First record of intersex in neotenic reproductives of the termite Cryptotermes brevis (Isoptera: Kalotermitidae)

Drywood termites of the family Kalotermitidae present a very flexible developmental pattern, in which pseudergates are totipotent and may become reproductives. In this study, three colonies of Cryptotermes brevis headed by neotenic reproductives were used: (i) colony A, with a primary king and a neotenic queen; (ii) colony B, with a primary queen and a neotenic king; and (iii) colony C, with neotenic king and queen. The primary king from colony A and the neotenic king from colony B presented well‐developed reproductive systems, with conspicuous testicular lobes and cysts containing spermatozoa also observed in the seminal vesicles. The neotenic queen of colony A and the primary queen of colony B had ovaries with several oocytes in early developmental stages with some of them already vitellogenic and terminal, which suggests egg‐laying activity. Both of these queens presented the spermathecae lumen filled with spermatozoa. The queen of colony C had vitellogenic oocytes; however, the oocytes were not terminal and the lumen of spermathecae was devoid of spermatozoa and secretions. The seminal vesicles of colony C neotenic king have no spermatozoa. This king displayed a previtellogenic oocyte among its testicular lobes, which suggested that this individual was an intersex.     

Head-butting as an Early Indicator of Reproductive Disinhibition in the Termite Zootermopsis nevadensis

In lower termites, functionally sterile larval helpers are totipotent—capable of becoming reproductively active with the loss of their colony’s king or queen. Full reproductive development may take several weeks, but initiation of this developmental response most likely occurs shortly after colony members detect when a reproductive-specific signal is missing. We investigated the early response of termite helpers to the removal of their king and queen in the basal termite species Zootermopsis nevadensis. Within 6–12 h after reproductives were removed, helpers displayed an increase in head-butting—a behavior associated with dominance in other termite species as well as in closely related roaches. The loss of just one reproductive, either the king or queen, was also sufficient to cause an increase in head-butting. We did not find evidence, however, that this response was sex-specific: males and females were equally likely to increase head-butting independent of the sex of the reproductive that was removed. Finally, we discovered that reproductive-specific compounds present on the cuticle of king and queen termites were also present in their feces, but the presence of the feces did not seem sufficient to inhibit the increased head-butting after the reproductives were removed. Collectively, these results indicate that termite workers readily detect the loss of reproductives in their colony and that they at least initially respond in a non sex-specific manner.     

The origins and relatedness of multiple reproductives in colonies of the termite Nasutitermes corniger

Colonies of the termite Nasutitermes corniger often contain multiple reproductive queens and kings. We used double-strand conformation polymorphism (DSCP) analysis of mitochondrial DNA (mtDNA) to determine the probable origins of co-occurring reproductives. Colonies differed in queen and king number, in the number of nests containing reproductives, and in the genetic relationships among reproductives. Most of the 44 colonies contained a single pair of maternally unrelated reproductives. In the two single-nest colonies with a pair of queens, the two queens differed in mtDNA haplotype, suggesting nest-founding by unrelated queens. In the seven single-nest colonies with larger numbers of reproductives (11–49), all reproductives shared the same haplotype, a pattern consistent with replacement of a single pair by several offspring. As predicted by theory, the number of coexisting queens was greater for replacement reproductives than for co-foundresses. Several complex colonies contained multiple queens of two or more haplotypes distributed among several interconnected nests. This indicates that several matrilines can persist within a colony through one or more generations of budding and replacement, a hypothesis confirmed by orphaning experiments. The various modes of termite colony formation rival the diversity seen in ant species and demonstrate the remarkable convergence of behaviours between the two groups.     

Extreme genetic mixing within colonies of the wood-dwelling termite Kalotermes flavicollis (Isoptera,Kalotermitidae)

The existence of altruism in social insects is commonly attributed to altruistic individuals gaining indirect fitness through kin selection. However, recent studies suggest that such individuals might also gain direct fitness through reproduction. Experimental studies on primitive wood-dwelling termites revealed that colony fusion often causes the death of primary reproductives (queen and king), allowing opportunities for workers to inherit the nest by developing into replacement reproductives (neotenics). Therefore, colony fusion has been proposed as an important factor that may have favoured sociality in termites. However, whether colony fusion occurs frequently in natural populations of wood-dwelling termites remains an open question. We analysed eleven colonies of the wood-dwelling termite Kalotermes flavicollis (Kalotermitidae), using two mitochondrial and five nuclear microsatellite markers. Nine of eleven colonies (82%) were mixed families, with offspring of three or more primary reproductives. To our knowledge, this result represents the highest frequency of mixed-family colonies ever reported in termites. Moreover, genetic mixing of colonies appeared extreme in two ways. First, the number of haplotypes per colony was exceptionally high (up to nine), indicating that colonies were composed of multiple queens' offspring. Second, some mixed-family colonies included individuals belonging to two highly divergent genetic lineages. F-statistics and relatedness values suggest that mixed-family colonies most likely result from colony fusion, giving support to the accelerated nest inheritance theory. These findings raise important questions about the mode of foundation of mixed-family colonies and the evolutionary forces that maintain them within populations.     

