated with detectable benefits for the offspring. By contrast, we found that the presence or absence of a tending mother did not shape the developmental rate of the inbred nymphs under restricted food conditions. This result is contrary to the prediction that mother–offspring competition exacerbates inbreeding depression, as it suggests that inbreeding may benefit to offspring (in terms of development) by reducing the costs of mother–offspring competition over the scarce, but essential, resources. Further studies exploring the behavioural mechanism mediating this differential effect of maternal presence on inbred versus noninbred offspring, as well as the fitness benefits associated with developmental time in earwigs will be required. Together with the result that sibling competition over resources did not show stronger effects of inbreeding on nymph development and survival, these findings indicate that family life has comparably weak effects on the expression of inbreeding depression in F. auricularia offspring.

The stronger inbreeding depression found in adult compared with young offspring suggest that inbreeding history has purged the most damaging recessive alleles (which typically shape the early life stage) from the studied population (Husband & Schemske, 1996; Glémin, 2003). This inbreeding history is unlikely to result from the laboratory rearing of the field-sampled parents, as these individuals were maintained for only one generation in a very large experimental population (1200 individuals), where they were allowed to freely mate with a large number of unrelated partners. By contrast, some degree of recurrent inbreeding is possible in natural populations of F. auricularia, as adults are gregarious and exhibit a relatively low dispersal rate (Moerkens et al., 2010). Further molecular studies (e.g. using microsatellites) exploring the relatedness between group-living adults and the genetic structure of the studied population would help to better understand the evolutionary processes that limited the detrimental effects of inbreeding on nymph survival and development.

Sib mating substantially reduced the reproductive success of earwig male offspring, but had only little effects on the one of female offspring. This male-specific effect of inbreeding depression is in line with recent findings in seed-feeding beetle and fruit flies (Enders & Nunney, 2010; Fox et al., 2011; Okada et al., 2011), but contrasts with results in the flour beetle, Tribolium castaneum, where eight generations of inbreeding in the absence of male–male competition did not decrease correlates of male fertility (Michalczyk et al., 2010). Spermatogenesis is commonly known to be particularly vulnerable to inbreeding depression and could thus mediate the male-specific effect of inbreeding observed in F. auricularia. One negative effect of inbreeding known to affect spermatogenesis is to decrease the number of sperm in the ejaculates of inbred males (e.g. Zajitschek et al., 2009; Fox et al., 2011) and consequently reduce the number of eggs produced by the mating partners (Fox et al., 2011; Okada et al., 2011). Another potential negative effect is to reduce the quality of sperm in the ejaculates of inbred males (e.g. in terms of viability, abnormal shapes), which hampers embryonic development, lowers the hatching success of the eggs produced by females (e.g. Mehlis et al., 2012), as well as reduces the number of eggs that females are able to produce after having stored the sperm for a relatively long time (due to shorter sperm viability). Although our study did not directly estimate the quantity and quality of sperm produced in earwigs male ejaculates, our findings are in line with lower sperm quality in inbred males. In particular, the hatching success of first clutch eggs sired by inbred males was substantially lower than the one sired by noninbred males. Furthermore, females mated to inbred males produced a smaller number of eggs in their second clutch, which was laid on average 2 months after the production of the first clutch and without remating events in between.

The negative effects of sib mating on the reproductive success of male offspring raise important questions on the influence of inbreeding on the evolution of mating strategies in the European earwig. In particular, the cost of producing inbred adults (sons in particular) could select individuals to actively avoid mating with genetically related partners (sib mating avoidance; e.g. Lihoreau et al., 2008) or females to favour the sperm of unrelated mating partners to fertilize their eggs (e.g. Firman & Simmons, 2008; Welke & Schneider, 2009). Conversely, reduction in the reproductive success of females mating with inbred males could favour mechanisms allowing females to actively choose to mate with noninbred mating partners (e.g. Ilmonen et al., 2009; Okada et al., 2011). To date, the mating strategies of the European earwig are poorly known, beside that males and females have multiple mating partners (Guillet, 2000) and that male mating success is associated with forceps size (Tomkins & Simmons, 1998; Walker & Fell, 2001). Whether inbreeding avoidance, cryptic female choice and/or mate choice against inbred males occur in F. auricularia should thus be the aim of future experiments.

Overall, our results reveal that family life has limited effects on inbreeding depression in earwig offspring, a result which is contrary to a common assumption that maternal presence buffers the negative effects of inbreeding on offspring traits (Margulis, 1997, 1998; Avilés & Bukowski, 2006; Thünken et al., 2007). Our study also demonstrates that inbreeding effects measured during the juvenile period underestimates the total fitness costs of sib mating in terms of number of grand-offspring, which emphasizes the key role of long-term studies to better understand the effects of inbreeding on individual fitness (e.g. Cornell & Tregenza, 2007). Finally, our results showed that females exhibit a lower reproductive success when mating with inbred (and unrelated) males than outbred males. To date, few studies disentangled the influence of male and female inbreeding status on the reproductive success of females (Enders & Nunney, 2010; Michalczyk et al., 2010; Fox et al., 2011; Okada et al., 2011; Mattey et al., 2013), possibly because inbred individuals can restore outbred fitness by mating with unrelated partners (and thus regenerating heterozygosis in the offspring; Frankham et al., 2002) (but see Lehmann et al., 2007). The results of the present study in earwigs and the growing number of results in other species (Fox et al., 2011; Okada et al., 2011; Mattey et al., 2013) now emphasizes that mating with inbred individuals (here males) have continued fitness consequences independent from offspring heterozygosity and thus call for more consideration in future theoretical models and empirical studies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We would like to thank Per Smiseth and three anonymous reviewers for their fruitful comments on a previous version of this manuscript. This study was financially supported by the Swiss National Science Foundation (grant no. PP00-119190 to M.K.)

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jeb12217-sup-0001-TableS1-FigS1.docxWord document60K

Figure S1 Association between the mean number of adult offspring and the mean weight of female and male offspring.

Table S1 Summary of the sex-specific effects of inbreeding on the reproductive success of offspring.

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