A comprehensive review on the documented characteristics of four Reticulitermes termites (Rhinotermitidae, Blattodea) of China / Uma revisão abrangente sobre as características documentadas de quatro cupins Reticulitermes da China

Termites are known as social insects worldwide. Presently in China 473 species, 44 genera and 4 families of termites have been reported. Of them, 111 Reticulitermes species are widely spread in different zones of China. The dispersion flight season of these Chinese Reticulitermes species are usually started from February to June, but in some regions different species are distributed, sharing their boundaries and having overlapping flight seasons. These reasons become important sources of hybridization between two different heterospecific populations of termites. It was confirmed that the fertilized eggs and unfertilized eggs of some Reticulitermes termites have the capacity of cleavage. While the unfertilized eggs of R. aculabialis, R. chinensis and R. labralis cleaved normally and the only R. aculabialis unfertilized eggs develop in embryos. While, the R. flaviceps and R. chinensis were observed with their abnormal embryonic development, and not hatching of eggs parthenogenetically. They were reported more threatening to Chinese resources as they propagate with parthenogenesis, hybridization and sexual reproduction. Eggshell and macrophiles of eggs play important roles in species identification and control. Although, they are severe pests and cause a wide range of damages to wooden structures and products in homes, buildings, building materials, trees, crops, and forests in China's Mainland.

Os cupins são conhecidos como insetos sociais em todo o mundo. Atualmente na China foram relatadas 473 espécies, 44 gêneros e 4 famílias de cupins. Destas, 111 espécies de Reticulitermes estão amplamente distribuídas em diferentes zonas da China. A temporada de voo de dispersão dessas espécies chinesas de Reticulitermes geralmente começa de fevereiro a junho, mas em algumas regiões diferentes espécies são distribuídas, compartilhando seus limites e tendo temporadas de voo sobrepostas. Essas razões tornam-se importantes fontes de hibridização entre duas populações heteroespecíficas de cupins. Foi confirmado que os ovos fertilizados e não fertilizados de alguns cupins Reticulitermes possuem capacidade de clivagem. Já os ovos não fertilizados de R. aculabialis, R. chinensis e R. labralis clivaram normalmente, e os únicos ovos não fertilizados de R. aculabialis se desenvolvem em embriões. R. flaviceps e R. chinensis foram observados com desenvolvimento embrionário anormal, e não eclosão de ovos por partenogênese. Eles foram relatados como mais ameaçadores para os recursos chineses à medida que se propagam com partenogênese, hibridização e reprodução sexual. Casca de ovo e macrófilos de ovos desempenham papéis importantes na identificação e controle de espécies, embora sejam pragas graves e causem uma ampla gama de danos a estruturas e produtos de madeira em residências, edifícios, materiais de construção, árvores, plantações e florestas na China continental.

Caste development and sex ratio of the Ryukyu drywood termite Neotermes sugioi and its potential mechanisms.

Sex allocation is one of the most studied traits in evolutionary biology because its theoretical predictions match the empirical data. Here, using the Ryukyu dry-wood termite Neotermes sugioi, we investigated several factors that could bias the sex allocation in three populations (Okinawa, Ishigaki/Iriomote, and Yonaguni). Our survey showed that there were more queen-only colonies than king-only colonies in these populations, suggesting a longer lifespan of the queens than that of the kings. In this condition, sex-asymmetric reproductive value (SRV) theory predicts female bias, because even after the short-lived kings die, the long-lived queens can continue reproduction with their sons. However, sex allocation in this species seemed to be biased toward males. Furthermore, we examined the possibility of intrasexual competition among siblings (ICS). If ICS is the cause of the bias, the allocation is expected to change depending on the total investment in sexual offspring. However, the biomass of both male and female alates increased linearly with the increase in the total biomass of the alates in these populations. Thus, neither the SRV nor the ICS theory could explain the male-biased sex ratio of N. sugioi. On the basis of these results, we discuss the remaining possibilities in this species.

Growth, death, and resource competition in sessile organisms.

