Molecular Phylogeny Reveals the Past Transoceanic Voyages of Drywood Termites (Isoptera, Kalotermitidae).

Termites are major decomposers in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae form the second-largest termite family and are distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes of ∼120 species, about 27% of kalotermitid diversity, including representatives of 21 of the 23 kalotermitid genera. Our mitochondrial genome phylogenetic trees were corroborated by phylogenies inferred from nuclear ultraconserved elements derived from a subset of 28 species. We found that extant kalotermitids shared a common ancestor 84 Ma (75-93 Ma 95% highest posterior density), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the ∼40 disjunctions among biogeographic realms were dated at <50 Ma, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for critical taxonomic revision and future comparative analyses of Kalotermitidae.

Evidence of cospeciation between termites and their gut bacteria on a geological time scale.

Termites host diverse communities of gut microbes, including many bacterial lineages only found in this habitat. The bacteria endemic to termite guts are transmitted via two routes: a vertical route from parent colonies to daughter colonies and a horizontal route between colonies sometimes belonging to different termite species. The relative importance of both transmission routes in shaping the gut microbiota of termites remains unknown. Using bacterial marker genes derived from the gut metagenomes of 197 termites and one Cryptocercus cockroach, we show that bacteria endemic to termite guts are mostly transferred vertically. We identified 18 lineages of gut bacteria showing cophylogenetic patterns with termites over tens of millions of years. Horizontal transfer rates estimated for 16 bacterial lineages were within the range of those estimated for 15 mitochondrial genes, suggesting that horizontal transfers are uncommon and vertical transfers are the dominant transmission route in these lineages. Some of these associations probably date back more than 150 million years and are an order of magnitude older than the cophylogenetic patterns between mammalian hosts and their gut bacteria. Our results suggest that termites have cospeciated with their gut bacteria since first appearing in the geological record.

Impact of Wood Age on Termite Microbial Assemblages.

The decomposition of wood and detritus is challenging to most macroscopic organisms due to the recalcitrant nature of lignocellulose. Moreover, woody plants often protect themselves by synthesizing toxic or nocent compounds which infuse their tissues. Termites are essential wood decomposers in warmer terrestrial ecosystems and, as such, they have to cope with high concentrations of plant toxins in wood. In this paper, we evaluated the influence of wood age on the gut microbial (bacterial and fungal) communities associated with the termites Reticulitermes flavipes (Rhinotermitidae) (Kollar, 1837) and Microcerotermes biroi (Termitidae) (Desneux, 1905). We confirmed that the secondary metabolite concentration decreased with wood age. We identified a core microbial consortium maintained in the gut of R. flavipes and M. biroi and found that its diversity and composition were not altered by the wood age. Therefore, the concentration of secondary metabolites had no effect on the termite gut microbiome. We also found that both termite feeding activities and wood age affect the wood microbiome. Whether the increasing relative abundance of microbes with termite activities is beneficial to the termites is unknown and remains to be investigated. IMPORTANCE Termites can feed on wood thanks to their association with their gut microbes. However, the current understanding of termites as holobiont is limited. To our knowledge, no studies comprehensively reveal the influence of wood age on the termite-associated microbial assemblage. The wood of many tree species contains high concentrations of plant toxins that can vary with their age and may influence microbes. Here, we studied the impact of Norway spruce wood of varying ages and terpene concentrations on the microbial communities associated with the termites Reticulitermes flavipes (Rhinotermitidae) and Microcerotermes biroi (Termitidae). We performed a bacterial 16S rRNA and fungal ITS2 metabarcoding study to reveal the microbial communities associated with R. flavipes and M. biroi and their impact on shaping the wood microbiome. We noted that a stable core microbiome in the termites was unaltered by the feeding substrate, while termite activities influenced the wood microbiome, suggesting that plant secondary metabolites have negligible effects on the termite gut microbiome. Hence, our study shed new insights into the termite-associated microbial assemblage under the influence of varying amounts of terpene content in wood and provides a groundwork for future investigations for developing symbiont-mediated termite control measures.

The Trail-Following Communication in Stylotermes faveolus and S. halumicus (Blattodea, Isoptera, Stylotermitidae).

