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Tribolium
Tribolium A. Pai, Spelman College, Atlanta, GA, USA ã 2010 Elsevier Ltd. All rights reserved.
A Familiar Beetle
Stored Product Pest
The name ‘tribolium’ comes from the Latin verb for threshing, ‘tribulo.’ This is likely a reference to the fact that the first Tribolium beetle to be discovered, Tribolium castaneum, is a pest of stored grains and was found near where grain was threshed. Because of their association with stored grain products, these beetles are likely one of the most familiar beetles known to man. The various beetles of the genus Tribolium have a long history of being used as a model organism in laboratory studies in the areas of ecology, evolution, genetics, developmental biology, and animal behavior.
Of the dozens of species in this genus, as few as ten are storage pests associated with human grain stores and come from the castaneum, confusum, and brevicornis groups. Evidence from ancient Egypt indicates that the association between Tribolium and humans may be as old as 4000 years. These insects are incapable of attacking whole grain and are therefore secondary pests of grains such as wheat, maize, rice, barley, rye, oats. As their typical habitat is in stores of the flours of these grains, they are commonly called ‘flour beetles’ or ‘bran bugs.’ Interestingly, although Tribolium are regarded as a major pest of grain products, they may also be found in other types of foods such as beans, peas, nuts, chocolate, and even spices like ginger and red pepper. Because of their synanthropic nature, many of these pest beetles such as T. castaneum, T. confusum, T. destructor, and T. madens have a worldwide distribution. Others such as T. anaphe, T. audax, T. brevicornis, T. freemani, T. parallelus, and T. thusa have a more restricted range. The biology of the cosmopolitan storage pest beetles such as T. castaneum and T. confusum is the best understood and will be the main focus of this article.
Taxonomy and Diversity Tribolium beetles belong to the group of ‘darkling beetles’ or Tenebrionidae. As the name suggests, they have a characteristic dark body color (Figure 1). Like many other tenebrionids, Tribolium beetles are saprophagous, phytophagous, or mycophagous, and may be found in leaf litter, under the bark of trees or near fungi. In addition, like some of their close relatives of the tribe Triboliini, Tribolium beetles too may be associated with the nests of other species such as insects and some vertebrates. For example, some beetles such as T. myrmecophilum may be ecologically associated with insects’ nests, whereas others such as T. castaneum are associated with human dwellings and their stored grain. Thus, there is significant diversity in the ecological habitats of the three dozen species described in this group.
Evolutionary History Tribolium beetles may have diverged from other holometabolous insects as early as the beginning of the Cretaceous period. Since then, these beetles may have evolved in five separate lineages, each of which is associated with a particular region of the world. Each has distinctive morphology. Thus, the brevicornis group evolved in South America, the confusum group in Africa, the alcine group in Madagascar, the castaneum group in Indo-Australia, and the myrmicophilum group in Malay-East Indies. Of these, the confusum group is the most speciose with 14 known members and the myrmecophilum group is the smallest with only two known species.
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Morphology Adult Tribolium beetles are 3–5 mm in size. Some such as T. brevicornis (4 mm) and T. freemani (5 mm) are visibly larger than other species such as T. audax, T. anaphe, T. castaneum, and T. confusum (3 mm or smaller). Adults are sexually dimorphic. The sexes may be significantly different from each other in body size. In some species, such as T. brevicornis, males may be larger than females but in others including T. confusum, there may be a tendency for females to be larger. T. castaneum adult males are distinguishable from females because of the presence of setiferous glands (‘sex patches’) on their first pair of legs, which are absent in females. Similarly, in T. confusum, only males have these glands but they appear on each pair of appendages.
Life Cycle Beetle life cycles include four stages: egg, larva, pupa, and adult. The microscopic eggs of tenebrionids are ovoid and
Tribolium
Figure 1 Adult Tribolium castaneum in flour. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
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Figure 3 Pupae of Tribolium castaneum. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
almost 9 weeks for some species of flour beetles. Developmental time varies even among different strains of a species. In addition to genetics, environmental factors such as temperature, humidity, and food also influence development. Adults may live more than 3 years, though a life span of 1–6 months is more typical in laboratory settings. In general, the life span of virgin adults in laboratory conditions for T. castaneum and T. confusum beetles is 7–11 months.
Tribolium as a Model Organism
Figure 2 Larvae of Tribolium castaneum. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
bright white. In T. castaneum and T. confusum, the egg stage typically lasts 4–5 days in ideal laboratory conditions. The larvae are worm-like and yellowish (Figure 2). In optimal conditions, this stage is typically about 2–3 weeks for T. castaneum and T. confusum. The pupae are light colored, and either white or yellowish (Figure 3). Pupae are not capable of locomotion though they may wriggle (with the movement originating in the abdomen). The sexes are dimorphic and easily discernable as pupae, because the females have larger genital papillae compared to those found on the male pupae. In a favorable environment, the pupal stage may last 5–6 days in T. castaneum and T. confusum. The typical life cycle from egg to adult in laboratory strains of T. castaneum and T. confusum may be as short as 4 weeks in optimal conditions of food, temperature, and humidity. However, the rate of development from egg to adult varies vastly among species and may be as long as
Several features about their biology make flour beetles a convenient model system. Their affinity for stored grain products makes it easy to find and collect them from pantries, feed mills, feed stores, silos, etc. At these sites, beetles are easily detectable because they leave tunnel shaped tracks in the flour dust (Figure 4). When brought to the laboratory, beetles do not require much space and basically can be stored in glassware such as vials and jars because of their small size. They can be reared rather inexpensively in flour medium (typically a mixture of wheat flour and brewers’ yeast). Further, they can be indefinitely kept in dark conditions; this tolerance for dark facilitates their lives as storage pests in grain products. Tribolium cultures do not require constant maintenance, since all the life stages of these beetles can be grown in the same flour medium. Easy separation of the life stages also facilitates experimentation. Eggs and young larvae are significantly smaller than pupae and adults, and can be easily separated from each other by means of sifters with different mesh sizes. Although pupae and adults are of similar size, they are also easily sorted from each other, by taking advantage of the lack of mobility of the pupae. These insects are hardy and easily withstand being handled for experimental manipulation.