Population-level scaling in ecological systems arises from individual growth and death with competitive constraints. We build on a minimal dynamical model of metabolic growth where the tension between individual growth and mortality determines population size distribution. We then separately include resource competition based on shared capture area. By varying rates of growth, death, and competitive attrition, we connect regular and random spatial patterns across sessile organisms from forests to ants, termites, and fairy circles. Then, we consider transient temporal dynamics in the context of asymmetric competition, such as canopy shading or large colony dominance, whose effects primarily weaken the smaller of two competitors. When such competition couples slow timescales of growth to fast competitive death, it generates population shocks and demographic oscillations similar to those observed in forest data. Our minimal quantitative theory unifies spatiotemporal patterns across sessile organisms through local competition mediated by the laws of metabolic growth, which in turn, are the result of long-term evolutionary dynamics.

The making of the defensive caste: Physiology, development, and evolution of the soldier differentiation in termites.

Termites (Blattodea, Termitoidea, or Isoptera) constitute one of the major lineages of eusocial insects. In termite societies, multiple types of functional individuals, that is, castes, perform divisions of labors to coordinate social behaviors. Among other castes, the soldier caste is distinctive since it is sterile and exclusively specialized into defensive behavior with largely modified morphological features. Therefore, many of the previous studies have been focused on soldiers, in terms of ecology, behavior, and evolution as well as developmental and physiological mechanisms. This article overviews the accumulation of studies especially focusing on the developmental and physiological mechanisms underlying the soldier differentiation in termites. Furthermore, the evolutionary trajectories that have led the acquisition of soldier caste and have diversified the soldier characteristics in association with the social evolution are discussed.

Increased genetic diversity from colony merging in termites does not improve survival against a fungal pathogen.

In some species of social insects the increased genetic diversity from having multiple breeders in a colony has been shown to improve pathogen resistance. Termite species typically found colonies from single mated pairs and therefore may lack the flexibility to buffer pathogen pressure with increased genetic diversity by varying the initial number of reproductives. However, they can later increase group diversity through colony merging, resulting in a genetically diverse, yet cohesive, workforce. In this study, we investigate whether the increased group diversity from colony fusion benefits social immunity in the subterranean termite Reticulitermes flavipes. We confirm previous findings that colonies of R. flavipes will readily merge and we show that workers will equally groom nestmates and non-nestmates after merging. Despite this, the survival of these merged colonies was not improved after exposure to a fungal pathogen, but instead leveled to that of the more susceptible or the more resistant colony. Our study brings little support to the hypothesis that colony fusion may improve immunity through an increase of genetic diversity in R. flavipes. Instead, we find that following exposure to a lethal pathogen, one colony is heavily influential to the entire group's survival after merging.

Development and comparative morphology of the reproductive system in different aged males of the drywood termite Cryptotermes brevis (Blattaria, Isoptera, Kalotermitidae).

Termites are eusocial cockroaches, which have received great attention due to their diversity of reproductive strategies. Although these novelties allow new interpretations concerning the mating biology of these insects, studies highlighting the structure of the reproductive system are limited to some termite lineages. Here we provide the first comparative analysis of the reproductive system of a drywood termite, using different aged males of Cryptotermes brevis as models. This species represents an important structural pest in tropical regions, and most aspects of its reproductive biology remain unknown, especially on males. The reproductive apparatus of C. brevis is equipped with paired testes, composed of seven testicular lobes, in which developing spermatozoa are located. The basal portion of the lobes connects to the vasa deferentia and transport spermatozoa to a pair of enlarged chambers, the seminal vesicles. These structures join in a median ejaculatory duct, which opens to the external region through a retractile penis. Spermatozoa were observed in all C. brevis males, exhibiting elongated morphology and measuring about 10 µm in length/4 µm in width. Compared with last-instar nymphs and alates, functional kings showed enlarged testes and seminal vesicles, as well as an intense secretory activity towards the lumen of the latter structures. Histochemical tests evidenced strongly PAS and xylidine Ponceau positive reactions of the secretion only in functional kings, indicating the occurrence of glycoproteins. Thus, we suggest that morphophysiological changes establish during the maturation of the reproductive system in C. brevis.

Ergatoid reproductives in the Neotropical termite Nasutitermes aquilinus (Holmgren) (Blattaria: Isoptera: Termitidae): developmental origin, fecundity, and genetics.

