Functionality of the GAL4/UAS system in <Emphasis Type="Italic">Tribolium</Emphasis> requires the use of endogenous core promoters

Background  

The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.     

The effects of temperature,relative humidity,light, and resource quality on flight initiation in the red flour beetle,Tribolium castaneum

We investigated the environmental conditions that induce a flight response in the red flour beetle, Tribolium castaneumHerbst (Coleoptera: Tenebrionidae), including resource quality, temperature, relative humidity, and light. Over 72‐h trial periods, we observed the proportion of individuals emigrating by flight to range from 0.0 in extreme heat or cold to 0.82 with starvation. Resource quality, presence of a light source, and temperature all directly influenced the initiation of the flight response. We did not detect any effect of relative humidity or sudden change in temperature on the incidence of flight. We discuss our findings in the context of Tribolium ecology and evolution.     

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster

When ectotherms are exposed to low temperatures, they enter a cold‐induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill‐coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold‐induced perturbations. The metabolites of cold‐hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.     

Insertional mutagenesis screening identifies the <Emphasis Type="Italic">zinc finger homeodomain 2</Emphasis> (<Emphasis Type="Italic">zfh2</Emphasis>) gene as a novel factor required for embryonic leg development in <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

The genetic control of leg development is well characterized in the fly Drosophila melanogaster. These control mechanisms, however, must differ to some degree between different insect species to account for the morphological diversity of thoracic legs in the insects. The legs of the flour beetle Tribolium castaneum differ from the Drosophila legs in their developmental mode as well as in their specific morphology especially at the larval stage. In order to identify genes involved in the morphogenesis of the Tribolium larval legs, we have analyzed EGFP enhancer trap lines of Tribolium. We have identified the zfh2 gene as a novel factor required for normal leg development in Tribolium. RNA interference with zfh2 function leads to two alternative classes of leg phenotype. The loss of a leg segment boundary and the generation of ectopic outgrowths in one class of phenotype suggest a role in leg segmentation and segment growth. The malformation of the pretarsal claw in the second class of phenotype suggests a role in distal development and the morphogenesis of the claw-shaped morphology of the pretarsus. This suggests that zfh2 is involved in the regulation of an unidentified target gene in a concentration-dependent manner. Our results demonstrate that enhancer trap screens in T. castaneum have the potential to identify novel gene functions regulating specific developmental processes.     

A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum

 Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species. Received: 4 November 1998 / Accepted: 7 February 1999     

From development to biodiversity—Tribolium castaneum, an insect model organism for short germband development

Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.     

The consequences of parental age for development,body mass and resistance to stress in the red flour beetle

The performance of most animals deteriorates with age. Motivated by the inconsistency in the literature regarding the effect of parental age on offspring traits and performance, we studied how parental age affects offspring development time, body mass, and starvation and cold tolerance in the red flour beetle (Tribolium castaneum). Offspring of old parents pupated later and at a higher body mass, and there was a general positive correlation between body mass and starvation tolerance. Despite their higher body mass, offspring of old parents tolerated starvation less well than those of young parents, emphasizing the impaired performance of the former. However, parental age did not affect offspring thermal tolerance and offspring of old parents were not more sensitive to cold shock than those of young parents. We also examined how ageing affects body mass and cold tolerance in the parental generations. By contrast to the effect of ontogeny on thermal tolerance, which is better known, change in thermal tolerance with age is seldom studied and can take different shapes. Old beetles were more sensitive to cold shock than younger beetles. Similar to cold tolerance, body mass decreased with age. In summary, older beetles reflect a worse physiological condition than younger ones. Ageing leads to impaired cold tolerance, lower body mass, lower number of offspring reaching adulthood, and deteriorated performance of the offspring, expressed as a lower starvation tolerance and a longer development time of the offspring. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 305–314.     

Postgenomics of Tribolium: Targeting the endocrine regulation of diuresis

Coleopteran insects comprise a highly successful taxon, representing more than 25% of eukaryotic species, many of which are economically important. One of these, the red flour beetle (Tribolium castaneum H.), is a major pest of stored grain and cereal products and is also an excellent genetic model for the Coleoptera. The Tribolium genome sequencing project was recently completed, the first for an agricultural insect pest species. This genome sequence and annotation, in combination with the tractability of Tribolium for genetic dissection and functional genomic analysis, makes it one of the most useful subjects for studies of insect development, genetics and physiology. Tribolium has a number of interesting physiological adaptations, including desiccation tolerance associated with specialized cryptonephridial organs for active rectal absorption of atmospheric water. The study of endocrine regulation of diuresis in this species has led to the identification of a number of osmoregulatory peptides, including the novel arginine‐vasopressin‐like peptide.     