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were first discovered in the confused flour beetle in the 1990s. The Tribolium system has been used to study many major ideas in evolutionary biology such as mechanisms of reproductive isolation, group selection. In the last three decades, red flour beetles have proved to be invaluable as models for sexual selection studies and evolutionary–developmental biology (evo-devo) studies. The prolific literature on this genus provides a wealth of background information on the biology of these insects and has led to the development of new tools and methodologies that will further promote experimentation in this organism. Several authors have developed microsatellite markers in the red flour beetle, which can be applied to population genetics and parentage analysis studies. Also, the genome of T. castaneum was recently sequenced, which has positioned this species as one of the most significant models for evo-devo, genetics, and genomics studies.
Behavioral Repertoire of Tribolium Figure 4 Beetles-infested flour with tunnel shaped tracks. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
Flour beetles, like many pest species, have a high reproductive capacity and remain reproductive throughout the year. Not only do males and females readily mate in a laboratory setting, but also, because adults have a long life span, a single female may produce hundreds of eggs in her long reproductive life, which enables high number of experimental trials. Because of their relatively short life cycle, these insects are suitable for multigenerational studies that ecology and evolutionary biology necessitate. All of these listed attributes have made Tribolium a wellstudied insect on which there is a plethora of information.
History as Laboratory Model Organism The flour beetle first came to be used as a laboratory model organism in population ecology studies initiated by Chapman in the late 1920s. In the subsequent four decades, from the 1930s to 1970s, Thomas Park and colleagues at the University of Chicago used this insect extensively as a laboratory model system to examine questions in ecology, genetics, behavior, and evolution. Indeed, some classical works illustrating such fundamental concepts in ecology and evolution, as population regulation and interspecific competition have employed flour beetles as model organisms. Many notable discoveries in genetics have also been made in Tribolium. For example, selfish genetic elements
Aside from being one of the foremost model organisms for population processes and developmental genetics at present, these beetles also make an appealing animal in which to study behavioral ecology and behavioral evolution, given their remarkable repertoire of behaviors. This article discusses only a small subset of some of the more intriguing of these behaviors. Cannibalism Tribolium beetles at various life stages may eat their own kind. Because of this taxon’s use as a laboratory model of population regulation, cannibalism is one of the beststudied aspects of beetle behavior and serves as an example of interference competition within a species. In general, the inactive stages, egg and pupa, are cannibalized by the active stages, larva and adult. However, it is also possible for adults to feed on very young larvae and for the old larvae to feed on callow adults the exoskeleton of which is not sclerotized. Studies in the confused flour beetles report that adults may devour an egg in a matter of minutes. This behavior is thought to be of great adaptive significance because it may facilitate beetles’ colonizing a novel food environment. Cannibalism is known to be under polygenic control. Thus, the tendency for cannibalism is variable and strongly depends on the species as well as the genetic background of beetle strains. Cannibalism in various flour beetle species does not correlate with phylogenetic relatedness. Apparently, it has been shaped by the evolutionary–ecological history of each individual species. Similarly, because of differences in selective pressures on different life stages that practice cannibalism, the
Tribolium
larvae and the adults too show differences in cannibalism behavior. The larvae in general may feed preferentially on eggs, whereas the adults may feed preferentially on pupae. One explanation for this difference may be that larval nutritional requirements are more completely met by feeding on eggs, whereas adults may possibly eliminate competition in the near future by consuming pupae. Thus, each life stage may be maximizing their chance of survival through preferential cannibalism. Environmental variables such as amounts and quality of food, as well as population density, also influence cannibalism rates. Beetles tend to be more cannibalistic in environments with greater population densities and lower food quantity and quality. Therefore, cannibalism in flour beetles is a classic example of population regulation through density-dependent processes. Cannibalism of eggs and pupae has a significant impact in regulating population sizes. In the confused flour beetle for instance, population sizes would be tenfold larger without cannibalism. Competition Different species of flour beetles may occupy a similar niche in an ecosystem. Because they require similar types of abiotic conditions as well as feed on similar foods (stored grain products), they may co-occur within a space and consequently compete for resources in the same environment. Interestingly, the outcome of competitive interactions between the red flour beetle and confused flour beetle depends on abiotic factors such as temperature and humidity, as well as biotic factors such as initial densities of beetles, and presence or absence of parasites. Competition experiments on flour beetles conducted by Park and his colleagues are a classic study in ecology. Their experiments revealed that the confused flour beetle is likely to dominate in environments that are cool and arid, whereas the red flour beetle is likely to prevail at higher temperatures and in humid environments. Furthermore, parasitism also influenced the outcome of competition between the two flour beetle species. When beetle cultures were infected with a microparasite, Adelina, it was found that T. confusum, which is better able to withstand Adelina, was likely to ‘win’ in the interspecific competition even in abiotic conditions that typically favored T. castaneum. When a different parasite, Hymenolepis diminuta was used, the results were the opposite; when beetles were infected with the abovementioned rat tapeworm, the red flour beetle tended to be the superior competitor. One of the chief ways competing species of flour beetles interact with each other is preying on their competitor’s eggs and pupae. Beetles are able to distinguish between eggs and pupae from their own species and those of their competitors. Adult beetles may preferentially feed on the heterospecific eggs and pupae.