Termite colonies are usually headed by primary reproductives, which establish nests during the swarming season. However, secondary reproductives may develop in some species and become supplementary or replacement breeders, extending colony lifespan. Here we investigate the developmental origin, fecundity and genetic characterization of ergatoid reproductives in the Neotropical termite Nasutitermes aquilinus (Holmgren), using morphometrical and histological techniques, five microsatellite loci and the COI mitochondrial DNA. Twelve measurements performed on 208 apterous individuals of N. aquilinus revealed 10 groups, including ergatoid females, which developed from major workers through two successive molts, and were characterized by the presence of imaginal features such as eyes and wing buds. The differentiation of these features was correlated to physogastric development in these ergatoids. Histology revealed oocytes in all maturation stages in worker-derived reproductives of N. aquilinus, presence of nonflagellate spermatozoa inside the spermatheca, and royal fat body. Thus, ergatoid reproductives were reproductively functional. According to the genotypes of 221 individuals from 11 nests, and mitochondrial haplotypes of 43 ergatoids, 73% of the colonies were simple families, whereas 27% were extended families. Despite the occurrence of related reproductives, low inbreeding rates were detected within and among colonies. Such values could be explained given that sib mating itself cannot result in a higher inbreeding rate but depend on several factors discussed in detail. This is the first study to investigate the genetic structure of termite colonies influenced by the development of ergatoids, and further investigations are encouraged to understand the influence of these reproductives on colony lifespan.

Regulation of macronutrient intake in termites: A dietary self-selection experiment.

Many animals have been shown to select among nutritionally complementary foods to reach a specific balance of nutrients that optimizes key life history traits. Nutritional ecology theory, however, predicts that an animal with a diet that is very stable in its composition, and with nutritional requirements that do not vary in their balance through time, would not need to display such mechanisms of regulation. Here we use the Australian termite Nasutitermes exitiosus as a model to test this prediction for the first time. We used the nutritional geometric framework to investigate the regulation of carbohydrate and protein, as well as the effects on foraging behaviour of protein type and group caste composition and size. Our results support the prediction of nutritional ecology, as termites failed to actively defend a well-defined macronutrient ratio. Termites maintained food collection relatively constant across protein type and group composition, and only appear to vary their collection by avoiding diets too rich in protein.

Disease-free monoculture farming by fungus-growing termites.

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis.

Unique Morphogenesis in the Damp-Wood Termite: Abscission of the Stylus during Female Reproductive Caste Differentiation.

Eusocial insects organize their colonies based on division of labor and caste differentiation, in which caste-specific morphologies are sculpted during postembryonic development. In the differentiation between reproductive and sterile castes, characteristics related to mating and reproduction are developed in reproductives, and degenerated in neuters, although little is known about the developmental regulations during the differentiation. In some species of termites, a sensory protrusion at the posterior end of the abdomen, the stylus, is known to disappear in female reproductives. In the present study, we performed anatomical and histological analyses in the damp-wood termite Hodotermopsis sjostedti to elucidate the developmental process underlying the disappearance of the stylus during neotenic and alate differentiation. Although it was first hypothesized that styli were hidden beneath the enlarged seventh sternite, our observation results found out that the styli were completely lost in reproductive females. Further histological observations revealed that the stylus disappearance was not accomplished by degeneration process; rather, styli were separated from the abdomen and discarded with the exoskeleton (exuviae) during the molt into the reproductive caste. This phenomenon in which live tissues are discarded through developmental processes is suggested to be a case of abscission, as seen in plant leaves.

A lipocalin protein, Neural Lazarillo, is key to social interactions that promote termite soldier differentiation.

