Intraspecific competition studies in social insects have focused mainly at the colony level (Passera et al., 1996; Thomas et al., 2005; Sanada-Morimura et al., 2006; Cronin et al., 2012; Lloyd and Poulin, 2014; Blight et al., 2016) and at the population level (Holway et al., 1998; Adams and Tschinkel, 2001; Korb and Linsenmair, 2001; Boulay et al., 2007; Grohmann et al., 2010; Bourguignon et al., 2011; Perdereau et al., 2011; Tsuji, 2013; Pringle and Tarnita, 2017); however, the effects of intraspecific competition at the individual level are poorly studied.

Termites are eusocial species that live in colonies organized into three castes (Thorne, 1996; Korb and Hartfelder, 2008; Eggleton, 2011): (i) primary reproductives involved in reproduction, (ii) soldiers involved in defense against intruders, and (iii) true workers (or pseudergates) involved in nest keeping and taking care of juveniles and other castes and also, together with soldiers, in defense. Mandibles of termite soldiers are morphologically adapted to defense (Scholtz et al., 2008); hence, soldiers are unable to feed themselves and they depend on pseudergates or workers for survival (Haverty, 1977; Henderson, 1998). Consequently, investment in the production of soldiers involves a trade-off between colony maintenance costs and defense benefits (Noirot, 1989; Chouvenc et al., 2015). Pseudergates are undifferentiated and totipotent individuals who may develop into other castes such as soldiers or primary reproductives, or they may spend their whole life as pseudergates, depending on environmental conditions (Ogino et al., 1993; Roisin and Korb, 2011). Thus, environmental factors such as colony conditions (colony size, reproductive status, caste ratio, resource availability, etc.), temperature and seasonality significantly affect hormone and gene expressions (Scharf et al., 2007; Miura and Scharf, 2011) which trigger the differentiation from pseudergate to presoldier instar. This process may also be enhanced by ecological factors such as inter and/or intraspecific competition as a form of induced defense at the colony level (Passera et al., 1996; Aguilera-Olivares et al., 2017).

Termite species may be classified according to their nesting behavior in two main categories: separate-piece nesting termites, where foraging substrates and nesting substrate are different; and single-piece nesting termites, who spent their whole life in the same piece of wood (Abe, 1991; Shellman-Reeve, 1997). The colonies of separate-piece nesting termites are composed commonly by true workers and their size ranges from thousands to millions of individuals who can forage and move for thousands of square meters. On the other hand, colonies of single-piece nesting termites are characterized by the presence of pseudergates and colony size (usually no larger than a few thousand individuals) is limited by the volume of the piece of wood where they thrive (Abe, 1991; Shellman-Reeve, 1997; Korb and Hartfelder, 2008; Mizumoto and Bourguignon, 2021). If several colonies of single-piece nesting termites share the same substrate, inter and/or intra-specific competition is expected to occur, particularly when nesting resources become limited (Thorne et al., 2003; Ripa and Luppichini, 2004; Amarillo-Suárez et al., 2011).

Neotermes chilensis (Blattodea: Kalortermitidae) (Blanchard, 1851) is a single-piece nesting termite (i.e., it completes its life cycle within a finite resource) which uses dry scapes (stems of inflorescences) of the bromeliads Puya alpestris ssp. zoellneri [ex P. berteroniana—Zizka et al. (2013)] and P. chilensis (Molina, 1782) among its hosts (Aguilera-Olivares et al., 2015). In previous work at the same study site (Aguilera-Olivares et al., 2017), we reported that about half of the scapes were occupied by a single colony (unicolonial substrates) and the other half contained between 2 and 9 colonies (multicolonial substrates). Additionally, we observed galleries interconnecting two colonies within a scape which had been blocked with sawdust, probably following an agonistic interaction. Under this intraspecific competition scenario, the soldiers/non-soldiers ratio significantly increased. This implies that colonies exposed to competition have an inherently higher cost due to the increased production and maintenance of soldiers, and since resources are finite in this termite, an increased developmental stress is imposed on these soldiers. N. chilensis stands as an interesting species to study the effect of intraspecific competition in an eusocial insect because: 1) it lives on a limited resource; 2) it is not able to move to another resource, so it must compete when the nesting substrate is shared with other colonies; 3) due to low colony sizes (no more than 500 individuals) it is easier to study than other termite species with typical colony sizes in the thousands to millions of individuals; and 4) about 90% of individuals inside a colony are pseudergates, i.e. totipotential individuals which could develop as soldiers, alates or remain as pseudergates their whole life according environmental signals. Moreover, the soldier caste of N. chilensis appears as particularly relevant in terms of intraspecific competition at the individual level, especially the consequences of stress during development.