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Chemical Communication Beetles produce various chemicals used in communication with conspecifics as well as with other species. Flour beetles produce a host of chemicals that are secreted to their external environment; for example, both T. castaneum and T. confusum produce over half a dozen such chemicals. However, the function of these compounds is not very well understood. Some compounds such as 4–8 dimethly decanal (4–8 DMD) are produced only by males, others such as Z-2–9-propionate are produced by females, and still others, such as various toluquinones and benzoquinones are produced by both sexes. Possibly, the best-known compound is 4–8 DMD, which is an aggregation pheromone. It is a common observation that beetles in laboratory cultures and in feed mills are found in aggregations. Typically, beetle behavioral response to 4–8 DMD includes walking toward the source accompanied by a movement of the head and antennae. So strong is this behavioral response that many commercial lures use synthetically produced 4–8 DMD to trap beetles in stores of grain. While this is primarily an aggregation pheromone, it also functions to attract potential mates at closer ranges. Female mate choice in T. castaneum may partially depend on male pheromone cue at least in some populations, and male sperm precedence was shown to depend on female’s response to this olfactory signal among those populations. Toluquinones are among the suite of compounds produced by beetles and function as allomones. It is likely that they serve as a defense against the microbes found in the flour and predators in the environment. Defensive Behaviors In addition to chemical defense, beetles may resort to thanatosis or feigning death as means of defense against predators. Beetles often feign death by lying still for as few as a fraction of a second to up to a few minutes upon sensing a predator. This behavior is known to vary among different genetic backgrounds of flour beetles. Recent studies show that death feigning behavior is correlated with another type of predator avoidance behavior, fleeing. Beetles that feigned death for longer fled shorter distances than beetles that feigned death for shorter durations. This suggests that beetle strains may be genetically predisposed to one of the two alternative strategies to avoid danger from a predator, either feign death or flee. Death feigning behavior and its evolutionary significance warrants further investigation. Interactions with Parasites Flour beetles have been used as a model system to study host–parasite dynamics especially in the recent past.
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Tribolium
These beetles may get infected with a variety of parasites, including microparasites such as Adelina and Nosema, as well as macroparasites such as Hymenolepis. Tribolium and their parasites have been used to test several theories in the realm of host–parasite interactions. Several studies have examined the fitness consequences of parasitism for flour beetles. As expected from a typical host–parasite interaction, a general pattern with respect to flour beetles’ interactions with parasites is that the latter adversely affect the fitness of these beetles. Beetle fitness may be affected because of one or more factors such as reduction in attractiveness, reduced fecundity, reduced sperm precedence. A hypothesis is whether parasites manipulate intermediate host behavior to facilitate their transmission to the final hosts. This has been tested using the rat tapeworm H. diminuta and flour beetles. Beetles get infected with this parasite when they consume tapeworm egg-infested rat feces. The eggs develop into cysticercoids inside the beetles. The tapeworm completes its life cycle after infected beetles are consumed by the final host, the rat. Thus, beetles serve as the intermediate hosts of this parasite. It is likely that higher rate of surface seeking (going to the top of the flour) and emigration would make beetles more vulnerable to predation by rats, thereby improving the chances of parasite transmission. Studies show that when infected by the rat tapeworm, beetle behaviors are significantly altered. Interestingly, prevalence of parasites increased T. confusum beetles’ tendency for surface seeking and emigration compared to uninfected beetles, but had the opposite effect on T. castaneum beetles. Thus, it is clear that parasites alter beetle host behavior. The available data, though, do not support the idea that flour beetles’ behavioral changes enhance parasite transmission. Mating Behaviors Mating rates
In recent years, the red flour beetle has been extensively used as a model system for sexual selection studies. However, the mating behavior of the other flour beetles, including the confused flour beetle, is relatively less studied. The red flour beetle and confused flour beetle differ remarkably in their mating rates. Red flour beetles mate readily in the lab, whereas confused flour beetles mate at a lower frequency in laboratory observations. In the red flour beetle, both sexes are promiscuous and may mate multiple times with the same partner (repeated mating) or mate with different partners (multiple mating) in a relatively short time of few minutes. Typically, males approach the female to initiate copulation. Females may move away from the male rapidly or remain quiescent and allow the males to mount. Copulation durations range from a few seconds to half an hour.