Social communication among castes is a crucial component of insect societies. However, the genes involved in soldier determination through the regulation of inter-individual interactions are largely unknown. In an incipient colony of the damp-wood termite Zootermopsis nevadensis, the first larva to develop into a third instar always differentiates into a soldier via frequent trophallactic feeding from the reproductives. Here, by performing RNA-seq analysis of third instar larvae, a homologue of Neural Lazarillo (named ZnNLaz1) was found to be the most differentially expressed gene in these soldier-destined larvae, compared with worker-destined larvae. This gene encodes a lipocalin protein related to the transport of small hydrophobic molecules. RNAi-induced knockdown of ZnNLaz1 significantly inhibited trophallactic interactions with the queen and decreased the soldier differentiation rates. This protein is localized in the gut, particularly in the internal wall, of soldier-destined larvae, suggesting that it is involved in the integration of social signals from the queen through frequent trophallactic behaviours. Based on molecular phylogenetic analysis, we suggest that a novel function of termite NLaz1 has contributed to social evolution from the cockroach ancestors of termites. These results indicated that a high larval NLaz1 expression is crucial for soldier determination through social communication in termites.

A Crucial Caste Regulation Gene Detected by Comparing Termites and Sister Group Cockroaches.

Sterile castes are a defining criterion of eusociality; investigating their evolutionary origins can critically advance theory. In termites, the soldier caste is regarded as the first acquired permanently sterile caste. Previous studies showed that juvenile hormone (JH) is the primary factor inducing soldier differentiation, and treatment of workers with artificial JH can generate presoldier differentiation. It follows that a shift from a typical hemimetabolous JH response might be required for soldier formation during the course of termite evolution within the cockroach clade. To address this possibility, analysis of the role of JH and its signaling pathway was performed in the termite Zootermopsis nevadensis and compared with the wood roach Cryptocercus punctulatus, a member of the sister group of termites. Treatment with a JH analog (JHA) induced a nymphal molt in C. punctulatus RNA interference (RNAi) of JH receptor Methoprene tolerant (Met) was then performed, and it inhibited the presoldier molt in Z. nevadensis and the nymphal molt in C. punctulatus Knockdown of Met in both species inhibited expression of 20-hydroxyecdysone (20E; the active form of ecdysone) synthesis genes. However, in Z. nevadensis, several 20E signaling genes were specifically inhibited by Met RNAi. Consequently, RNAi of these genes were performed in JHA-treated termite individuals. Knockdown of 20E signaling and nuclear receptor gene, Hormone receptor 39 (HR39/FTZ-F1ß) resulted in newly molted individuals with normal worker phenotypes. This is the first report of the JH-Met signaling feature in termites and Cryptocercus JH-dependent molting activation is shared by both taxa and mediation between JH receptor and 20E signalings for soldier morphogenesis is specific to termites.

TGFß signaling related genes are involved in hormonal mediation during termite soldier differentiation.

A working knowledge of the proximate factors intrinsic to sterile caste differentiation is necessary to understand the evolution of eusocial insects. Genomic and transcriptomic analyses in social hymenopteran insects have resulted in the hypothesis that sterile castes are generated by the novel function of co-opted or recruited universal gene networks found in solitary ancestors. However, transcriptome analysis during caste differentiation has not been tested in termites, and evolutionary processes associated with acquiring the caste are still unknown. Termites possess the soldier caste, which is regarded as the first acquired permanently sterile caste in the taxon. In this study, we performed a comparative transcriptome analysis in termite heads during 3 molting processes, i.e., worker, presoldier and soldier molts, under natural conditions in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Although similar expression patterns were observed during each molting process, more than 50 genes were shown to be highly expressed before the presoldier (intermediate stage of soldier) molt. We then performed RNA interference (RNAi) of the candidate 13 genes, including transcription factors and uncharacterized protein genes, during presoldier differentiation induced by juvenile hormone (JH) analog treatment. Presoldiers induced after RNAi of two genes related to TGFß (Transforming growth factor beta) signaling were extremely unusual and possessed soldier-like phenotypes. These individuals also displayed aggressive behaviors similar to natural soldiers when confronted with Formica ants as hypothetical enemies. These presoldiers never molted into the next instar, presumably due to the decreased expression levels of the molting hormone (20-hydroxyecdysone; 20E) signaling genes. These results suggest that TGFß signaling was acquired for the novel function of regulating between JH and 20E signaling during soldier differentiation in termites.

Variations in the relative abundance of Wolbachia in the gut of Nasutitermes arborum across life stages and castes.

