By Zhuang-Dong Bai

Zhuang-Dong’s study, exploring the evolution of a harmonious behavioral strategy to reduce conflict over reproduction in the lower termite Reticulitermes labralis can be found here. Like it -> check him out on ResearchGate.

Eusocial insects such as ants and termites represent an important biomass on Earth and ecologically dominate many ecosystems (Tuma et al. 2020; Wilson 1990). One key to this success is the reproductive division of labor among colony individuals, meaning that a minority of specialist individuals monopolize reproduction, whereas workers and soldiers raise the offspring of these breeders as helpers (Wilson 1971). In some species, when the primary queens are too old or lost, other colony individuals have the capability to develop into replacement queens soon after to undertake reproduction. In these species, the differentiation of reproductive individuals in the colony is particularly important for its prosperity.
However, my supervisor put forward a simple but profound question: “If most or all colony individuals have the same reproductive potential to differentiate into queens, conflicts are difficult to avoid because there will be a similar reproductive interest among colony members, are there any behavioral cues performed by individuals to indicate that they have priority to become replacement reproductives?”
By reviewing the literature, we found that research of this question has mainly focused on the primitive eusocial wasp Ropalidia marginata, in which, when the wasp queen is lost or removed, one and only one of the workers becomes hyper-aggressive and is then considered as the next queen of the colony. Surprisingly, this species has not just one designated heir, but a long reproductive queue and these workers successively take over the role of egg laying (Bang and Gadagkar 2012; Bhadra and Gadagkar 2008). In the termite Cryptotermes secundus, it has been first reported that butting and proctodeal trophallaxis (anal feeding) are the ‘dominance’ behaviors that indicate which workers inherit the breeding position when reproductives are absent (Hoffmann and Korb 2011). Both examples suggest that some differences of an individuals’ behavioral profile may prevent overt conflict in colonies during the replacement of reproductives. However, these behavioral traits are all shown by workers in the absence of the queen. Do workers also show some behavioral traits in the presence of the queen and do these predict differential outcomes when they have a chance to differentiate in the future?
Here, to study this aspect, we used the lower termite species Reticulitermes labralis as experimental subject, whose workers may develop into replacement queens when queens are artificially removed or dead (Su et al., 2017). First, we collected five colonies of Reticulitermes labralis from northern Qinling Mountains, Xi’an, Shaanxi Province, China (108° 46´ E, 34° 00´ N). Then, we put 100 termites (96 workers and 4 soldiers) into a Petri dish with moist filter paper at the bottom. In total, we set up 15 groups from five colonies, and all these 15 groups were reared in a controlled climate chamber to produce replacement reproductives. After one month, one or more replacement reproductives were differentiated in each group. We randomly picked out
50 individuals (48 workers, one soldier, and one replacement queen) from each of the 15 groups and kept them in another Petri dish for 2 days without disturbance to allow the termites to adapt to their new environment. Then, workers were marked with an individual color code consisting of one or two dots of paint on the abdomen and/or on the thorax.

After two days, we recorded termite behaviors of these 15 groups with a camera for one hour for subsequent analysis. See an example of 10-seconds recording for two groups here:

Then, we removed the replacement queens from the Petri dishes and continued to observe the individuals until a new replacement queen appeared in each group. In the video, we tracked what the behavior of the replacement queen (when it was still at worker status) was towards other individuals in the presence of the queen. Moreover, we selected three female workers that did not differentiate into queens in each group as control. We kept observing for 3 days after the appearance of the first replacement queen to be sure that this worker who differentiated into a replacement queen was the only first one to differentiate. The behaviors measured were (i) the number of butting (one worker moves repeatedly backwards and forwards to contact another worker), (ii) the number of antennation with other workers (contacting other workers with antennae), (iii) the number of antennation with the queen (contacting the queen with antennae), (iv) the number of allogrooming by other workers, (v) the number of mouth-to-mouth feeding occurrences (as a receiver), (vi) the number of anus-to-mouth feeding occurrences (as a donor), and (vii) the locomotion time.
Our results showed that when the queen was present, the workers who successfully replaced queens in the future had three different behavioral profiles compared to workers which did not develop into queens. That is, in a group with a queen present,
the workers who differentiated into replacement queens moved less, performed more anal feeding, and were groomed more than others.
Our study revealed that the significant differences in the behaviors exhibited by workers in a group with a queen present may give priority to these workers to differentiate into replacement queens when the queen is removed. And this could be considered as an important behavioral mechanism to reduce intra-colony reproduction conflicts. Anal feeding, allogrooming, and weak mobility of workers might function as the notable behaviors indicating their commitment in the differentiation pathway. These specific workers may be regarded as the “cryptic heir” designated to be the next queen (Bhadra & Gadagkar, 2008). Moreover, these could reduce the reproductive competition among workers, like the reproductive queue without overt conflict in the primitively eusocial wasp Ropalidia marginata (Bang & Gadagkar, 2012). Instead, these workers ensure that the colony will be quickly headed by a new queen in case of the sudden death of the original one.

References
Bang A, Gadagkar R, 2012. Reproductive queue without overt conflict in the primitively eusocial wasp ropalidia marginata. P Natl Acad Sci USA 109:14494-14499.
Bhadra A, Gadagkar R, 2008. We know that the wasps ‘know’: Cryptic successors to the queen in ropalidia marginata. Biol Lett 4:634-637.
Hoffmann K, Korb J, 2011. Is there conflict over direct reproduction in lower termite colonies? Anim Behav 81:265-274.
Su X, Yang X, Li J, Xing L, Liu H, et al., 2017. The transition path from female workers to neotenic reproductives in the termite reticulitermes labralis. Evol Dev 19:218-226.
Tuma J, Eggleton P, Fayle TM, 2020. Ant-termite interactions: An important but under-explored ecological linkage. Biol Rev 95:555-572.
Wilson EO, 1971. The insect societies. Cambridge, Massachusetts: Harvard University Press.