Mate choice
Both sexes of the red flour beetle exhibit precopulatory mate choice and preferred traits likely vary among populations. Some male traits known to be attractive to females include large body size and certain pheromone cues. Males may show preference for virgin females and also favor novel mates in comparison with females they have previously mated with. In addition, males may avoid mating with callow females. Evidence from studies examining the correlation between male traits, female response, and sperm precedence suggests that females continue to asses male quality even after the initial decision to mate is made and that females bias paternity in favor of preferred males. Beetle males that stroke their partners more vigorously with forelegs while in copula (a form of copulatory courtship) have a higher-than-expected share of paternity. A clever experiment that altered only female perception of male quality but not male quality itself showed that male share of paternity was significantly affected by female perception of male quality. Female red flour beetles have the capacity to control sperm movement in their reproductive tract, which may be the mechanism for such a postcopulatory female mate choice. Male–male interactions
While a red flour beetle pair is in copula, rival males may interfere by butting into the pair or climbing over them. In some cases, males may not be able to complete intromission because of physical interference by rivals. Success and duration of copulation may be influenced by these male–male interactions. When copulating red flour beetles are disturbed by rival males, the copulations typically last longer, possibly because physical disturbance from rivals cause males to require additional time for a successful sperm transfer. Because of the high mating rates of both sexes in the red flour beetles, sperm from different males may cooccur in a female’s reproductive tract. Hence, sperm competition and sperm precedence is a well-studied aspect of flour beetle biology. Typically, the last male to mate sires the majority of a female’s offspring. Just as there are female controlled postcopulatory processes, male controlled postcopulatory processes also have been documented in the red flour beetle. Aside from mating order and female preference, other factors may also influence the outcome of sperm competition in terms of male sperm precedence. One facet of intrasexual competition in this beetle is male ability to displace a rival male’s sperm by means of their intromittent organ (the aedeagus). This may in fact be a significant aspect of sperm competition in this species, but has not been studied in much detail. In a small percentage of cases with sperm displacement, males end up translocating their rival’s sperm, which lingers on their intromittent
Tribolium
organ, when they subsequently seek to mate with other partners. This phenomenon, when males gain a share of paternity of offspring from females they themselves have not mated with because of sperm translocation by rivals, has been described as fertilization by ‘proxy.’ Another relatively unexplored aspect of male–male interactions is the phenomenon of homosexual copulations. Homosexual copulations between males are quite common in beetle aggregations. Males may mount other males and even deposit a spermatophore in the course of a homosexual copulation. The adaptive significance of this behavior, if any, is unclear. Male–female interactions
The high level of promiscuity in red flour beetles causes a conflict of interest between the sexes in this species. For example, males are selected to maximize their paternity, whereas females may be selected to choose the highest quality sire for their offspring. Conflicts of interest may lead to antagonistic co-evolution between the sexes. One example of a conflict of interest between the sexes may relate to the mating frequency. Males may benefit from mating frequently or for a prolonged duration, because this may help them to maximize their sperm transfer and facilitate mate guarding, thereby increasing their share of paternity. The same may not be true for females that may incur costs, such as expenditure of time, energy, and/or loss of other mating opportunities. Thus, in beetle populations that show a high female mating rate, it is possible that the females are coerced into copulating. That females may be resisting male attempts at frequent copulation is suggested by some of their behaviors during mating. For example, they may exhibit swift movement away from a male that is attempting to mount. Even after the male manages to mount a female, they may move backwards to cause a mounted male to dislodge, or move extremely fast, another tactic that may cause the mounted male to dislodge. Females are also usually the first to walk away from copula. In contrast to these resistance behaviors, female cooperation is evident when they remain quiescent and allow a male to mount. Multiple studies have demonstrated that populations of red flour beetles differ with respect to their mating related traits such as mating frequency, oviposition rates, mating duration. Possibly, this stems from not just differences in natural selection and drift, but also because of differences in patterns of male–female co-evolution in different populations. Thus, this insect is probably one of the best model systems to study male–female co-evolution.
Conclusion Overall this humble group of pest species has served as an incomparable laboratory model, which has yielded
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valuable insights into the evolution of various behaviors. Many behaviors described here are only partially understood and merit further study. In addition, with the advent of new tools in molecular biology and genomics, this genus will continue to be a useful organism in which to explore questions on evo-devo and functional genomics. See also: Intermediate Host Behavior; Invertebrates: The Inside Story of Post-Insemination, Pre-Fertilization Reproductive Interactions; Isolating Mechanisms and Speciation; Mating Signals; Microevolution and Macroevolution in Behavior; Parasite-Modified Vector Behavior; Parasites and Sexual Selection; Propagule Behavior and Parasite Transmission; Reproductive Behavior and Parasites: Invertebrates; Signal Parasites; Social Recognition; Social Selection, Sexual Selection, and Sexual Conflict.
Further Reading Alabi T, Michaud JP, Arnaud L, et al. (2008) A comparative study of cannibalism and predation in seven species of flour beetle. Ecological Entomology 33: 716–726. Angelini DR and Jockusch EL (2008) Relationships among pest flour beetles of the genus Tribolium (Tenebrionidae) inferred from multiple molecular markers. Molecular Phylogenetics and Evolution 46: 127–141. Beeman RW, Friesen KS, and Denell RE (1992) Maternal-effect, selfish genes in flour beetles. Science 256: 89–92. Bonneton F (2008) The beetle by the name of Tribolium, typology and etymology of Tribolium castaneum Herbst, 1797. Insect Biochemistry and Molecular Biology 38: 377–379. Fedina T and Lewis SM (2008) An integrative view of sexual selection in Tribolium flour beetles. Biological Reviews of the Cambridge Philosophical Society 83: 151–171. Mertz DB (1972) The Tribolium model and the mathematics of population growth. Annual Review of Ecology and Systematics 3: 51–78. Miyatake T, Tabuchi K, Sasaki K, et al. (2008) Pleiotropic antipredator strategies, fleeing and feigning death, correlated with dopamine levels in Tribolium castaneum. Animal Behaviour 75: 113–121. Nakakita H (1983) Rediscovery of Tribolium freemani Hinton: A stored product insect unexposed to entomologists for the past 100 years. JARQ 16: 239–245. Pai A and Bernasconi G (2008) Polyandry and female control: The red flour beetle Tribolium castaneum as a case study. Journal of Experimental Zoology Part B, Molecular and Developmental Evolution 310: 148–159. Park T (1962) Beetle, competition and populations. Science 138: 1369–1375. Sokoloff A (1972, 1974, 1977) The Biology of Tribolium vols. 1–3. Oxford: Clarendon Press and Oxford University Press. Tribolium Sequencing Consortium (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452: 949–955. Wade MJ, Patterson H, Chang NW, et al. (1994) Postcopulatory, prezygotic isolation in flour beetles. Heredity 72: 163–167. Yan G, Stevens L, Goodnight C, et al. (1997) Effects of a tapeworm parasite on the competition of Tribolium beetles. Ecology 79: 1093–1103. Yan G, Stevens L, and Schall JS (1994) Behavioral changes in Tribolium beetles infected with tapeworm: Variation in effects between beetle species and among genetic strains. The American Naturalist 143: 830–847.
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Relevant Websites http://www.ars.usda.gov – USDA Agricultural Research Service. http://www.beetlebase.org – Tribolium Genome Database.
http://www.hgsc.bcm.tmc.edu – Baylor College of Medicine, Human Genome Sequencing Center. http://www.pherobase.com – The Pherobase.