Stylotermitidae appear peculiar among all termites, feeding in trunks of living trees in South Asia only. The difficulty to collect them limits the ability to study them, and they thus still belong to critically unknown groups in respect to their biology. We used a combination of microscopic observations, chemical analysis and behavioural tests, to determine the source and chemical nature of the trail-following pheromone of Stylotermes faveolus from India and S. halumicus from Taiwan. The sternal gland located at the 5th abdominal segment was the exclusive source of the trail-following pheromone in both S. faveolus and S. halumicus, and it is made up of class I, II and III secretory cells. Using gas chromatography coupled mass spectrometry, (3Z)-dodec-3-en-1-ol (DOE) was identified as the trail-following pheromone which elicits strong behavioural responses in workers at a threshold around 10- 4 ng/cm and 0.1 ng/gland. Our results confirm the switch from complex aldehyde trail-following pheromones occurring in the basal groups to simpler linear alcohols in the ancestor of Kalotermitidae and Neoisoptera.

Termite dispersal is influenced by their diet.

Termites feed on vegetal matter at various stages of decomposition. Lineages of wood- and soil-feeding termites are distributed across terrestrial ecosystems located between 45°N and 45°S of latitude, a distribution they acquired through many transoceanic dispersal events. While wood-feeding termites often live in the wood on which they feed and are efficient at dispersing across oceans by rafting, soil-feeders are believed to be poor dispersers. Therefore, their distribution across multiple continents requires an explanation. Here, we reconstructed the historical biogeography and the ancestral diet of termites using mitochondrial genomes and δ13C and δ15N stable isotope measurements obtained from 324 termite samples collected in five biogeographic realms. Our biogeographic models showed that wood-feeders are better at dispersing across oceans than soil-feeders, further corroborated by the presence of wood-feeders on remote islands devoid of soil-feeders. However, our ancestral range reconstructions identified 33 dispersal events among biogeographic realms, 18 of which were performed by soil-feeders. Therefore, despite their lower dispersal ability, soil-feeders performed several transoceanic dispersals that shaped the distribution of modern termites.

Using ultraconserved elements to reconstruct the termite tree of life.

The phylogenetic history of termites has been investigated using mitochondrial genomes and transcriptomes. However, both sets of markers have specific limitations. Mitochondrial genomes represent a single genetic marker likely to yield phylogenetic trees presenting incongruences with species trees, and transcriptomes can only be obtained from well-preserved samples. In contrast, ultraconserved elements (UCEs) include a great many independent markers that can be retrieved from poorly preserved samples. Here, we designed termite-specific baits targeting 50,616 UCE loci. We tested our UCE bait set on 42 samples of termites and three samples of Cryptocercus, for which we generated low-coverage highly-fragmented genome assemblies and successfully extracted in silico between 3,426 to 42,860 non-duplicated UCEs per sample. Our maximum likelihood phylogenetic tree, reconstructed using the 5,934 UCE loci retrieved from upward of 75% of samples, was congruent with transcriptome-based phylogenies, demonstrating that our UCE bait set is reliable and phylogenetically informative. Combined with non-destructive DNA extraction protocols, our UCE bait set provides the tool needed to carry out a global taxonomic revision of termites based on poorly preserved specimens such as old museum samples. The Termite UCE database is maintained at: https://github.com/oist/TER-UCE-DB/.

Termite evolution: mutualistic associations, key innovations, and the rise of Termitidae.

Termites are a clade of eusocial wood-feeding roaches with > 3000 described species. Eusociality emerged ~ 150 million years ago in the ancestor of modern termites, which, since then, have acquired and sometimes lost a series of adaptive traits defining of their evolution. Termites primarily feed on wood, and digest cellulose in association with their obligatory nutritional mutualistic gut microbes. Recent advances in our understanding of termite phylogenetic relationships have served to provide a tentative timeline for the emergence of innovative traits and their consequences on the ecological success of termites. While all "lower" termites rely on cellulolytic protists to digest wood, "higher" termites (Termitidae), which comprise ~ 70% of termite species, do not rely on protists for digestion. The loss of protists in Termitidae was a critical evolutionary step that fostered the emergence of novel traits, resulting in a diversification of morphology, diets, and niches to an extent unattained by "lower" termites. However, the mechanisms that led to the initial loss of protists and the succession of events that took place in the termite gut remain speculative. In this review, we provide an overview of the key innovative traits acquired by termites during their evolution, which ultimately set the stage for the emergence of "higher" termites. We then discuss two hypotheses concerning the loss of protists in Termitidae, either through an externalization of the digestion or a dietary transition. Finally, we argue that many aspects of termite evolution remain speculative, as most termite biological diversity and evolutionary trajectories have yet to be explored.