There are multiple forms of interactions between termites and bacteria. In addition to their gut microbiota, which has been intensively studied, termites host intracellular symbionts such as Wolbachia. These distinct symbioses have been so far approached independently and mostly in adult termites. We addressed the dynamics of Wolbachia and the microbiota of the eggs and gut for various life stages and castes of the wood-feeding termite, Nasutitermes arborum, using deep-sequencing of the 16S rRNA gene. Wolbachia was dominant in eggs as expected. Unexpectedly, it persisted in the gut of nearly all stages and castes, indicating a wide somatic distribution in termites. Wolbachia-related sequences clustered into few operational taxonomic units, but these were within the same genotype, acquired maternally. Wolbachia was largely dominant in DNA extracts from the guts of larvae and pre-soldiers (59.1%-99.1% of reads) where gut-resident lineages were less represented and less diverse. The reverse was true for the adult castes. This is the first study reporting the age-dependency of the relative abundance of Wolbachia in the termite gut and its negative correlation with the diversity of the microbiota. The possible mechanisms underlying this negative interaction are discussed.

Molting site fidelity accounts for colony elimination of the Formosan subterranean termites (Isoptera: Rhinotermitidae) by chitin synthesis inhibitor baits.

Site fidelity by molting termites in Formosan subterranean termite, Coptotermes formosanus Shiraki colonies is a new addition to our understanding of lower termites' behavior and biology. Our previous studies indicated that workers moved to the central nest to molt in the presence of eggs and reproductives. The current study showed that noviflumuron-affected workers also return to the central nest and died in the vicinity of reproductives and eggs. The aversion to the dead and decaying workers caused reproductives and brood to leave the original central nest site in a colony and refuge at newer sites every few days in response to newly dead workers near them. Because mortality was an event observed only in workers undergoing molting under the effect of noviflumuron- a CSI, the death of molting individuals was observed only around reproductives and brood. This study reveals a previously undiscovered behavior of molting termites and the mechanics behind a successful arsenal; noviflumuron baits used against subterranean termites.

Evidence from the gut microbiota of swarming alates of a vertical transmission of the bacterial symbionts in Nasutitermes arborum (Termitidae, Nasutitermitinae).

Studies on termite symbiosis have revealed that significant symbiont lineages are maintained across generations. However, most studies have focused only on the worker caste. Little is known about the gut microbiota of reproductives, the most probable vectors for transmitting these lineages to offspring. Using 16S rRNA gene-based Illumina MiSeq sequencing, we compared the gut microbiota of swarming alates of the higher termite Nasutitermes arborum with those of their nestmates from the parental colony. The OTU-based alpha diversity indices showed that the gut microbiota of the alates was at least as diverse as those of non-reproductive adults. It was largely dominated by Spirochaetes mostly of the Treponema I cluster (63.1% of reads), the same dominant taxa found in soldiers and workers of this species and in workers of closely related Nasutitermes species. The termite-specific lineages also included other representative taxa such as several clusters of Bacteroidetes and Fibrobacteres-TG3 group. The microbiota of alates was dominated by a core set of host-specific lineages (87% of reads, 77.6% of OTUs), which were always present across all castes/stages. This first comprehensive survey of the microbiota of the founding reproductives of these xylophagous higher termites shows that the bulk of the host endogenous symbionts, mostly taxa that cannot thrive outside the gut, are brought from the parent colony. The royal pair therefore seems to be a key player in the transmission of symbionts across generations and thereby in host-symbiont codiversification. The high proportion of fiber-degrading lineages in their gut suggests a wood-rich diet unlike the larval stages.

Secondary queens in the parthenogenetic termite Cavitermes tuberosus develop through a transitional helper stage.