A Familiar Beetle
Stored Product Pest
The name ‘tribolium’ comes from the Latin verb for threshing, ‘tribulo.’ This is likely a reference to the fact that the first Tribolium beetle to be discovered, Tribolium castaneum, is a pest of stored grains and was found near where grain was threshed. Because of their association with stored grain products, these beetles are likely one of the most familiar beetles known to man. The various beetles of the genus Tribolium have a long history of being used as a model organism in laboratory studies in the areas of ecology, evolution, genetics, developmental biology, and animal behavior.
Of the dozens of species in this genus, as few as ten are storage pests associated with human grain stores and come from the castaneum, confusum, and brevicornis groups. Evidence from ancient Egypt indicates that the association between Tribolium and humans may be as old as 4000 years. These insects are incapable of attacking whole grain and are therefore secondary pests of grains such as wheat, maize, rice, barley, rye, oats. As their typical habitat is in stores of the flours of these grains, they are commonly called ‘flour beetles’ or ‘bran bugs.’ Interestingly, although Tribolium are regarded as a major pest of grain products, they may also be found in other types of foods such as beans, peas, nuts, chocolate, and even spices like ginger and red pepper. Because of their synanthropic nature, many of these pest beetles such as T. castaneum, T. confusum, T. destructor, and T. madens have a worldwide distribution. Others such as T. anaphe, T. audax, T. brevicornis, T. freemani, T. parallelus, and T. thusa have a more restricted range. The biology of the cosmopolitan storage pest beetles such as T. castaneum and T. confusum is the best understood and will be the main focus of this article.
Taxonomy and Diversity Tribolium beetles belong to the group of ‘darkling beetles’ or Tenebrionidae. As the name suggests, they have a characteristic dark body color (Figure 1). Like many other tenebrionids, Tribolium beetles are saprophagous, phytophagous, or mycophagous, and may be found in leaf litter, under the bark of trees or near fungi. In addition, like some of their close relatives of the tribe Triboliini, Tribolium beetles too may be associated with the nests of other species such as insects and some vertebrates. For example, some beetles such as T. myrmecophilum may be ecologically associated with insects’ nests, whereas others such as T. castaneum are associated with human dwellings and their stored grain. Thus, there is significant diversity in the ecological habitats of the three dozen species described in this group.
Evolutionary History Tribolium beetles may have diverged from other holometabolous insects as early as the beginning of the Cretaceous period. Since then, these beetles may have evolved in five separate lineages, each of which is associated with a particular region of the world. Each has distinctive morphology. Thus, the brevicornis group evolved in South America, the confusum group in Africa, the alcine group in Madagascar, the castaneum group in Indo-Australia, and the myrmicophilum group in Malay-East Indies. Of these, the confusum group is the most speciose with 14 known members and the myrmecophilum group is the smallest with only two known species.
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Morphology Adult Tribolium beetles are 3–5 mm in size. Some such as T. brevicornis (4 mm) and T. freemani (5 mm) are visibly larger than other species such as T. audax, T. anaphe, T. castaneum, and T. confusum (3 mm or smaller). Adults are sexually dimorphic. The sexes may be significantly different from each other in body size. In some species, such as T. brevicornis, males may be larger than females but in others including T. confusum, there may be a tendency for females to be larger. T. castaneum adult males are distinguishable from females because of the presence of setiferous glands (‘sex patches’) on their first pair of legs, which are absent in females. Similarly, in T. confusum, only males have these glands but they appear on each pair of appendages.
Life Cycle Beetle life cycles include four stages: egg, larva, pupa, and adult. The microscopic eggs of tenebrionids are ovoid and
Tribolium
Figure 1 Adult Tribolium castaneum in flour. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
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Figure 3 Pupae of Tribolium castaneum. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
almost 9 weeks for some species of flour beetles. Developmental time varies even among different strains of a species. In addition to genetics, environmental factors such as temperature, humidity, and food also influence development. Adults may live more than 3 years, though a life span of 1–6 months is more typical in laboratory settings. In general, the life span of virgin adults in laboratory conditions for T. castaneum and T. confusum beetles is 7–11 months.
Tribolium as a Model Organism
Figure 2 Larvae of Tribolium castaneum. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
bright white. In T. castaneum and T. confusum, the egg stage typically lasts 4–5 days in ideal laboratory conditions. The larvae are worm-like and yellowish (Figure 2). In optimal conditions, this stage is typically about 2–3 weeks for T. castaneum and T. confusum. The pupae are light colored, and either white or yellowish (Figure 3). Pupae are not capable of locomotion though they may wriggle (with the movement originating in the abdomen). The sexes are dimorphic and easily discernable as pupae, because the females have larger genital papillae compared to those found on the male pupae. In a favorable environment, the pupal stage may last 5–6 days in T. castaneum and T. confusum. The typical life cycle from egg to adult in laboratory strains of T. castaneum and T. confusum may be as short as 4 weeks in optimal conditions of food, temperature, and humidity. However, the rate of development from egg to adult varies vastly among species and may be as long as
Several features about their biology make flour beetles a convenient model system. Their affinity for stored grain products makes it easy to find and collect them from pantries, feed mills, feed stores, silos, etc. At these sites, beetles are easily detectable because they leave tunnel shaped tracks in the flour dust (Figure 4). When brought to the laboratory, beetles do not require much space and basically can be stored in glassware such as vials and jars because of their small size. They can be reared rather inexpensively in flour medium (typically a mixture of wheat flour and brewers’ yeast). Further, they can be indefinitely kept in dark conditions; this tolerance for dark facilitates their lives as storage pests in grain products. Tribolium cultures do not require constant maintenance, since all the life stages of these beetles can be grown in the same flour medium. Easy separation of the life stages also facilitates experimentation. Eggs and young larvae are significantly smaller than pupae and adults, and can be easily separated from each other by means of sifters with different mesh sizes. Although pupae and adults are of similar size, they are also easily sorted from each other, by taking advantage of the lack of mobility of the pupae. These insects are hardy and easily withstand being handled for experimental manipulation.