Asexual queen succession in the subterranean termite Reticulitermes virginicus

Termite colonies are founded by a pair of primary reproductives. In many species, including subterranean termites (family Rhinotermitidae), the primary king and queen can be succeeded by neotenic reproductives that are produced from workers or nymphs within the colony. It is generally believed that these neotenics inbreed within the colony, sometimes for many generations. Here, we show that primary queens of the North American subterranean termite, Reticulitermes virginicus, are replaced by numerous parthenogenetically produced female neotenics. We collected functional female neotenics from five colonies of R. virginicus in North Carolina and Texas, USA. Genetic analysis at eight microsatellite loci showed that 91-100% of the neotenics present within a colony were homozygous at all loci, indicating that they were produced through automictic parthenogenesis with terminal fusion. In contrast, workers, soldiers and alates were almost exclusively sexually produced by mating between the female neotenics and a single king. This is the second termite species shown to undergo asexual queen succession, a system first described in the Japanese species, Reticulitermes speratus. Thus, the conditional use of sexual and asexual reproduction to produce members of different castes may be widespread within Reticulitermes and possibly other subterranean termites.     

Workers do not mediate the inhibitory power of queens in a termite, <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Isoptera,Rhinotermitidae)

In social insects, the caste systems are based on reproductive division of labor; queens specialize in reproduction and workers primarily maintain the colony. Recently, a volatile pheromone containing n-butyl-n-butyrate and 2-methyl-1-butanol was identified as a termite queen pheromone that inhibits the differentiation of female neotenic reproductives (secondary queens). Although this volatile inhibitory pheromone regulates caste differentiation directly, the method by which it reaches members without direct contact with the queen in large colonies is not well understood. Therefore, additional mechanisms of indirect communication must exist, such as worker-mediated queen signal transport. We found that workers exposed to female reproductives did not mediate queens’ inhibitory signal in a termite Reticulitermes speratus. The experiment assessed worker transfer from direct to indirect contact groups and determined that the differentiation of new female reproductives in the indirect contact groups was not influenced by the direct contact groups, whereas direct contact with functional female reproductives and artificial queen pheromone did suppress neotenic differentiation. This suggests that worker transfer of the queen signal is unlikely and that for colony-wide inhibition direct contact by the majority of infertile members with reproductives or eggs, which emit the same volatiles as female reproductives, is necessary within a certain time interval.     

Queen pheromone regulates egg production in a termite

In social insects, resource allocation is a key factor that influences colony survival and growth. Optimal allocation to queens and brood is essential for maximum colony productivity, requiring colony members to have information on the total reproductive power in colonies. However, the mechanisms regulating egg production relative to the current labour force for brood care remain poorly known. Recently, a volatile chemical was identified as a termite queen pheromone that inhibits the differentiation of new neotenic reproductives (secondary reproductives developed from nymphs or workers) in Reticulitermes speratus. The same volatile chemical is also emitted by eggs. This queen pheromone would therefore be expected to act as an honest message of the reproductive power about queens. In this study, we examined how the queen pheromone influences the reproductive rate of queens in R. speratus. We compared the number of eggs produced by each queen between groups with and without exposure to artificial queen pheromone. Exposure to the pheromone resulted in a significant decrease in egg production in both single-queen and multiple-queen groups. This is the first report supporting the role of queen pheromones as a signal regulating colony-level egg production, using synthetically derived compounds in a termite.     

Termite colony ontogeny: a long-term assessment of reproductive lifespan, caste ratios and colony size in Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Thirty Reticulitermes flavipes (Kollar) colonies established by alates collected from two separate field sites were raised in the laboratory for eight years. Twenty-one of the colonies were founded by alates from one field source and nine from another, providing demographic data from two unrelated parental lineages. Colony totals ranged from 3620 to 11641 individuals, with no significant difference in size between lineages. Soldier caste proportion of the colony total and mean wet weights for workers, soldiers and kings were significantly different between the two lineages. This suggests that at least a portion of the variability observed in caste ratios and body size may be heritable. One founding reproductive had died in five of the colonies (17%); none lost both parents. The queenless colonies contained exclusively female replacement reproductives (neotenics); the kingless colony contained a female-skewed mixture of male and female neotenics. All the nests that lost a founding parent contained significantly more pre-alate nymphs than the nests with both a king and a queen. Comparisons with published reports of ontogenetic patterns in other termites and social insects are discussed.     