Evidence for reduced immune gene diversity and activity during the evolution of termites.

The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well-studied immune systems of cells and individuals, little is known about the origins of immunity during the transition to eusociality, a major evolutionary transition comparable to the evolution of multicellular organisms from single-celled ancestors. We aimed to tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We find that key transitions in termite sociality are correlated with immune gene family contractions. In cross-species comparisons of immune gene expression, we find evidence for a caste-specific social defence system in termites, which appears to operate at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity may have entailed a fundamental reshaping of the immune system optimized for group over individual defence.

Termites Are Associated with External Species-Specific Bacterial Communities.

All termites have established a wide range of associations with symbiotic microbes in their guts. Some termite species are also associated with microbes that grow in their nests, but the prevalence of these associations remains largely unknown. Here, we studied the bacterial communities associated with the termites and galleries of three wood-feeding termite species by using 16S rRNA gene amplicon sequencing. We found that the compositions of bacterial communities among termite bodies, termite galleries, and control wood fragments devoid of termite activities differ in a species-specific manner. Termite galleries were enriched in bacterial operational taxonomic units (OTUs) belonging to Rhizobiales and Actinobacteria, which were often shared by several termite species. The abundance of several bacterial OTUs, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus, was reduced in termite galleries. Our results demonstrate that both termite guts and termite galleries harbor unique bacterial communities.IMPORTANCE As is the case for all ecosystem engineers, termites impact their habitat by their activities, potentially affecting bacterial communities. Here, we studied three wood-feeding termite species and found that they influence the composition of the bacterial communities in their surrounding environment. Termite activities have positive effects on Rhizobiales and Actinobacteria abundance and negative effects on the abundance of several ubiquitous genera, such as Bacillus, Clostridium, Corynebacterium, and Staphylococcus Our results demonstrate that termite galleries harbor unique bacterial communities.

Trail-Following Pheromones in the Termite Subfamily Syntermitinae (Blattodea, Termitoidae, Termitidae).

Trail-following behavior is a key to ecological success of termites, allowing them to orient themselves between the nesting and foraging sites. This behavior is controlled by specific trail-following pheromones produced by the abdominal sternal gland occurring in all termite species and developmental stages. Trail-following communication has been studied in a broad spectrum of species, but the "higher" termites (i.e. Termitidae) from the subfamily Syntermitinae remain surprisingly neglected. To fill this gap, we studied the trail-following pheromone in six genera and nine species of Syntermitinae. Our chemical and behavioral experiments showed that (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol is the single component of the pheromone of all the termite species studied, except for Silvestritermes euamignathus. This species produces both (3Z,6Z)-dodeca-3,6-dien-1-ol and neocembrene, but only (3Z,6Z)-dodeca-3,6-dien-1-ol elicits trail-following behavior. Our results indicate the importance of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol, the most widespread communication compound in termites, but also the repeated switches to other common pheromones as exemplified by S. euamignathus.

Historical biogeography of the termite clade Rhinotermitinae (Blattodea: Isoptera).

Termites are the principal decomposers in tropical and subtropical ecosystems around the world. Time-calibrated molecular phylogenies show that some lineages of Neoisoptera diversified during the Oligocene and Miocene, and acquired their pantropical distribution through transoceanic dispersal events, probably by rafting in wood. In this paper, we intend to resolve the historical biogeography of one of the earliest branching lineages of Neoisoptera, the Rhinotermitinae. We used the mitochondrial genomes of 27 species of Rhinotermitinae to build two robust time-calibrated phylogenetic trees that we used to reconstruct the ancestral distribution of the group. Our analyses support the monophyly of Rhinotermitinae and all genera of Rhinotermitinae. Our molecular clock trees provided time estimations that diverged by up to 15.6 million years depending on whether or not 3rd codon positions were included. Rhinotermitinae arose 50.4-64.6 Ma (41.7-74.5 Ma 95% HPD). We detected four disjunctions among biogeographic realms, the earliest of which occurred 41.0-56.6 Ma (33.0-65.8 Ma 95% HPD), and the latest of which occurred 20.3-34.2 Ma (15.9-40.4 Ma 95% HPD). These results show that the Rhinotermitinae acquired their distribution through a combination of transoceanic dispersals and dispersals across land bridges.

Molecular Identity of Holomastigotes (Spirotrichonymphea, Parabasalia) with Descriptions of Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp.