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were first discovered in the confused flour beetle in the 1990s. The Tribolium system has been used to study many major ideas in evolutionary biology such as mechanisms of reproductive isolation, group selection. In the last three decades, red flour beetles have proved to be invaluable as models for sexual selection studies and evolutionary–developmental biology (evo-devo) studies. The prolific literature on this genus provides a wealth of background information on the biology of these insects and has led to the development of new tools and methodologies that will further promote experimentation in this organism. Several authors have developed microsatellite markers in the red flour beetle, which can be applied to population genetics and parentage analysis studies. Also, the genome of T. castaneum was recently sequenced, which has positioned this species as one of the most significant models for evo-devo, genetics, and genomics studies.
Behavioral Repertoire of Tribolium Figure 4 Beetles-infested flour with tunnel shaped tracks. (Photo credit: Mark Lee, DOP images, Atlanta, GA).
Flour beetles, like many pest species, have a high reproductive capacity and remain reproductive throughout the year. Not only do males and females readily mate in a laboratory setting, but also, because adults have a long life span, a single female may produce hundreds of eggs in her long reproductive life, which enables high number of experimental trials. Because of their relatively short life cycle, these insects are suitable for multigenerational studies that ecology and evolutionary biology necessitate. All of these listed attributes have made Tribolium a wellstudied insect on which there is a plethora of information.
History as Laboratory Model Organism The flour beetle first came to be used as a laboratory model organism in population ecology studies initiated by Chapman in the late 1920s. In the subsequent four decades, from the 1930s to 1970s, Thomas Park and colleagues at the University of Chicago used this insect extensively as a laboratory model system to examine questions in ecology, genetics, behavior, and evolution. Indeed, some classical works illustrating such fundamental concepts in ecology and evolution, as population regulation and interspecific competition have employed flour beetles as model organisms. Many notable discoveries in genetics have also been made in Tribolium. For example, selfish genetic elements
Aside from being one of the foremost model organisms for population processes and developmental genetics at present, these beetles also make an appealing animal in which to study behavioral ecology and behavioral evolution, given their remarkable repertoire of behaviors. This article discusses only a small subset of some of the more intriguing of these behaviors. Cannibalism Tribolium beetles at various life stages may eat their own kind. Because of this taxon’s use as a laboratory model of population regulation, cannibalism is one of the beststudied aspects of beetle behavior and serves as an example of interference competition within a species. In general, the inactive stages, egg and pupa, are cannibalized by the active stages, larva and adult. However, it is also possible for adults to feed on very young larvae and for the old larvae to feed on callow adults the exoskeleton of which is not sclerotized. Studies in the confused flour beetles report that adults may devour an egg in a matter of minutes. This behavior is thought to be of great adaptive significance because it may facilitate beetles’ colonizing a novel food environment. Cannibalism is known to be under polygenic control. Thus, the tendency for cannibalism is variable and strongly depends on the species as well as the genetic background of beetle strains. Cannibalism in various flour beetle species does not correlate with phylogenetic relatedness. Apparently, it has been shaped by the evolutionary–ecological history of each individual species. Similarly, because of differences in selective pressures on different life stages that practice cannibalism, the
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larvae and the adults too show differences in cannibalism behavior. The larvae in general may feed preferentially on eggs, whereas the adults may feed preferentially on pupae. One explanation for this difference may be that larval nutritional requirements are more completely met by feeding on eggs, whereas adults may possibly eliminate competition in the near future by consuming pupae. Thus, each life stage may be maximizing their chance of survival through preferential cannibalism. Environmental variables such as amounts and quality of food, as well as population density, also influence cannibalism rates. Beetles tend to be more cannibalistic in environments with greater population densities and lower food quantity and quality. Therefore, cannibalism in flour beetles is a classic example of population regulation through density-dependent processes. Cannibalism of eggs and pupae has a significant impact in regulating population sizes. In the confused flour beetle for instance, population sizes would be tenfold larger without cannibalism. Competition Different species of flour beetles may occupy a similar niche in an ecosystem. Because they require similar types of abiotic conditions as well as feed on similar foods (stored grain products), they may co-occur within a space and consequently compete for resources in the same environment. Interestingly, the outcome of competitive interactions between the red flour beetle and confused flour beetle depends on abiotic factors such as temperature and humidity, as well as biotic factors such as initial densities of beetles, and presence or absence of parasites. Competition experiments on flour beetles conducted by Park and his colleagues are a classic study in ecology. Their experiments revealed that the confused flour beetle is likely to dominate in environments that are cool and arid, whereas the red flour beetle is likely to prevail at higher temperatures and in humid environments. Furthermore, parasitism also influenced the outcome of competition between the two flour beetle species. When beetle cultures were infected with a microparasite, Adelina, it was found that T. confusum, which is better able to withstand Adelina, was likely to ‘win’ in the interspecific competition even in abiotic conditions that typically favored T. castaneum. When a different parasite, Hymenolepis diminuta was used, the results were the opposite; when beetles were infected with the abovementioned rat tapeworm, the red flour beetle tended to be the superior competitor. One of the chief ways competing species of flour beetles interact with each other is preying on their competitor’s eggs and pupae. Beetles are able to distinguish between eggs and pupae from their own species and those of their competitors. Adult beetles may preferentially feed on the heterospecific eggs and pupae.