圆唇散白蚁补充生殖蚁的类型与建群能力

摘要: 【目的】为了探讨圆唇散白蚁Reticulitermes labralis补充生殖蚁对巢群稳定和发展的作用。【方法】对野外巢群进行调查研究, 及对婚飞成虫通过雌雄配对与补充生殖蚁隔离巢群进行人工饲养的对比研究。【结果】在野外巢群只发现1对原始蚁王蚁后, 而补充生殖蚁的数量最多可达到689头/巢。圆唇散白蚁有3种类型的补充生殖蚁, 即由工蚁转化来的无翅型补充生殖蚁、若蚁转化来的翅芽型补充生殖蚁和末龄若蚁羽化来的拟成虫型补充生殖蚁。实验室条件下婚飞配对群体和隔离群体建群1个月后的存活率分别为64%和96%。建巢初期婚飞配对群体的子代数目增长缓慢, 2个月时的群体数量为6.3±1.54, 10个月时的群体数量也仅为8.4±1.47; 而隔离建群补充生殖蚁2个月时的群体数量为52.4±6.44, 10个月时的群体数量为164.3±20.85, 都高于婚飞配对群体。 此外, 野外巢群的补充蚁后跟原始蚁后一样都具有发达的卵巢。【结论】在圆唇散白蚁中补充生殖蚁是白蚁巢群主要的繁殖力量, 也是建立新巢群的重要繁殖品级。     

Repeated copulation and testes enlargement in Macrotermes michaelseni

ABSTRACT. The minimal copulation frequency of the reproductives of Macrotermes michaelseni (Sjöstedt) was investigated by removing the king from incipient laboratory colonies and from mature field colonies. Laboratory females stopped producing fertile eggs 56–154 days, and physogastric queens from field colonies less than 13 days after the removal of the king. Compared with young laboratory male reproductives, kings from mature field colonies are reported to have enormously enlarged testes, larger corpora allata (CA) and a higher juvenile hormone (JH) titre. However, JH application to young males did not increase the volume of the testes. The testes volumes only increased if the males were kept with additional workers or if their incipient colonies were allowed to grow continuously under unrestricted space conditions. These males also had bigger CA, and the soil in their midgut was replaced by food from the workers. It is concluded that in a growing colony the king is differently fed by the increasing number of workers and that trophic change in combination with higher CA activity induce enlargement of the testes. These enlarged testes are able to produce the sperm to fertilize the large number of eggs laid by a physogastric queen.     

Clinal variation in colony breeding structure and level of inbreeding in the subterranean termites Reticulitermes flavipes and R. grassei

Social insects exhibit remarkable variation in their colony breeding structures, both within and among species. Ecological factors are believed to be important in shaping reproductive traits of social insect colonies, yet there is little information linking specific environmental variables with differences in breeding structure. Subterranean termites (Rhinotermitidae) show exceptional variation in colony breeding structure, differing in the number of reproductives and degree of inbreeding; colonies can be simple families headed by a single pair of monogamous reproductives (king and queen) or they can be extended families headed by multiple inbreeding neotenic reproductives (wingless individuals). Using microsatellite markers, we characterized colony breeding structure and levels of inbreeding in populations over large parts of the range of the subterranean termites Reticulitermes flavipes in the USA and R. grassei in Europe. Combining these new data with previous results on populations of both species, we found that latitude had a strong effect on the proportion of extended‐family colonies in R. flavipes and on levels of inbreeding in both species. We examined the effect of several environmental variables that vary latitudinally; while the degree of inbreeding was greatest in cool, moist habitats in both species, seasonality affected the species differently. Inbreeding in R. flavipes was most strongly associated with climatic variables (mean annual temperature and seasonality), whereas nonclimatic variables, including the availability of wood substrate and soil composition, were important predictors of inbreeding in R. grassei. These results are the first showing that termite breeding structure is shaped by local environmental factors and that species can vary in their responses to these factors.     

Preliminary studies on the control of caste formation in the harvester termiteHodotermes mossambicus (Hagen)

The control of caste formation in the harvester termiteHodotermes mossambicus (Hagen) was investigated.Normal laboratory colonies contained a primary reproductive pair, between 6.59 and 24.98% workers, 0.39 and 3.45% soldiers and 74.64 and 92.06% larvae. Supplementary reproductives did not develop in colonies deprived of their king and queen. Continuous removal of soldiers stimulated soldier production. We have suggested that a pheromone produced by the soldiers plays a major role in controlling the soldier population of a colony. A similar phermone system appreared to control the production of workers.Evidence was also obtained that the primary reproductive pair influenced the production of workers and soldiers.     

