A rare experiment with cooperatively breeding birds in the wild reveals high costs of reproduction but only in groups with few helpers.
Every breath we take damages our cells a little. This inevitable cost of metabolism, oxidative damage, accumulates with time and can contribute to pathology and senescence1. The potential role of oxidative stress in explaining inter-individual differences in life history trajectories and fitness has not gone unnoticed by evolutionary ecologists. Researchers have proposed that the accumulation of oxidative damage is expected to occur at greater rates during periods of heavy reproductive investment and thus may be a key physiological mechanism mediating life history trade-offs between current and future reproduction, and between reproduction and survival2,3. Empirical studies, however, have produced conflicting results4. The failure of some to show the expected relationship between reproduction and oxidative damage has been attributed to the lack of experimental manipulation of reproductive effort or to the fact that many studies were conducted in laboratory conditions where subjects are removed from ecological constraints5.
Writing in Proceedings of the Royal Society B: Biological Sciences, Cram and colleagues6 overcome some previous methodological issues by conducting a clutch-removal experiment with a cooperatively breeding bird in the Kalahari. The white-browed sparrow weaver, Plocepasser mahali, nests in territorial groups of 2-12 individuals, in which one dominant pair monopolises within-group reproduction and the remaining subadults help raise the offspring. To assess how reproductive effort affects oxidative status, Cram and colleagues monitored 20 groups of weavers until a full clutch of eggs was laid in each nest. They then captured all birds (except the incubating dominant females), weighed them and collected blood samples. In 9 of the groups, they removed all eggs. About a month after clutch-removal, Cram and colleagues re-captured the birds in all groups and collected follow-up measurements on condition and oxidative stress to compare if the birds with chicks differed from those without.
Contrary to expectations, there was no difference in weight loss between birds in groups that had their eggs removed and birds in groups that kept their eggs but had many helpers (group size 5-8 birds). Reproductive effort seemed to be cost-free. Individuals in small groups (2-4 birds) that raised chicks and had few helpers, however, lost more weight compared to the birds that underwent clutch removal. The data on oxidative stress paralleled the findings on weight loss.
Birds in small groups, rearing chicks, experienced increased levels of oxidative damage, compared to the birds whose eggs were removed. Breeding birds in large groups with chicks did not differ in oxidative damage from the birds not raising any chicks. Importantly, these effects were not due to differences in clutch size. Rather, it was the number of helpers available in large groups that acted as a buffer against the costs of increased reproductive
effort. This interpretation was further supported by the observation that among chick-raising birds, rates of provisioning were negatively associated with body mass – individual investment in reproductive effort carried significant energetic costs for the helpers. Although the level of oxidative damage was not associated with rates of provisioning, the level of total antioxidant protection was. Birds with higher provisioning rates exhibited lower antioxidant protection, making them more vulnerable to oxidative damage.
This study measured only one marker of oxidative damage, lipid peroxidation, in plasma samples. Its conclusions are thus tentative, as oxidative damage to other biomolecules and in other tissues may reveal different patterns. Nevertheless, the contribuion of this work is important – not only because it is one of the few demonstrations of the oxidative costs of reproduction in the wild but because, uniquely, it reveals the role of cooperative breeding as a mechanism for mitigating these costs.
Costantini, D. Oxidative Stress and Hormesis in Evolutionary Ecology and Physiology. (Springer Berlin Heidelberg, 2014). doi:10.1007/978-3-642-54663-1
Metcalfe, N. B. & Alonso-Alvarez, C. Oxidative stress as a life-history constraint: the role of reactive oxygen species in shaping phenotypes from conception to death. Functional Ecology 24, 984–996 (2010).
Monaghan, P., Metcalfe, N. B. & Torres, R. Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation. Ecology Letters 12, 75–92 (2009).
Blount, J. D., Vitikainen, E., Stott, I. & Cant, M. A. Oxidative shielding and the cost of reproduction. Biological Reviews DOI: 10.1111 / brv, (2015).