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Chemical Communication Beetles produce various chemicals used in communication with conspecifics as well as with other species. Flour beetles produce a host of chemicals that are secreted to their external environment; for example, both T. castaneum and T. confusum produce over half a dozen such chemicals. However, the function of these compounds is not very well understood. Some compounds such as 4–8 dimethly decanal (4–8 DMD) are produced only by males, others such as Z-2–9-propionate are produced by females, and still others, such as various toluquinones and benzoquinones are produced by both sexes. Possibly, the best-known compound is 4–8 DMD, which is an aggregation pheromone. It is a common observation that beetles in laboratory cultures and in feed mills are found in aggregations. Typically, beetle behavioral response to 4–8 DMD includes walking toward the source accompanied by a movement of the head and antennae. So strong is this behavioral response that many commercial lures use synthetically produced 4–8 DMD to trap beetles in stores of grain. While this is primarily an aggregation pheromone, it also functions to attract potential mates at closer ranges. Female mate choice in T. castaneum may partially depend on male pheromone cue at least in some populations, and male sperm precedence was shown to depend on female’s response to this olfactory signal among those populations. Toluquinones are among the suite of compounds produced by beetles and function as allomones. It is likely that they serve as a defense against the microbes found in the flour and predators in the environment. Defensive Behaviors In addition to chemical defense, beetles may resort to thanatosis or feigning death as means of defense against predators. Beetles often feign death by lying still for as few as a fraction of a second to up to a few minutes upon sensing a predator. This behavior is known to vary among different genetic backgrounds of flour beetles. Recent studies show that death feigning behavior is correlated with another type of predator avoidance behavior, fleeing. Beetles that feigned death for longer fled shorter distances than beetles that feigned death for shorter durations. This suggests that beetle strains may be genetically predisposed to one of the two alternative strategies to avoid danger from a predator, either feign death or flee. Death feigning behavior and its evolutionary significance warrants further investigation. Interactions with Parasites Flour beetles have been used as a model system to study host–parasite dynamics especially in the recent past.
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These beetles may get infected with a variety of parasites, including microparasites such as Adelina and Nosema, as well as macroparasites such as Hymenolepis. Tribolium and their parasites have been used to test several theories in the realm of host–parasite interactions. Several studies have examined the fitness consequences of parasitism for flour beetles. As expected from a typical host–parasite interaction, a general pattern with respect to flour beetles’ interactions with parasites is that the latter adversely affect the fitness of these beetles. Beetle fitness may be affected because of one or more factors such as reduction in attractiveness, reduced fecundity, reduced sperm precedence. A hypothesis is whether parasites manipulate intermediate host behavior to facilitate their transmission to the final hosts. This has been tested using the rat tapeworm H. diminuta and flour beetles. Beetles get infected with this parasite when they consume tapeworm egg-infested rat feces. The eggs develop into cysticercoids inside the beetles. The tapeworm completes its life cycle after infected beetles are consumed by the final host, the rat. Thus, beetles serve as the intermediate hosts of this parasite. It is likely that higher rate of surface seeking (going to the top of the flour) and emigration would make beetles more vulnerable to predation by rats, thereby improving the chances of parasite transmission. Studies show that when infected by the rat tapeworm, beetle behaviors are significantly altered. Interestingly, prevalence of parasites increased T. confusum beetles’ tendency for surface seeking and emigration compared to uninfected beetles, but had the opposite effect on T. castaneum beetles. Thus, it is clear that parasites alter beetle host behavior. The available data, though, do not support the idea that flour beetles’ behavioral changes enhance parasite transmission. Mating Behaviors Mating rates
In recent years, the red flour beetle has been extensively used as a model system for sexual selection studies. However, the mating behavior of the other flour beetles, including the confused flour beetle, is relatively less studied. The red flour beetle and confused flour beetle differ remarkably in their mating rates. Red flour beetles mate readily in the lab, whereas confused flour beetles mate at a lower frequency in laboratory observations. In the red flour beetle, both sexes are promiscuous and may mate multiple times with the same partner (repeated mating) or mate with different partners (multiple mating) in a relatively short time of few minutes. Typically, males approach the female to initiate copulation. Females may move away from the male rapidly or remain quiescent and allow the males to mount. Copulation durations range from a few seconds to half an hour.