Theories of cell ageing: a case of mistaken identity

In social insects which reproduce by colony fission there is often only one queen in each swarm (e.g. honeybees, army ants) and the number of males greatly exceeds the number of new queens produced at swarming time. This seems to contradict Fisher's principle that there should be equal investment in male and female reproductives. Hamilton 1975 has suggested that the principle can be saved by counting the investment in the swarm as part of the investment in female reproductives. Craig 1980, on the other hand, argues that few queens are produced because any further investment in queens would be wasted since a queen without a swarm is valueless; on this view the sex ratio is male-biased because of local competition between queens for swarms. The present paper investigates from first principles how many males should be produced by a species which reproduces by colony fission and how the workers should divide themselves between the new colonies. The results of the analysis do not support Hamilton's conjecture but show that a number of factors are involved in the evolution of male production, including kin selection and the relationship between the “fitness” of a colony and the number of workers in it.     

Size and composition of swarming colonies in Provespa anomala (Hymenoptera, Vespidae), a nocturnal social wasp

Summary: Ninety-eight nests of various growing stages of Provespa anomala were found in the lowlands of Sumatra, Indonesia, from August to January. Its colony cycle is considered to be annual, with nests built throughout the year. There were two types of early nests before the emergence of workers, one with a worker number of less than 63 and the other of more than 231. The former were new nests, each containing a new queen and were built by reproductive swarming, whereas each of the latter was possibly re-built by an old queen and an absconding swarm. Ten swarming colonies were attracted by an oil-lamp light; these consisted of a copulated queen with immature oocytes in the ovary and 55 workers on average. No males, or workers stylopized by Xenos provesparum were observed either in colonies before worker emergence or in swarms coming flying into the light. Differences between swarming in Provespa, polygyny and nest relocation in Vespa, and those in swarm-founding Polistinae are discussed.     

The artificial insemination of bumblebee queens

Summary: Artificial insemination (AI) is a technique to transfer instrumentally sperm from the male into the female's reproductive system. AI is widespread in vertebrates for economical animal breeding and for conservation biology. However, in invertebrates only a few cases of successful AI have been reported. In this paper we describe a new technique to artificially inseminate bumblebee queens (Bombus spp.). Males were dissected and the accessory testes were removed and washed in insect ringer. They were then opened and the outflowing sperm was picked up with a glass capillary mounted on a syringe. For the sperm transfer into the queen we adapted a standard apparatus used for honeybee inseminations. The queen was anaesthetized with CO₂, held in place by a queen holder and the sting chamber was opened using two hooks. The sperm containing glass capillary was introduced into the queen's sexual tract. The sperm was released into the bursa copulatrix very near the opening of the spermathecal duct of the queen. Inseminated queens were hibernated for 2 weeks and produced a normal colony under field conditions. Multiple inseminations proved to be successful since different patrilines could be detected in the worker offspring. Successful inseminations were performed for B. terrestris, B. lucorum and B. hypnorum.     

Queen-worker conflicts over male production and sex allocation in a primitively eusocial wasp

Abstract In a colony headed by a single monandrous foundress, theories predict that conflicts between a queen and her workers over both sex ratio and male production should be intense. If production of males by workers is a function of colony size, this should affect sex ratios, but few studies have examined how queens and workers resolve both conflicts simultaneously. We conducted field and laboratory studies to test whether sex-ratio variation can be explained by conflict over male production between queen and workers in the primitively eusocial wasp Polistes chinensis antennalis.
Worker oviposition rate increased more rapidly with colony size than did queen oviposition. Allozyme and micro-satellite markers revealed that the mean frequency of workers' sons among male adults in queen-right colonies was 0.39 ± 0.08 SE (n = 22). Genetic relatedness among female nestmates was high (0.654–0.796), showing that colonies usually had a single, monandrous queen. The mean sex allocation ratio (male investment/male and gyne investments) of 46 queen-right colonies was 0.47 ± 0.02, and for 25 orphaned colonies was 0.86 ± 0.04. The observed sex allocation ratio was likely to be under queen control. For queen-right colonies, the larger colonies invested more in males and produced reproductives protandrously and/or simultaneously, whereas the smaller colonies invested more in females and produced reproductives protogynously. Instead of positive relationships between colony size and worker oviposition rate, the frequency of workers' sons within queen-right colonies did not increase with colony size. These results suggest that queens control colony investment, even though they allow worker oviposition in queen-right colonies. Eggs laid by workers may be policed by the queen and/or fellow workers. Worker oviposition did not influence the outcome of sex allocation ratio as a straightforward function of colony size.     