Developmental stability is the ability of an organism to produce its developmentally programed phenotype despite epigenetic perturbations; contrastingly, developmental instability occurs when an organism is unable to buffer those perturbations (Markow, 1995). In species with symmetric morphological traits, the developmentally programed phenotype is one with perfect symmetry of such traits; and any random deviations from this symmetry on individuals inside a population leads to fluctuating asymmetry (Palmer, 1994; Graham et al., 2010). These deviations may be associated with genetic stress such as hybridization (Graham and Felley, 1985; Handy et al., 2004), and with environmental stress such as food limitation (Swaddle and Witter, 1994), heat shock (Hosken et al., 2000), and intraspecific competition (Witter and Swaddle, 1994). In insects, there is evidence that high levels of larval density constitute an environmental stressor inducing fluctuating asymmetry (Clarke and McKenzie, 1992; Gibbs and Breuker, 2006; Beasley et al., 2013), which may be caused by the effects of intraspecific competition due to a reduction of food availability (Hunt and Allen, 2000). Nevertheless, the effect on fluctuating asymmetry of another scenario of intraspecific competition caused by a restriction in the use of a nesting substrate, as occurs when two or more insect colonies share the same nest, has not been assessed.

On the other hand, body size is a life-history trait which has been used in insects as an indicator of developmental stress (Warren et al., 2006; Couret and Benedict, 2014). Thus, the effect of factors such as altitude (Cushman et al., 1993; Smith et al., 2007; Hoiss et al., 2012), temperature (Bochdanovits and De Jong, 2003), and competition (Heinrich, 1993; Warren et al., 2006; Amarillo-Suárez et al., 2011; Wills et al., 2014; Korallo-Vinarskaya et al., 2015) during insect development are important predictors of body size outcome. When interference competition occurs, body size generally determines who will dominate the resources; thus, larger individuals are frequently more successful than smaller ones (Heinrich and Bartholomew, 1979; Otronen, 1988; Heinrich, 1993; Zobel and Paxton, 2007; Bespalova and Helms, 2014). In termites, significantly higher fluctuating asymmetry has been detected in soldiers of Coptotermes formosanus Shiraki, which develop within incipient colonies (instable and stressful scenario) in comparison with soldiers which develop within mature colonies (more stable environment) (Chouvenc et al., 2014a). Furthermore, studies in three Reticulitermes (R. speratus, R. virginicus, and R. flavipes) and two Cryptotermes (C. secundus and C. domesticus) termite species have shown increased female-to-male ratio because female soldiers are larger than male soldiers and thus have a better chance to win in a physical encounter (Matsuura, 2006; Muller and Korb, 2008). In summary, intraspecific competition is a stressful scenario for termite colonies which could affect body size and symmetry properties of developing individuals and hence their performance.

The aim of this work was to study the effects of intraspecific competition at the individual level in an eusocial insect. If competition in the context of finite resources induces colonies to invest in larger and/or more numerous soldiers, it would directly impose a developmental stress on these individuals. We hypothesized that soldiers of N. chilensis developing in colonies that share a nesting resource with other colonies (high intraspecific competition scenario) would show significantly higher values of fluctuating asymmetry and they would be larger than soldiers whose colonies occur singly within a nesting resource (low intraspecific competition scenario).

Many abiotic and biotic factors may affect the normal ontogenetic development of insects (Palmer, 1994; Markow, 1995; Graham et al., 2010; Couret and Benedict, 2014). In this work, termite colonies were collected from a common site; hence, those present in unicolonial and multicolonial substrates were exposed to the same abiotic factors. Additionally, N. chilensis was the only termite species present in the study site and N. chilensis termites were the only insects found inside the scapes. Hence, intraspecific competition operating on a termite colony as a whole is expected to be the most important stress factor that may differentially affect unicolonial and multicolonial soldier individual development. All the indexes and analyses used in this work showed significantly higher levels of fluctuating asymmetry in soldiers developed in multicolonial substrates than soldiers developed in unicolonial substrates. Thus, our results showed that the presence of neighboring colonies generated developmental instability in soldier differentiation from pseudergates.

When two or more colonies share a nesting substrate, several scenarios could follow, e.g., their galleries could meet and colonies could fuse, they could fight against each other with the possible consequence of the elimination of one of them, or they could avoid each other. In exotic invasive species, nestmate recognition mechanisms generally fail because genetic diversity in the population is low and relatedness among nestmates is high. This may lead to colony fusion, as has been shown in termites (Perdereau et al., 2010, 2011; Lee et al., 2019) and ants (Holway et al., 1998; Eyer et al., 2018). On the other hand, in native species, where genetic diversity in the population is high and relatedness between colonies is low, colony fusion is rare (Shelton and Grace, 1996; Deheer and Vargo, 2004; Smith et al., 2012). In the population of the native species N. chilensis under study, the mean relatedness of nestmate pairs was shown to be 0.465 ± 0.0085 (mean ± sem), corresponding closely to that expected for full siblings (r = 0.50) and the relatedness between non-nestmate pairs was close to zero (Aguilera-Olivares et al., 2015). This suggests that if two or more colonies meet within the nesting substrate, colony fusion would be rare or inexistent. In fact, nestmate recognition was reported in N. chilensis, soldiers being more aggressive toward non-nestmates than toward nestmates from different castes (Aguilera-Olivares et al., 2016a,b). These observations suggest that individuals from two colonies of N. chilensis within a scape either fight and eliminate opponent colonies or avoid them. Additionally, the colony of origin had a very slight if any effect when its inclusion or exclusion as random factor was considered in the models; thus, despite the fact that individuals inside a colony of N. chilensis are closely related, there is enough variation among soldiers to be considered independent samples, suggesting that the response to intraspecific competition in our analyses is independent of their degree of relatedness.