Mate choice
Both sexes of the red flour beetle exhibit precopulatory mate choice and preferred traits likely vary among populations. Some male traits known to be attractive to females include large body size and certain pheromone cues. Males may show preference for virgin females and also favor novel mates in comparison with females they have previously mated with. In addition, males may avoid mating with callow females. Evidence from studies examining the correlation between male traits, female response, and sperm precedence suggests that females continue to asses male quality even after the initial decision to mate is made and that females bias paternity in favor of preferred males. Beetle males that stroke their partners more vigorously with forelegs while in copula (a form of copulatory courtship) have a higher-than-expected share of paternity. A clever experiment that altered only female perception of male quality but not male quality itself showed that male share of paternity was significantly affected by female perception of male quality. Female red flour beetles have the capacity to control sperm movement in their reproductive tract, which may be the mechanism for such a postcopulatory female mate choice. Male–male interactions
While a red flour beetle pair is in copula, rival males may interfere by butting into the pair or climbing over them. In some cases, males may not be able to complete intromission because of physical interference by rivals. Success and duration of copulation may be influenced by these male–male interactions. When copulating red flour beetles are disturbed by rival males, the copulations typically last longer, possibly because physical disturbance from rivals cause males to require additional time for a successful sperm transfer. Because of the high mating rates of both sexes in the red flour beetles, sperm from different males may cooccur in a female’s reproductive tract. Hence, sperm competition and sperm precedence is a well-studied aspect of flour beetle biology. Typically, the last male to mate sires the majority of a female’s offspring. Just as there are female controlled postcopulatory processes, male controlled postcopulatory processes also have been documented in the red flour beetle. Aside from mating order and female preference, other factors may also influence the outcome of sperm competition in terms of male sperm precedence. One facet of intrasexual competition in this beetle is male ability to displace a rival male’s sperm by means of their intromittent organ (the aedeagus). This may in fact be a significant aspect of sperm competition in this species, but has not been studied in much detail. In a small percentage of cases with sperm displacement, males end up translocating their rival’s sperm, which lingers on their intromittent
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organ, when they subsequently seek to mate with other partners. This phenomenon, when males gain a share of paternity of offspring from females they themselves have not mated with because of sperm translocation by rivals, has been described as fertilization by ‘proxy.’ Another relatively unexplored aspect of male–male interactions is the phenomenon of homosexual copulations. Homosexual copulations between males are quite common in beetle aggregations. Males may mount other males and even deposit a spermatophore in the course of a homosexual copulation. The adaptive significance of this behavior, if any, is unclear. Male–female interactions
The high level of promiscuity in red flour beetles causes a conflict of interest between the sexes in this species. For example, males are selected to maximize their paternity, whereas females may be selected to choose the highest quality sire for their offspring. Conflicts of interest may lead to antagonistic co-evolution between the sexes. One example of a conflict of interest between the sexes may relate to the mating frequency. Males may benefit from mating frequently or for a prolonged duration, because this may help them to maximize their sperm transfer and facilitate mate guarding, thereby increasing their share of paternity. The same may not be true for females that may incur costs, such as expenditure of time, energy, and/or loss of other mating opportunities. Thus, in beetle populations that show a high female mating rate, it is possible that the females are coerced into copulating. That females may be resisting male attempts at frequent copulation is suggested by some of their behaviors during mating. For example, they may exhibit swift movement away from a male that is attempting to mount. Even after the male manages to mount a female, they may move backwards to cause a mounted male to dislodge, or move extremely fast, another tactic that may cause the mounted male to dislodge. Females are also usually the first to walk away from copula. In contrast to these resistance behaviors, female cooperation is evident when they remain quiescent and allow a male to mount. Multiple studies have demonstrated that populations of red flour beetles differ with respect to their mating related traits such as mating frequency, oviposition rates, mating duration. Possibly, this stems from not just differences in natural selection and drift, but also because of differences in patterns of male–female co-evolution in different populations. Thus, this insect is probably one of the best model systems to study male–female co-evolution.
Conclusion Overall this humble group of pest species has served as an incomparable laboratory model, which has yielded
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valuable insights into the evolution of various behaviors. Many behaviors described here are only partially understood and merit further study. In addition, with the advent of new tools in molecular biology and genomics, this genus will continue to be a useful organism in which to explore questions on evo-devo and functional genomics. See also: Intermediate Host Behavior; Invertebrates: The Inside Story of Post-Insemination, Pre-Fertilization Reproductive Interactions; Isolating Mechanisms and Speciation; Mating Signals; Microevolution and Macroevolution in Behavior; Parasite-Modified Vector Behavior; Parasites and Sexual Selection; Propagule Behavior and Parasite Transmission; Reproductive Behavior and Parasites: Invertebrates; Signal Parasites; Social Recognition; Social Selection, Sexual Selection, and Sexual Conflict.
Further Reading Alabi T, Michaud JP, Arnaud L, et al. (2008) A comparative study of cannibalism and predation in seven species of flour beetle. Ecological Entomology 33: 716–726. Angelini DR and Jockusch EL (2008) Relationships among pest flour beetles of the genus Tribolium (Tenebrionidae) inferred from multiple molecular markers. Molecular Phylogenetics and Evolution 46: 127–141. Beeman RW, Friesen KS, and Denell RE (1992) Maternal-effect, selfish genes in flour beetles. Science 256: 89–92. Bonneton F (2008) The beetle by the name of Tribolium, typology and etymology of Tribolium castaneum Herbst, 1797. Insect Biochemistry and Molecular Biology 38: 377–379. Fedina T and Lewis SM (2008) An integrative view of sexual selection in Tribolium flour beetles. Biological Reviews of the Cambridge Philosophical Society 83: 151–171. Mertz DB (1972) The Tribolium model and the mathematics of population growth. Annual Review of Ecology and Systematics 3: 51–78. Miyatake T, Tabuchi K, Sasaki K, et al. (2008) Pleiotropic antipredator strategies, fleeing and feigning death, correlated with dopamine levels in Tribolium castaneum. Animal Behaviour 75: 113–121. Nakakita H (1983) Rediscovery of Tribolium freemani Hinton: A stored product insect unexposed to entomologists for the past 100 years. JARQ 16: 239–245. Pai A and Bernasconi G (2008) Polyandry and female control: The red flour beetle Tribolium castaneum as a case study. Journal of Experimental Zoology Part B, Molecular and Developmental Evolution 310: 148–159. Park T (1962) Beetle, competition and populations. Science 138: 1369–1375. Sokoloff A (1972, 1974, 1977) The Biology of Tribolium vols. 1–3. Oxford: Clarendon Press and Oxford University Press. Tribolium Sequencing Consortium (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452: 949–955. Wade MJ, Patterson H, Chang NW, et al. (1994) Postcopulatory, prezygotic isolation in flour beetles. Heredity 72: 163–167. Yan G, Stevens L, Goodnight C, et al. (1997) Effects of a tapeworm parasite on the competition of Tribolium beetles. Ecology 79: 1093–1103. Yan G, Stevens L, and Schall JS (1994) Behavioral changes in Tribolium beetles infected with tapeworm: Variation in effects between beetle species and among genetic strains. The American Naturalist 143: 830–847.
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Relevant Websites http://www.ars.usda.gov – USDA Agricultural Research Service. http://www.beetlebase.org – Tribolium Genome Database.
http://www.hgsc.bcm.tmc.edu – Baylor College of Medicine, Human Genome Sequencing Center. http://www.pherobase.com – The Pherobase.