Clustered male production by workers in the stingless bee Melipona subnitida Ducke (Apidae, Meliponinae)

Summary: In stingless bees brood cells are sequentially filled with liquid larval food (mass-provisioning), upon which the queen lays an egg. Thereafter the cell is closed by a worker. This study showed that during these processes workers of Melipona subnitida regularly laid eggs that served as food for the queen. Occasionally cells were oviposited in and immediately closed by a worker. These cells always rendered males. Some of these reproductive workers were seen to lay a trophic egg as well. Cells which were exclusively oviposited in by the physogastric queen gave rise to workers and queens only. In one colony it could be verified that three workers alone, which differed in age by one day, laid 15 male-producing eggs within a period of two successive weeks. Among them the number of ovipositions was positively related to the order in which workers eclosed - the oldest worker laying most eggs - and inversely related to the number of times they closed cells oviposited in exclusively by the queen. Apparently the physogastric queen was not able to stop certain workers from reproducing. We therefore conclude that some workers in M. subnitida temporarily dominated their queens in egg-laying.     

Effects of weather on activity and sleep in brooding king penguins (Aptenodytes patagonicus)

Avian sleep is sensitive to thermal challenges. Brooding king penguins (Aptenodytes patagonicus) defending a territory are exposed to wide daily fluctuations in ambient temperature and wind conditions. We studied the daytime behavioural time budget of 89 groups of territorial adults brooding a 1- to 5-week-old chick on Crozet Island during summer 1998. Scans were performed every 10 min and each bird was categorized as either active, resting or sleeping. Three ranges of ambient temperatures (T1=5-9°C, T2=10-13°C, T3=14-19°C) and wind conditions (calm-light, moderate, strong) were distinguished. Wind conditions did not affect the behavioural time budget of king penguins during summer. Resting, which represented about half of the daytime behavioural time budget, increased by 17% when ambient temperature increased from T1 to T3, mostly at the expense of active behaviours. The percentage of time spent sleeping was low, but was reduced by 66% when ambient temperature increased over 10°C. Thus, behavioural sleep was mainly observed in a range of temperatures within which resting metabolic rate of adult king penguins is minimal, i.e. between -1 and 11°C. It is also interesting to note that the range of ambient temperatures in which sleeping was high coincides with the most common microclimatology prevailing at the colony during summer, i.e. ambient temperatures between 5 and 10°C.     

Queen mating frequency and maternity of males in the stingless bee Trigona carbonaria Smith

Summary. Aspects of the sociobiology of the native Australian stingless bee Trigona carbonaria (Hymenoptera, Meliponini) were investigated using highly variable microsatellite markers. The queen mating frequency was estimated by examining genotypes of samples of workers from five colonies across three to five microsatellite loci. In each case, results were consistent with the workers being progeny of a queen mated with a single male. Microsatellite analysis of haploid males from two of the colonies suggested they predominantly arose from queen-laid eggs. As workers are more related to sons-of-workers than to sons-of-queens in monandrous colonies, this is somewhat surprising, and suggests that there may be colony-level costs associated with worker reproduction, or that queens are able to regulate worker reproduction via the ritualized cell provisioning and oviposition process. Males from another T. carbonaria colony were found to be diploid.     

Genetic structure of colonies and a male aggregation in the stingless bee Scaptotrigona postica,as revealed by microsatellite analysis

Summary: Stingless bee queens have for long been assumed to mate once on a nuptial flight, early in life. To evaluate critically monandry in one stingless bee, Scaptotrigona postica, worker offspring (adults or brood) were genetically analysed with microsatellite loci, five of which were developed specifically for the species. Marker loci were highly variable; unbiased estimates of heterozygosity were > 0.5. "Foreign" workers, either those having drifted from other colonies (circa 2%) or those of a replacement queen, were identified with the genetic markers and removed from further analysis. Worker genotypes were consistent with some queens having mated once and others having mated with up to six different males. Scaptotrigona postica queens are therefore facultatively polyandrous. Effective mating frequencies, m_e, were generally lower than the number of patrilines observed. Relatedness estimates of nestmates from individual colonies concurred with those derived from direct counts of the number of patrilines and their proportional representation. Putative genotypes of a colony's queen and her mates were deduced from those of her workers. Queens were generally not related to their mates. For one polyandrous queen, her six mates were related to each other, possibly because of numerically biased representation of males from different colonies at mating sites. However, males at an aggregation outside a colony came from numerous colonies.