Metcalfe, N. B. & Monaghan, P. Does reproduction cause oxidative stress? An open question. Trends in Ecology & Evolution 28, 347–350 (2013).
Cram, D. L., Blount, J. D. & Young, A. J. The oxidative costs of reproduction are group-size dependent in a wild cooperative breeder. Proceedings of the Royal Society B: Biological Sciences 282, 20152031–20152031 (2015).
Picture the scene. You are trying to see which male is mating with this very attractive and sexually receptive female chimpanzee. She’s perched high up in the canopy above. The only place where you can get a good look at the action through all the foliage happens to be directly below the branch on which she is currently standing on. The male approaches briskly from behind. You peer through your binoculars with your neck craned up and aching. Then through the limited field of vision of your binoculars you see something falling down straight at you. It appears to have fallen out of the female at the moment that the male mated. You duck and dive and narrowly miss an unnecessarily close encounter with what, you later learn from the more experienced chimp-watchers around you, was a ‘sperm plug’. Up to that moment, sometime in the summer of 2006 in Kibale National Park, Uganda, I knew next to nothing about these structures. This was easily the most exciting personal ‘discovery’ of my first year in graduate school.
So why do chimpanzees (as opposed to any other primate or animal) have sperm plugs (more accurately called ‘copulatory plugs’) and what are they for? Were these things common in other primates, too? I was quite sure they were not part of human reproductive biology but I wasn’t too certain about the rest of the primate order.
I wasn’t the first person to ask these questions. Taking a wide-angle view, via comparative analysis, Dixson and Anderson rated the level of seminal coagulation of the ejaculates of 40 primate species on a 4-point scale. Species with no visible signs of coagulation received a score of 1 and those whose ejaculates coagulated to the point of forming a firm plug received a score of 4. Clearly there were other primates, besides chimps that had copulatory plugs.
If the function of sperm coagulation and plug formation was to aid males in competing indirectly for fertilizations (via post-copulatory sperm competition within the reproductive tracts of females), the authors predicted that species, in which females commonly mate with multiple males, would receive higher coagulation scores. And they did. On average, species with multiple-male mating had a coagulation score of 3.64, while those, in which females usually mated with only one male had a mean score 2.09. As a point of reference, according to this classification, the ejaculates of humans and gorillas both got a score of 2 (‘semen becomes gelatinous and remains semifluid but there is no distinct coagulum’), most macaques and baboons got a 3 (‘semen coagulates so that the coagulate forms a whitish, non-fluid, non-gelatinous mass, but not a compact plug’) and chimpanzees scored top marks, at 4 (‘there is a distinct compact, rubbery or semi-solid copulatory plug, retaining its shape and molded to the contours of the female’s tract’).
Another bit of evidence supporting the important role of sperm coagulation and plugs for the process of sperm competition comes from genetics. Dorus and colleagues looked at the relationship between the rate of evolution of a major seminal protein gene and the levels of female promiscuity among 12 primate species. They found that species with greater levels of promiscuity (e.g. chimps) were characterized by much higher rates of evolution of that particular gene. The species with the highest rates of molecular evolution also scored higher on the ‘sperm coagulation scale’ used by Dixson and Anderson. The genetic evidence thus suggested that selection for specific traits had occurred – traits that enabled males to do better when it came down to sperm competition.
These observations, however, do not provide conclusive evidence of the reproductive benefits that males who produce ‘better’ or ‘bigger’ sperm plugs might enjoy. Obtaining more solid ‘proof’ however could be tricky in species that are long-lived, slow-reproducing and, very often, endangered in their wild habitats (i.e. most primates). This is why a lot of the most revealing studies of sperm plugs have been carried out in other animals. Such as small rodents. They are fast-reproducing, amenable to clever experimental manipulations and overwhelmingly not at risk of extinction in the wild (which allows somewhat more invasive methodological approaches).
The function of copulatory plugs in house mice
In a study published in Behavioral Ecology this August, Sutter and colleagues wanted to test the hypothesis that copulatory plugs in mice had a role in the process of sperm competition. To do that they needed to document how the presence of plugs affects the subsequent sexual interactions of the female and whether variation in plug size affected the reproductive success of the males who produced those plugs.
