Kin selection theory predicts that individuals will show less aggression and more altruism towards relatives1,2. However, recent theoretical developments suggest that with limited dispersal, competition between relatives can override the effects of relatedness3,4,5,6,7,8,9. The predicted and opposing influences of relatedness and competition are difficult to approach experimentally because conditions that increase average relatedness among individuals also tend to increase competition. Polyembryonic wasps in the family Encyrtidae are parasites whose eggs undergo clonal division to produce large broods10. These insects have also evolved a caste system: some embryos in a clone develop into reproductive larvae that mature into adults, whereas others develop into sterile soldier larvae that defend siblings from competitors11,12,13,14. In a brood from a single egg, reproductive altruism by soldiers reflects clone-level allocation to defence at the cost of reproduction, with no conflict between individuals. When multiple eggs are laid into a host, inter-clone conflicts of interest arise. Here we report that soldier aggression in Copidosoma floridanum is inversely related to the genetic relatedness of competitors but shows no correlation with the level of resource competition.
Polyembryonic encyrtids are small (1 mm) parasitoid wasps that oviposit into moth (host) eggs10,11. After parasitism the host egg hatches and the larva develops to its final (fifth) instar (Fig. 1). During this period, the wasp egg proliferates clonally, producing multiple embryos in an assemblage called a polymorula. Several species, including C. floridanum, produce thousands of embryos per egg. Some embryos develop during the host's early instars into obligately sterile soldier (precocious) larvae with fighting mandibles and an elongate body10,11,12. The remaining embryos develop during the host's final instar into reproductive larvae with tiny mandibles and rounded bodies. These reproductive larvae consume the host, pupate and emerge as adult wasps. Soldier larvae always die after the reproductive larvae consume the host10.
Hamilton's rule specifies that altruism is favoured when rb - c > 0, where c is the fitness cost to the altruist, b is the fitness benefit to the recipient and r is their genetic relatedness1,2. Conditions favouring the most extreme form of altruism, that is, sterile castes, include high relatedness and/or factors that make b > c. Key elements in the evolution of sterile soldier larvae by polyembryonic wasps are thus likely to be: (1) clonal development in a confined space (high r) and (2) the benefits, b, of defending a nutrient-rich but limiting resource (the host) from competitors relative to the costs, c, in lost reproduction10,11. Previous studies indicate that soldiers attack and frequently sacrifice themselves in defence of their clone-mates against other species of parasitoids13,14. In laboratory experiments, 96% of females with no previous oviposition experience (N = 50) oviposited into hosts that were parasitized by another wasp (superparasitism). Females superparasitized hosts irrespective of when the first female oviposited or the relatedness of the resident clone. Screening individual wasps from broods (56 broods with 20 same sex wasps per brood) collected in Georgia, using polymorphic allozymes such as glucosephosphate isomerase, further indicated that some broods contain progeny of at least two genotypes (D.G., K. G. Ross and M.R.S., unpublished results). Given that females also often lay two eggs per host (see below), these data collectively indicate that: (1) different kin classes of C. floridanum frequently compete within the same host, and (2) relatedness of potential competitors ranges from full siblings to distant relatives. This provides an opportunity for experimentally separating the effects of relatedness and competition under conditions of limited dispersal while using soldier behaviour to quantify conflicts of interest.
Like most Hymenoptera C. floridanum is haplodiploid, with unfertilized eggs producing males and fertilized eggs producing females. Mothers produce all-male or all-female broods by laying one egg per host, and mixed broods by laying two eggs (one male and one female)15. Almost 1,400 wasps per host emerge from each brood type16,17. A developmental asymmetry, however, exists between males and females: female broods produce about 30–50 soldiers by the host's fourth instar and male broods produce almost none15,16,17,18. Correspondingly, soldiers in mixed broods are almost exclusively female. Female soldiers have been shown to attack male embryos which results in mixed broods having strongly female-biased sex ratios16,19. The resolution of conflict in mixed broods in favour of females could be due to relatedness asymmetries, because soldiers are predicted to value reproductive, clone-mate sisters (r = 1) more than brothers (r ∼ 0.25), particularly as only a few of the latter are needed for sib mating16. The indirect pay-off (inclusive fitness) to soldier larvae through a brother's post-dispersal matings may, however, provide some check on siblicidal behaviour19.
If soldiers are able to assess their relatedness to other individuals in the host, we might expect the aggressive behaviour of soldiers to be negatively correlated with relatedness to a competing clone. The highest levels of inter-clone aggression should thus be observed when unrelated mothers lay eggs into the same host. The completely local level of competition for limiting host resources could, however, override effects of relatedness3,4,5,6,7,8,9, in which case soldier behaviour towards other clones is predicted to correlate with the severity of resource competition. This will increase with the number of competitors and/or reductions in host size, irrespective of relatedness. An additional consideration is that female clones might benefit from the presence of future mating partners if mating opportunities after dispersal are scarce. If so, soldiers might exhibit lower levels of aggression towards non-relative males than females.
