Two Males, One Female: Rare Triplet Mating Observed in a Swiss Parasitoid Wasp

On a humid early afternoon in July 2019, on a woodpile of cut spruce logs below Läheweid near Lenk in the Swiss Canton of Bern, researchers watched something they had never seen in decades of studying parasitoid wasps: a female Darwin wasp, Xorides ater, in copula with two males at the same time. Over roughly two hours of observation they recorded three such triplet-style matings, while only two ordinary pairings involving a single male and a single female occurred during the same period.

The setting was an exposed woodpile of cut and partly split spruce logs, Picea abies, originally up to about 20 centimetres in diameter before splitting. It lay about 600 metres northwest of the Bettelberg cable car station at roughly 1510 metres altitude, measured approximately 10 by 2 by 1 metres, and was covered with corrugated iron. From the insect species present and their activity, the observers deduced that the woodpile had been standing for at least two years. Most of the insect activity was on the shaded northern side, where conditions were a cool 23 degrees Celsius, and that is where the observations were made between 13:00 and 15:00.

The level of ichneumonoid activity was striking. At one point the researchers counted more than 40 males in flight in front of the shaded side of the woodpile. Most of the individuals belonged to Xorides ater, with fewer of the similarly sized braconid Helcon nunciator, a koinobiont endoparasitoid, also present. At least some of the female X. ater were clearly freshly emerged, as matings were underway, though other females rested unattended and may or may not have already mated. Males were mostly seen flying close to the woodpile, apparently searching for females, but without forming evident aggregations. In the braconid, about three matings were observed, each between one male and one female.

Xorides ater belongs to the Xoridinae, a subfamily considered the putative sister group to all other Darwin wasps. It is a solitary idiobiont ectoparasitoid, meaning it kills or permanently paralyses its host at oviposition, and by analogy with related congeners it is presumed to develop on the body of a beetle larva, prepupa or pupa. Neither the host range nor the developmental biology of X. ater is properly understood, and before this report there was no account at all of its courtship and mating behaviour.

What made the triplet matings remarkable was how unremarkable they seemed to the participants. The three-wasp assemblages were as quiescent as the normal copulae seen at the same time and appeared to persist for an equivalent period, at least five to ten minutes, with separation occurring only when the insects were collected. There was no evident jostling and no attempt by one male to dislodge the other; the males seemed extremely relaxed, with legs twitching and often held outstretched and clear of everything, usually with at least one hind leg touching the substrate but sometimes with all legs completely free. The females, too, appeared unperturbed and made no attempt to dislodge either male. No courtship or commencement of copulation was witnessed, so whether the two males attach simultaneously or sequentially remains unknown.

The copulatory stance itself was unusual for the genus. In most Hymenoptera the male genitalia are orthandrous, producing a mating stance in which the male is mounted on top of the female, head-to-head, with the apex of his metasoma curled below hers. In the X. ater pairings observed, whether involving one or two males, the male instead fell back and flipped through 180 degrees, ending up with the heads looking in opposite directions and the male on his back, often with only his genitalia attached to anything. This orientation has been reported in the tryphonine subgenus Netelia, though another species of the same subgenus shows the more typical male-on-top stance, suggesting plasticity within the group. Mating behaviour has been described for only two other Xorides species, both in the laboratory, and in both the male was mounted head-to-head on the female, a stance that differs markedly from what was seen here.

The researchers note that this reversed position may be one factor facilitating matings involving two males, since more space around the female’s external genitalia is exposed than in a male-on-top stance. The visible genital attachment appeared normal: the single paramere that could be seen seemed to be normally deployed, attached to the female’s sixth sternite, presumably by the usual pincer action of the cuspis and digitus of the volsella. However, the other side of the male’s genitalia was not visible, and how it was deployed, and indeed whether insemination would be possible in the triplets, could not be deduced. Whether sperm transfer from one or both males takes place is a key question the study cannot resolve.

Beyond the mating observations, the report adds ecological information drawn from museum collections. All 71 specimens of X. ater found in Swiss collections had been collected above 980 metres altitude, and a narrow flight period from late June to early August indicates that the species is univoltine. Three of the 26 collection events included at least a dozen males, while females were always collected only one or two at a time, supporting the field impression of high male abundance. The few and mostly unconfirmed host records in the literature all involve cerambycid beetles in conifers. The only suitably sized adult beetles seen at the site were three species of Callidium that occur in Switzerland, but there was no direct evidence that any of these was serving as a host. Female wasps seen were rather uniform in size, whereas males varied substantially, suggesting that hosts of different sizes had been accepted, though whether of the same or different species is unknown.

Why triplet matings occur at all remains open. The observers found several non-mating females on the woodpile that attracted little attention from the numerous males, and they saw nothing resembling scramble competition, so there is no reason to believe the triplet matings were a unique result of high male density and a shortage of females. If sperm transfer from both males takes place, the authors speculate that it might be a female strategy to secure a higher genetic mix for her progeny or to offset the risk of mating with an infertile male, or that simultaneous mating could reduce the time and predation costs of mating successively with several males. From the male side, it might simply be an unintended consequence of the scramble competition frequently observed in parasitoids of wood-inhabiting insects, or a trade-off in avoiding conflict. If instead triplet matings are sterile, they could constitute a fitness impediment by producing a higher proportion of males than would be optimal.

The authors caution that additional observations under natural circumstances are needed to assess whether triplet-style matings are common in the genus or restricted to X. ater, and further work is required to establish whether sperm transfer from one or both males is successfully achieved and whether any morphological modifications might facilitate it. For now, the report stands as the first documented case of more than one male achieving simultaneous copulation with a single female in any species of Ichneumonoidea outside a brief mention of Rhyssa persuasoria under conditions of disturbance, and a striking illustration of how much basic biology remains to be discovered even in familiar insect groups.

Subject of Research: Triplet-style mating behaviour in the Darwin wasp Xorides ater observed in the Swiss Alps

Article Title: Two males, one female: triplet-style mating behaviour in the Darwin wasp Xorides ater (Gravenhorst, 1829) ( Hymenoptera , Ichneumonidae , Xoridinae ) in the Swiss Alps

Article References: Shaw, M. R., Giannotta, M., Herrera-Flórez, A. F., & Klopfstein, S. (2021). Two males, one female: triplet-style mating behaviour in the Darwin wasp Xorides ater (Gravenhorst, 1829) (Hymenoptera, Ichneumonidae, Xoridinae) in the Swiss Alps. Alpine Entomology, 5, 15-22. https://doi.org/10.3897/alpento.5.64803

Image Credits: AI Generated

DOI: 10.3897/alpento.5.64803

Keywords: Xorides ater, Darwin wasps, Ichneumonidae, mating behaviour, Swiss Alps, parasitoid, entomology, triplet mating, wood-boring beetles, Picea abies, haplo-diploidy, field observation