So they rounded up some laboratory domestic mice and got them to mate in a carefully orchestrated series of experiments. Naturally, they recorded all the action on video. When males mated successively, the plug they produced the second time around was significantly smaller than the first one by about 19.2 mg. The duration between successive copulations also seemed to matter: males who had more time to recover after sex (up to 56 hours) tended to produce larger plugs during their second copulation than those who had little time to rest between matings (as little as a 2 hours). The size of the plug was affected by ejaculate depletion and thus time since last mating could be used as a proxy for plug size.
Next, the researchers wanted to examine how plug size affected paternity. Female mice produce a lot of embryos at once and multiple paternity is common (i.e. not all embryos are sired by the same male). In this study, females mated with two different males and the goal was to establish how the size of the copulatory plug deposited by the first male affected the proportion of embryos that the second male got to sire. To figure out who the sire was the females who were part of the experiment were sacrificed and their embryos extracted. Genetic testing established the paternity of the embryos. The size of the copulatory plug deposited by the first male was, indeed, important for his reproductive success: second-maters sired a greater proportion of embryos (within the same female) when the male that mated before them had recently mated himself (i.e. the plug he left inside the female would have been smaller). If the preceding male had not mated recently (i.e. was able to produce a larger plug), the proportion of embryos sired by the second-mating male was significantly lower. So there was evidence that sperm plugs have an important function in sperm competition! Larger plugs were better at preventing other males from siring more offspring.
Although this second experiment relied on the sexual restedness of the first male (i.e. the number of days since his last copulation) as a proxy indicator of plug size, rather than directly measuring the size of the plug (the plug had to remain in place to assess its function) – in combination with the previous experiment, which showed the relationship of sexual restedness to plug size – this study does demonstrates the adaptive function of sperm plugs in species with multiple mating.
Statistically speaking, however, the effect of inferred sperm plug size (sexual restedness of the first male) on the proportion of offspring sired by a second male was reduced (still negative, but non-significant) when another variable was included in the same analysis – the ease of mating by the second male. Second-mating males who mated with greater ‘ease’ (those who were quicker to ejaculate and achieved more ejaculations) secured a greater share of the embryos that were conceived. The study also showed that ease of mating for the second male was negatively affected by the sexual restedness of the first-mating male (as a proxy of plug size). This effect, however, was small and non-significant. So not entirely an open-and-shut case.
The relatively smaller effect of sperm plug size on the paternity share of the second male and its confounding by ease of mating of the second male is not too surprising as the sample size used in the analysis of paternity distribution was not very large. There were a total of 14 pregnant females who copulated with both the first and the second male (5 other females got pregnant but when they mated with the second male he failed to ejaculate). These females produced 99 embryos (between 5 and 9 per mother) and multiple paternities were observed in 57% of all cases (only 6 females had embryos, which were all sired by the same male, in 4 cases – by the first-mating male).
The mechanism behind the finding that males who were more sexually rested managed to sire more of the embryos in a female after she mated with another male is not entirely clear but could be one of two things. First, males who were more sexually rested were also able to produce more sperm (which we known from previous studies in this and other mammals to be true) so they were more able to outcompete the sperm of the second-maters. And second, these males produced larger plugs (which this study showed), which in combination with greater sperm numbers – might have helped to reduce the proportion of embryos sired by second-maters. Either way, the evidence from this and other studies shows that sperm plugs are a product of indirect male-male competition (sperm competition, to be precise) and their role in mediating male reproductive success can be significant.
What about primates?
The mouse study of Sutter and colleagues alerted me to a large body of fascinating literature that people have produced on the topic of copulatory plugs in mice and other non-primate species. Primatologists, by comparison, are at a disadvantage when it comes to investigating the functional effects of plugs on the sexual behaviour and the reproductive success of their study subjects, for both logistical and ethical reasons. Nevertheless, detailed observational studies are feasible and would make a large contribution to our understanding of this relatively under-studied aspect of indirect sexual competition in primate males.