To evaluate the importance of relatedness asymmetries, resource competition and sex of the competitor on soldier aggression, we conducted two complementary experiments. In the first experiment, we injected a labelled polymorula that was either a full sibling (brother or sister) or a non-relative (male or female) into a host containing an all-female brood (see Methods). Note that introducing different kin classes into hosts containing a female brood varied relatedness (and sex) of the potential competitors. However, the intensity of resource competition among treatments was approximately the same because each host was almost identical in size and host resources for the resident clone would be similarly reduced by injection of a second polymorula regardless of its relatedness. Hosts containing a female brood were also injected with a labelled tissue (gonad) collected from a non-parasitized host larva. This tissue would have no impact on relatedness, resource competition or future mating opportunities, and controlled for the possibility that soldiers attack any foreign entity introduced into the host. In all treatments, attack of the target by resident soldiers was scored as: (1) the proportion of hosts from each treatment that contained at least one soldier with fluorescent tracer in its gut, and (2) the mean proportion of soldiers per host with tracer in their gut. Both measures revealed a strong association between soldier aggression and relatedness of the competitor (Table 1). Attack rates were highest towards non-relative female or male clones, intermediate towards brothers and lowest towards full sisters or host tissue. Resource competition did not seem to affect attack rate because a sister clone would reduce available host resources whereas host tissue (gonad) would not, but resident soldiers rarely attacked either. In contrast, soldiers attacked non-relative clones much more frequently than sisters even though all introduced competitors would similarly reduce host resources for the resident clone. Soldier aggression also did not correlate with the sex of the competitor and future mating opportunities, given that soldiers attacked males as frequently as non-relative females. Direct observation of soldier larvae in vitro corroborated our in vivo results by showing that non-relative clones and brothers were attacked significantly more often than sisters (P < 0.0001) (Table 2). Combined, these results clearly indicate that C. floridanum soldiers differentially attack competitors as a function of relatedness. Recent studies further reveal that soldiers distinguish kin from non-kin by cues from the extraembryonic membrane that surrounds each individual20. This membrane is also essential for protecting C. floridanum from the host's immune response, suggesting that the cues used in kin recognition may be maintained in part by selection for resistance against the host.
In our second experiment, we simultaneously assessed the effect of resource competition and relatedness on soldier aggression by comparing attacks towards competitors of different relatedness in large versus small hosts. Large hosts were naturally parasitized larvae (unstarved) as used in experiment 1, whereas small hosts were created by depriving parasitized larvae of food for 48 h. Hosts always survived transient starvation but their average maximum size (286 mg) was reduced by almost 50% relative to unstarved hosts (507 mg) (see Methods). Host starvation clearly intensified resource competition among progeny of the resident clone, because the number of adult progeny produced in starved hosts (485 ± 74 (mean ± s.e.m.), N = 20) was significantly smaller than in unstarved hosts (mean = 1,206 ± 117, N = 20; P < 0.0001 (t-test)) (see Methods). By extension, the intensity of resource competition after introduction of a second polymorula would likewise be much higher between kin classes in a starved host compared with an unstarved host. We therefore injected labelled sister, brother, non-relative female (Wisconsin) or non-relative male polymorulae into starved or unstarved hosts containing a Georgia female clone (N = 30 for each treatment). When we compared the outcomes among only starved hosts, we found that soldier aggression remained strongly associated with the relatedness of the competitor (G2 = 53.87; d.f. = 4; P < 0.0001). This indicates that soldiers differentially attacked competitors as a function of relatedness even when host resources were more limited than in experiment 1. Moreover, we found no differences in soldier attack rates towards sisters (unstarved, 0.18; starved, 0.12; P > 0.5), brothers (unstarved, 0.52; starved, 0.5; P > 0.8), non-relative females (unstarved, 0.76; starved, 0.80; P > 0.9) or non-relative males (unstarved, 0.77; starved, 0.76; P > 0.9) when we compared outcomes in starved hosts versus unstarved hosts. This indicated that levels of soldier aggression are negatively correlated with relatedness of the potential competitor, but do not change with the severity of resource competition.
Hamilton originally proposed that the evolution of altruism could arise through either direct kin recognition or limited dispersal (population viscosity) which would have the effect of elevating local relatedness sufficiently to favour generalized altruism towards neighbours1,2. Our results with C. floridanum indicate that altruism extends beyond clone-mates to close relatives like non-identical sisters. This response is unlikely to reflect generalized altruism due to high neighbour–neighbour relatedness because females do not benignly share resources with brothers or non-relatives. Recent theory further suggests that altruism is unlikely to evolve in purely viscous populations because viscosity increases neighbour–neighbour competition for resources as much as it elevates relatedness3,4,5,6,7,8,9. Potential benefits of sharing a host with future mating partners also seem to be relatively unimportant in C. floridanum because soldiers usually attack males. Instead, our data support the alternative conclusion that altruism in C. floridanum operates by means of kin recognition1,2. Theory suggests that the evolution of kin-recognition based altruism may be suppressed in viscous populations unless periodic dispersal (alternating viscosity) occurs, which allows the beneficiaries of altruism to compete for resources against non-relatives3,4,6. Within the Georgia field population that was used in this study, the frequency of homozygous broods for the polymorphic allozyme glucosephosphate isomerase depart significantly from Hardy–Weinberg expectations (P < 0.002; Fisher's exact test) suggesting a high level of inbreeding (D.G., K. G. Ross and M.R.S., unpublished results). However, adult dispersal and subsequent resource competition between non-relatives is also likely because winged adult females must disperse to find new hosts and winged adult males are likewise able to disperse and can potentially mate with females from other hosts15,16. Our observations of sister–sister (r ∼ 0.75) altruism but sister–brother (r ∼ 0.25) conflict within hosts further suggest that opportunities exist for surviving males from mixed broods to disperse and mate with non-sisters16. In contrast, a comparative study of fig wasps suggests that local competition between males for mates is severe enough to obviate the benefits of kin selection8.