Holomastigotes is a protist genus (Parabasalia: Spirotrichonymphea) that resides in the hindguts of "lower" termites. It can be distinguished from other parabasalids by spiral flagellar bands that run along the entire length of the cell, an anterior nucleus, a reduced or absent axostyle, the presence of spherical vesicles inside the cells, and the absence of ingested wood particles. Eight species have been described based on their morphology so far, although no molecular data were available prior to this study. We determined the 18S rRNA gene sequences of Holomastigotes from the hindguts of Hodotermopsis sjostedti, Reticulitermes flavipes, Reticulitermes lucifugus, and Reticulitermes tibialis. Phylogenetic analyses placed all sequences in an exclusive and well-supported clade with the type species, Holomastigotes elongatum from R. lucifugus. However, the phylogenetic position of Holomastigotes within the Spirotrichonymphea was not resolved. We describe two new species, Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp., inhabiting the hindguts of R. flavipes and R. tibialis, respectively.

Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites, Ecosystem Engineers of the Tropics.

The higher termites (Termitidae) are keystone species and ecosystem engineers. They have exceptional biomass and play important roles in decomposition of dead plant matter, in soil manipulation, and as the primary food for many animals, especially in the tropics. Higher termites are most diverse in rainforests, with estimated origins in the late Eocene (∼54 Ma), postdating the breakup of Pangaea and Gondwana when most continents became separated. Since termites are poor fliers, their origin and spread across the globe requires alternative explanation. Here, we show that higher termites originated 42-54 Ma in Africa and subsequently underwent at least 24 dispersal events between the continents in two main periods. Using phylogenetic analyses of mitochondrial genomes from 415 species, including all higher termite taxonomic and feeding groups, we inferred 10 dispersal events to South America and Asia 35-23 Ma, coinciding with the sharp decrease in global temperature, sea level, and rainforest cover in the Oligocene. After global temperatures increased, 23-5 Ma, there was only one more dispersal to South America but 11 to Asia and Australia, and one dispersal back to Africa. Most of these dispersal events were transoceanic and might have occurred via floating logs. The spread of higher termites across oceans was helped by the novel ecological opportunities brought about by environmental and ecosystem change, and led termites to become one of the few insect groups with specialized mammal predators. This has parallels with modern invasive species that have been able to thrive in human-impacted ecosystems.

Exclusive Gut Flagellates of Serritermitidae Suggest a Major Transfaunation Event in Lower Termites: Description of Heliconympha glossotermitis gen. nov. spec. nov.

The guts of lower termites are inhabited by host-specific consortia of cellulose-digesting flagellate protists. In this first investigation of the symbionts of the family Serritermitidae, we found that Glossotermes oculatus and Serritermes serrifer each harbor similar parabasalid morphotypes: large Pseudotrichonympha-like cells, medium-sized Leptospironympha-like cells with spiraled bands of flagella, and small Hexamastix-like cells; oxymonadid flagellates were absent. Despite their morphological resemblance to Pseudotrichonympha and Leptospironympha, a SSU rRNA-based phylogenetic analysis identified the two larger, trichonymphid flagellates as deep-branching sister groups of Teranymphidae, with Leptospironympha sp. (the only spirotrichosomid with sequence data) in a moderately supported basal position. Only the Hexamastix-like flagellates are closely related to trichomonadid flagellates from Rhinotermitidae. The presence of two deep-branching lineages of trichonymphid flagellates in Serritermitidae and the absence of all taxa characteristic of the ancestral rhinotermitids underscores that the flagellate assemblages in the hindguts of lower termites were shaped not only by a progressive loss of flagellates during vertical inheritance but also by occasional transfaunation events, where flagellates were transferred horizontally between members of different termite families. In addition to the molecular phylogenetic analyses, we present a detailed morphological characterization of the new spirotrichosomid genus Heliconympha using light and electron microscopy.

Molecular Mechanism of the Two-Component Suicidal Weapon of Neocapritermes taracua Old Workers.

In termites, as in many social insects, some individuals specialize in colony defense, developing diverse weaponry. As workers of the termite Neocapritermes taracua (Termitidae: Termitinae) age, their efficiency to perform general tasks decreases, while they accumulate defensive secretions and increase their readiness to fight. This defensive mechanism involves self-sacrifice through body rupture during which an enzyme, stored as blue crystals in dorsal pouches, converts precursors produced by the labial glands into highly toxic compounds. Here, we identify both components of this activated defense system and describe the molecular basis responsible for the toxicity of N. taracua worker autothysis. The blue crystals are formed almost exclusively by a specific protein named BP76. By matching N. taracua transcriptome databases with amino acid sequences, we identified BP76 to be a laccase. Following autothysis, the series of hydroquinone precursors produced by labial glands get mixed with BP76, resulting in the conversion of relatively harmless hydroquinones into toxic benzoquinone analogues. Neocapritermes taracua workers therefore rely on a two-component activated defense system, consisting of two separately stored secretions that can react only after suicidal body rupture, which produces a sticky and toxic cocktail harmful to opponents.