One of the few non-comparative studies relying on careful observation comes from the work of Joyce Parga and colleagues, who collected 9 plugs that fell out of ring-tailed lemur females when males displaced the plug left by the females’ previous sexual partners with their penis. Parga et al. used high-resolution X-ray computed tomography scanning to measure their volume and surface area. They then related the behaviour observed in the field to these measures. One question about the role of the plugs in sperm competition was whether larger ones were more difficult to displace by the next male. The data, however, showed they were not. The difficulties of collecting copulatory plugs in the field limited the statistical power of this lemur study to test an important functional hypothesis. There is still a lot of work left if we really want to reach the level of understanding of mating plugs that researchers working with rodents have.
Blue-footed boobies (Sulla nebouxii) are relatively large and long-lived for a bird and it takes a lot of food for the adults to raise their chicks to independence. They nest in large colonies along the west coast of the Americas and feed on fish, caught in the tropical waters of the Pacific. Both parents care for the young and feed them for up to 6 months after hatching. As the chicks grow, so does the demand they place on their parents, who must make ever more frequent trips out to the ocean to find fish for the brood.
A new study published in Animal Behaviour investigated the physiological costs that the parents incur during this critical stage of their lives. Researchers monitored the behaviour of both male and female boobies in a large colony on
a Mexican island, where approximately 3,000 booby pairs nest at a density of 0.26 nests/m2. They also collected blood from the adults to measure their physiological condition and conducted an experiment to manipulate the need for provisioning. The experiment consisted of swapping chicks of different ages between nests. Chicks that were 1 week old were placed in the nests of parents who had 2-week old chicks and vice versa. The result of this manipulation was that parents who had younger chicks were suddenly faced with the significantly greater nutritional needs of the older and larger 2-week chicks (these older chicks need up to twice as much food). Conversely, the original parents of the older (2-week) chicks got a break and had a ‘new’ brood, which was younger, smaller, and less demanding.
As a measure of the costs of caring for their chicks – the researchers analysed blood samples to quantify several compounds. Several blood plasma metabolites were used to assess changes in levels of body reserves and physical exertion, associated with muscle activity and foraging. An additional measure (the ratio between two types of blood cells, heterophils and lymphocytes) was used as an index of stressors that might adversely affect immune function (e.g. inflammation, infectious disease, parasite infestation, food or water deprivation as well as temperature extremes). Blood was collected from the birds before and after the experimental manipulation (chick-swapping) so that any changes in these parameters could be related to the change in provisioning demands the parents experienced. The researchers also collected data on body weight for a more general assessment of their condition.
When parents experienced an increase in provisioning demand (i.e. thy were given bigger chicks) – both of them increased the number of trips to sea they made looking for food and they spent less time at their nest. When provisioning demand decreased (i.e. when parents were given younger, less needy chicks), surprisingly, the parents did not decrease their foraging effort. Although overall body condition was not affected by changes in provisioning demand, some of the blood plasma markers were: greater demand resulted in higher levels of biological markers indicative of physical exertion and potential stress on the immune system. Parents that experienced a decrease in provisioning demand had lower levels of these biomarkers, relative to control subjects suggesting that physiological condition tracked adjustments in foraging effort.
The costs associated with raising a brood can thus affect the physiological condition and possibly the future reproductive effort of the parent boobies. Yet, adults increased their effort to make sure that their chicks were well fed and had a better chance of reaching maturity. The flexibility in provisioning behaviour shown by the parent birds may be important given that the seas they inhabit can be very unpredictable in terms of food availability.
González-Medina, E., Castillo-Guerrero, J. A., Santiago-Quesada, F., Villegas, A., Masero, J. A., Sánchez-Guzmán, J. M., & Fernández, G. (2015). Regulation of breeding expenditure in the blue-footed booby, Sula nebouxii: an experimental approach. Animal Behaviour, 108(C), 9–16. http://doi.org/10.1016/j.anbehav.2015.06.025