Body size is a life-history trait that has been used in insects as an indicator of developmental stress (Warren et al., 2006; Couret and Benedict, 2014). Intraspecific competition as a stress factor has been described in literature (Heinrich, 1993; Warren et al., 2006; Amarillo-Suárez et al., 2011; Wills et al., 2014; Korallo-Vinarskaya et al., 2015). In territorial species where interference competition occurs with physical encounters displayed, larger individuals have better chances to win a fight and monopolize resources than smaller individuals (Price et al., 2011; Bespalova and Helms, 2014; Holland et al., 2021). In our study, we demonstrated that soldiers from multicolonial substrates had significantly larger heads than those from unicolonial substrates, strongly suggesting that in a competitive scenario, colonies produce larger soldiers which increase the chance to win fights and monopolize resources. Furthermore, Muller and Korb (2008) showed that females of Zootermopsis nevadensis (Hagen, 1858), a single-piece nesting termite, develop more often into soldiers than males because they are significantly larger, which is an advantage when blocking holes/galleries or fighting with other individuals (Zobel and Paxton, 2007). In our work, quitinized traits of N. chilensis that showed differences in size between uni- and multicolonial substrates are closely related to defensive tasks. Thus, head traits (head area, head perimeter, head length, and head maximum width) could be related to the blocking of galleries with the head and to more developed muscles to produce stronger bites (Bespalova and Helms, 2014). When the scape used by two or more colonies is depleted and the tunnels dug by members of different colonies finally meet, all of these traits could improve the chance to win a fight in a hypothetical scenario of interference intraspecific competition (Heinrich and Bartholomew, 1979; Otronen, 1988; Heinrich, 1993; Zobel and Paxton, 2007; Bespalova and Helms, 2014).

Soldiers totally depend on workers or pseudergates for survival (Haverty, 1977; Henderson, 1998); thus, the soldier production represent a trade-off at colony level between benefits (colony defense) and costs (investment in soldier nurturing) (Noirot, 1989; Chouvenc et al., 2015). Additionally, in incipient colonies some larvae must develops into soldiers instead of workers in order to maintain a relatively stable soldier proportion, which could have consequences in colony growth as a result of a reduction of worker number (Chouvenc et al., 2015). In Coptotermes species, a subterranean and separate-piece nesting termite, incipient colonies with low numbers of workers and hence a reduced nurturing capacity produce smaller and more asymmetric soldiers (i.e., low quality soldiers produced at low cost) as an strategy to keep a stable caste ratio which produces a delay on the growth of colonies (Chouvenc and Su, 2014b; Chouvenc et al., 2015, 2017); while soldiers from mature colonies which contain high number of workers and hence exhibit high nurturing capacity, are larger and symmetric (high quality soldiers produced at high cost) (Chouvenc and Su, 2014b; Chouvenc et al., 2017). In single-piece nesting termites, such as N. chilensis, colony survival is directly related with its capacity to get and defend resources; thus, despite the high cost to produce large soldiers in small colonies, larger soldier production could be an adaptative strategy to avoid being outcompeted.

This study shows for first time the effect of intraspecific competition at the individual level in an eusocial insect. Soldiers of N. chilensis which developed from pseudergates in a high intraspecific competition scenario (multicolonial substrates) were larger and exhibited more asymmetric traits than soldiers which developed in a low intraspecific competition scenario (unicolonial substrates). While the large body size in a soldier could improve its chance to win a physical contest with a non-nestmate opponent, the effects of deviations from perfect symmetry on soldier performance are not clear. In single-piece nesting termites, such as N. chilensis, colony survival is directly related with its capacity to get and defend resources; thus, despite the high cost to produce large soldiers in small colonies, larger soldier production could be an adaptative strategy to avoid being outcompeted. Having shown the effects of intraspecific competition on body size and asymmetry of soldiers, it would be interesting to test the fitness consequences of larger body size and fluctuating asymmetry in soldiers of N. chilensis and its consequences for colony life. Additionally, the effects could be tested in other single-piece nesting termites such as the worldwide distributed (except in Asia) Cryptotermes brevis (Walker, 1853) and in Z. nevadensis, where multicolonial substrates have been reported.

The original contributions presented in this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

DA-O, LF-P, BT-G, and HN: study conception and design. BT-G and DA-O: material preparation and data collection. AA, BT-G, and DA-O: statistical analyses. DA-O: writing first draft of the manuscript. All authors commented on previous versions of the manuscript and read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.