Breaking the cipher: ant eavesdropping on the variational trail pheromone of its termite prey.

Predators may eavesdrop on their prey using innate signals of varying nature. In regards to social prey, most of the prey signals are derived from social communication and may therefore be highly complex. The most efficient predators select signals that provide the highest benefits. Here, we showed the use of eusocial prey signals by the termite-raiding ant Odontoponera transversaO. transversa selected the trail pheromone of termites as kairomone in several species of fungus-growing termites (Termitidae: Macrotermitinae: Odontotermes yunnanensis, Macrotermes yunnanensis, Ancistrotermes dimorphus). The most commonly predated termite, O. yunnanensis, was able to regulate the trail pheromone component ratios during its foraging activity. The ratio of the two trail pheromone compounds was correlated with the number of termites in the foraging party. (3Z)-Dodec-3-en-1-ol (DOE) was the dominant trail pheromone component in the initial foraging stages when fewer termites were present. Once a trail was established, (3Z,6Z)-dodeca-3,6-dien-1-ol (DDE) became the major recruitment component in the trail pheromone and enabled mass recruitment of nest-mates to the food source. Although the ants could perceive both components, they revealed stronger behavioural responses to the recruitment component, DDE, than to the common major component, DOE. In other words, the ants use the trail pheromone information as an indication of suitable prey abundance, and regulate their behavioural responses based on the changing trail pheromone component. The eavesdropping behaviour in ants therefore leads to an arms race between predator and prey where the species specific production of trail pheromones in termites is targeted by predatory ant species.

The evolutionary history of termites as inferred from 66 mitochondrial genomes.

Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153-196 Ma 95% confidence interval [CI]), that modern Termitidae arose 54 Ma (46-66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35-49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events.

Oceanic dispersal, vicariance and human introduction shaped the modern distribution of the termites Reticulitermes, Heterotermes and Coptotermes.

Reticulitermes, Heterotermes and Coptotermes form a small termite clade with partly overlapping distributions. Although native species occur across all continents, the factors influencing their distribution are poorly known. Here, we reconstructed the historical biogeography of these termites using mitochondrial genomes of species collected on six continents. Our analyses showed that Reticulitermes split from Heterotermes + Coptotermesat 59.5 Ma (49.9-69.5 Ma 95% CI), yet the oldest split within Reticulitermes(Eurasia and North America) is 16.1 Ma (13.4-19.5 Ma) and the oldest split within Heterotermes + Coptotermesis 36.0 Ma (33.9-40.5 Ma). We detected 14 disjunctions between biogeographical realms, all of which occurred within the last 34 Ma, not only after the break-up of Pangaea, but also with the continents in similar to current positions. Land dispersal over land bridges explained four disjunctions, oceanic dispersal by wood rafting explained eight disjunctions, and human introduction was the source of two recent disjunctions. These wood-eating termites, therefore, appear to have acquired their modern worldwide distribution through multiple dispersal processes, with oceanic dispersal and human introduction favoured by the ecological traits of nesting in wood and producing replacement reproductives.

Sphinganine-like biogenesis of (E)-1-nitropentadec-1-ene in termite soldiers of the genus Prorhinotermes.

In 1974, (E)-1-nitropentadec-1-ene, a strong lipophilic contact poison of soldiers of the termite genus Prorhinotermes, was the first-described insect-produced nitro compound. However, its biosynthesis remained unknown. In the present study, we tested the hypothesis that (E)-1-nitropentadec-1-ene biosynthesis originates with condensation of amino acids with tetradecanoic acid. By using in vivo experiments with radiolabeled and deuterium-labeled putative precursors, we show that (E)-1-nitropentadec-1-ene is synthesized by the soldiers from glycine or L-serine and tetradecanoic acid. We propose and discuss three possible biosynthetic pathways.

Crystal structure of blue laccase BP76, a unique termite suicidal defense weapon.

Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N. taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N. taracua workers, providing old workers with an efficient defensive weapon to protect their colony.