In termite species with asexual queen succession (AQS), parthenogenetically produced immatures mostly differentiate into secondary queens, called "neotenics." In order to elucidate the ontogenetic origin of neotenics in Cavitermes tuberosus (Termitidae: Termitinae), a neotropical termite with AQS, we investigated developmental pathways of offspring according to their sex and genetic origin using both morphometric and genetic tools. The caste system of C. tuberosus follows the classical pathway of Termitidae. After the first larval instar, there is a bifurcation between two developmental lines. The apterous line is composed of a second larval instar, several worker instars, presoldiers, and soldiers. Workers display a consistent male bias and soldiers are female-only. The nymphal line is composed of five nymphal instars and the imago stage. We highlight that neotenic queens derive from third and fourth instar nymphs displaying peculiar morphological traits, here termed "aspirants," most of which are produced by parthenogenesis. Aspirants are present in all nests and perform worker tasks while waiting for the queen's death to differentiate into neotenic queens. Aspirants can successfully be used to demonstrate the occurrence of parthenogenesis in termite species whose reproductive cores are difficult to access.

Molting Site Fidelity in Workers of Formosan Subterranean Termites (Isoptera: Rhinotermitidae).

Spatial assessment of molting in workers of Coptotermes formosanus Shiraki was conducted in laboratory-reared colonies using extended foraging arenas. Workers at a premolt stage were found concentrated in the nest or in a planar arena near the nest. However, molting individuals were found exclusively in the central nest and they stayed inside or near the central nest for at least 36 h postmolting. The absence of premolt workers at foraging sites suggests that the workers have an affinity to the nest for molting and the second study on nest-fidelity evaluation suggested that the workers molt in the proximity of eggs. The molting site fidelity by workers in a colony ensures that speeding up the time for mortality induced by chitin synthesis inhibitor (CSI) baits will not result in an inhibitory cascade of dead termites around the bait stations. Thus, speeding up the elimination of a C. formosanus colony using CSI baits with the addition of molt-accelerating compounds will not lead to secondary repellency. Reasons for the molting-site fidelity amongst workers in a colony are discussed.

The transition path from female workers to neotenic reproductives in the termite Reticulitermes labralis.

Termite workers are characterized by unique flexibility in that a worker can develop in one of three ways: remain a worker, become a soldier within two successive moults, or become a neotenic reproductive (NR) within a single moult. However, is it true that workers can transform into NRs within a single moult? Actually, the developmental pathways of workers turning into NRs remains unclear. In this study, we show for the first time that the female workers of Reticulitermes labralis develop into NRs after a pre-NRs stage. We found that a female worker became a NR after two successive moults, whereas the male workers copulated directly with queens without undergoing any moults. After the first moult led the female workers into the pre-NR stage, the length of their abdomens, seventh sternites and ovaries significantly increased. After the second moult from the pre-NRs stage into NRs, the follicle cells returned to normal, and a few oocytes and follicle cells underwent apoptosis. These results demonstrated that the female pre-NR type was a transitional type during the development of female workers to the NR caste, and the starting point for oogenesis resumption was the NR stage. We confirmed there were fundamental differences in the reproductive pathway of the male and female workers. Therefore, we determined that the transformation process of the female NRs from workers may be a very complex process, and the reproductive biology of the workers has great potential to provide important and spectacular insights into the evolution strategy of termites.

An American termite in Paris: temporal colony dynamics.

Termites of the genus Reticulitermes are widespread invaders, particularly in urban habitats. Their cryptic and subterranean lifestyle makes them difficult to detect, and we know little about their colony dynamics over time. In this study we examined the persistence of Reticulitermes flavipes (Kollar) colonies in the city of Paris over a period of 15 years. The aim was (1) to define the boundaries of colonies sampled within the same four areas over two sampling periods, (2) to determine whether the colonies identified during the first sampling period persisted to the second sampling period, and (3) to compare the results obtained when colonies were delineated using a standard population genetic approach versus a Bayesian clustering method that combined both spatial and genetic information. Herein, colony delineations were inferred from genetic differences at nine microsatellite loci and one mitochondrial locus. Four of the 18 identified colonies did not show significant differences in their genotype distributions between the two sampling periods. While allelic richness was low, making it hard to reliably distinguish colony family type, most colonies appeared to retain the same breeding structure over time. These large and expansive colonies showed an important ability to fuse (39% were mixed-family colonies), contained hundreds of reproductives and displayed evidence of isolation-by-distance, suggesting budding dispersal. These traits, which favor colony persistence over time, present a challenge for pest control efforts, which apply treatment locally. The other colonies showed significant differences, but we cannot exclude the possibility that their genotype distributions simply changed over time.