The critical interplay between relatedness and competition in the evolution of altruism is potentially best understood in organisms that develop in non-standard ways, like clonal species that form assemblages of potentially competing relatives and non-relatives21. Like C. floridanum, several other clonally developing animals show a strong ability to recognize close relatives22,23,24. In contrast, examples of one clone exploiting another when clones mix are rare, and may reflect either the inability of some species to suppress conflict, or that a sufficiently large benefit to cost ratio (b > c) favours cooperation in the absence of high relatedness25,26. In C. floridanum, soldier attack clearly depends on relatedness, even under severe resource competition.
Two populations of C. floridanum were established in the laboratory from field-collected broods obtained in the southern (Georgia) and northern (Wisconsin) US. Each culture was maintained separately as large randomly mating populations on the host Trichoplusia ni. Host larvae were reared on an artificial diet at 27 °C and in a 16 h light/8 h dark photoperiod as previously described27. Experimental conditions were manipulated to favour production of all-male and all-female broods. Ovipositions were observed to determine that wasps laid one male or female egg to produce all-female or all-male broods17. Identifying the composition of broods in advance was possible, because females exhibit different behaviours when laying a fertilized or an unfertilized egg14. Hosts containing full sisters were produced by mating clonal females from one brood with a male from another. Hosts containing brothers were produced by allowing unmated females from the same brood to oviposit.
Male or female polymorulae containing on average 500 embryos were collected from fourth instar hosts and labelled with carboxyfluorocein diacetate succinimidyl ester (CFSE) using previously established methods14,16. These previous studies confirmed that soldiers that attack CFSE-labelled tissues ingest the label which is then clearly visible when larvae are examined by epifluorescent microscopy. In experiment 1, CFSE-labelled sister, brother, non-relative Georgia female, non-relative Wisconsin female and non-relative Wisconsin male polymorulae were injected into fifth instar hosts containing a female brood from the Georgia population using a glass needle mounted on a micromanipulator. As a control, testes from fourth instar T. ni, which are similar in size to a polymorula, were labelled and individually injected. Hosts were then dissected 24 h after injection and the number of soldiers with label in their gut were determined. In vitro assays were conducted in 1-ml culture wells containing TC-100 medium (Sigma)14,16. Soldiers and polymorulae from relative and non-relative broods were collected from fourth instar hosts immediately before the experiment. One soldier larva was placed in the culture well with a full sister, brother, non-relative female or non-relative male polymorula. The proportion of each type of polymorula that was attacked during a 2-h bioassay period was then recorded. An attack was defined as the larva gripping a polygerm with its mandibles for more than 1 min. When this occurred, consumption of tissue by the soldier was readily visible.
In experiment 2, hosts containing female broods were starved by removing them on the first day of the fifth instar from the food source to cups containing moist cotton wool. Each larva was returned to food 24 or 48 h after starvation and reared until completion of C. floridanum development. The average maximum size of hosts treated in this manner declined linearly from 507 ± 56 mg (mean ± s.e.m., N = 20) for unstarved controls to 398 ± 42 mg (N = 20) and 286 ± 38 mg (N = 20) for hosts starved for 24 or 48 h, respectively. Previous studies indicated that the total number of wasp progeny produced per polymorula is established at the end of the host fourth instar11,18. Thus, any reduction in the number of progeny produced in starved, fifth instar hosts is due to mortality caused by increased competition for more limited host resources rather than a change in the development of the polymorula itself. To assess the effect of host starvation on the resident clone, parasitized hosts containing a female brood were starved for 48 h, returned to food and then reared until completion of development. The total number of wasp progeny per host was then counted. For comparisons of soldier aggression in starved and unstarved hosts, one cohort of hosts was starved for 48 h, returned to food and then injected 24 h later with a CFSE-labelled sister, brother, non-relative female (Wisconsin) or non-relative male polymorula. The second, unstarved cohort was treated identically except that hosts were continuously provided with food. Hosts in both cohorts were dissected 24 h after injection and the number of hosts containing soldiers with label in their gut was determined as described in experiment 1.
The association between soldier aggression, relatedness, resource competition or gender of the competitor were analysed by likelihood ratio chi-square tests using the JMP, v.3.0 statistical package.
This work was supported in part by the Natural Environment Research Council (UK), the National Science Foundation (US), the University of Georgia Experiment Station, and the Conseil General de la Region (France).
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Development Genes and Evolution (2009)