Selection for rapid and slow recovery from chill‐ and heat‐coma in Drosophila melanogaster

To assess the trade‐offs associated with cold and heat tolerance, selection experiments were conducted on the rate of recovery from chill‐ and heat‐coma using Drosophila melanogaster. Flies were treated with cold and heat to induce coma, and those that showed rapid or slow recovery from coma were selected. The lines selected for rapid (or slow) recovery from chill‐coma also showed rapid (slow) recovery from heat‐coma, although such a correlation was not observed in the lines selected for the rate of recovery from heat‐coma. On the other hand, survival after cold was enhanced in both lines selected for rapid and slow recovery from chill‐coma, and survival after heat was enhanced in both lines selected for rapid and slow recovery from heat‐coma. It was assumed that cold and heat treatments to induce coma caused some damages to flies and those that were tolerant to cold or heat were unintentionally selected in the present coma‐based selection. Only a weak trade‐off was observed between survival‐based cold and heat tolerance. On the other hand, developmental time was prolonged and desiccation resistance, walking speed, and longevity were reduced in the lines selected for rapid and slow recovery from chill‐ and/or heat‐coma, suggesting that these resistance and life‐history traits are under trade‐offs with cold and/or heat tolerance. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 72–80.     

Stress tolerance in a novel system: Genetic and environmental sources of (co)variation for cold tolerance in the butterfly Eurema smilax

The physiological ability to survive climatic extremes, such as low temperature, is a major determinant of species distribution. Research suggests that tropically restricted insect populations may possess low to zero variation in stress tolerance, thereby limiting any potential to adapt to colder climates. This paradigm derives largely from contrasts among Drosophila populations and species along the tropical–temperate cline of eastern Australia. Butterfly groups, such as the variously distributed representatives of the genus Eurema, offer opportunities to test the taxonomic breadth of this paradigm. We contribute here by investigating plasticity, repeatability and heritability (h²) for cold tolerance in Eurema smilax. This continentally widespread species (extending from the Torres Strait to the south coast of Victoria) offers an important comparative basis for evaluating stress tolerance in geographically restricted congenerics. We reared two generations of E. smilax under laboratory conditions and measured recovery from a chill‐coma assay, which is one of the commonly used methods for characterizing adult cold stress tolerance. Trials on F2s conducted over three consecutive days revealed individual repeatability (r = 0.405). However, recovery time decreased systematically across trials, which is characteristic of a phenotypically plastic ‘hardening’ response to prior cold exposure. Generalized linear modelling, wherein genetic variance was estimated via an ‘animal model’ approach, indicated no difference between sexes and no effect of body size, but a significant additive genetic term, corresponding to a heritability estimate of h² = 0.414 ± 0.100. These data suggest significant adaptive potential for cold tolerance in E. smilax but show that individuals may also respond directly to extremes of cold via phenotypic plasticity. This indicates the potential to adapt to varied thermal extremes, which would be expected for a broadly distributed species that is resilient to climate change.     

CHILL-COMA TOLERANCE, A MAJOR CLIMATIC ADAPTATION AMONG DROSOPHILA SPECIES

Abstract.— Most drosophilid species can be classified either as temperate or tropical. Adults of species were submitted to a cold treatment (0°C) and then brought back to ambient temperature. They generally exhibited a chill coma and the time needed to recover was measured. We found in a set of 26 temperate species that recovery was rapid (average 1.8 min, range 0.15–4.9). In contrast, a long recovery time (average 56 min, range 24–120) was observed for 48 tropical species. A few species, like Drosophila melanogaster, are cosmopolitan and can proliferate under temperate and tropical climates. In 9 of 10 such species, slight genetic differences were found: a shorter recovery in temperate than in tropical populations. Comparing physiological data to phylogeny suggests that chill‐coma tolerance has been a recurrent adaptation that is selected for in cold climates but tends to disappear under a permanently warm environment. This major climatic adaptation, evidenced in drosophilids, seems to occur in other insect groups also.     

Impact of different acclimation temperatures and duration on the chill coma temperature and oxygen consumption in the tenebrionid beetle Alphitobius diaperinus

When the ambient temperature is lowered to an insect's lower thermal limit, the insect enters into chill coma. Chill coma temperature and chill coma recovery can vary within species as a result of thermal acclimation, although the physiological basis of the onset of chill coma remains poorly understood. The present study investigates how the temperature of acclimation (0, 5, 10, 15 and 20 °C for 2 or 7 days) affects chill coma temperature and oxygen consumption in adult Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae). It is hypothesized that the threshold decline in metabolic rate corresponds to the entry into chill coma. Oxygen consumption (as a proxy of metabolism) is measured across the chill coma temperature threshold, and a strong decline in oxygen consumption is expected at entry into chill coma. The acclimation decreases the chill coma temperature significantly from 6.6 ± 1.1 °C in control insects to 3.1 ± 0.7 °C in those acclimated to 10 °C. The change in metabolic rate (Q₁₀) after acclimation to temperatures ranging from 10 to 20 °C is 3.7. Despite acclimation, the metabolic rate of A. diaperinus conforms to Arrhenius kinetics, suggesting that the response of this beetle does not show metabolic compensation. The data suggest the existence of a threshold decline in metabolic rate during cooling that coincides with the temperature at which an insect goes into chill coma.     

The <Emphasis Type="Italic">Tribolium spineless</Emphasis> ortholog specifies both larval and adult antennal identity

The morphology of insect antennae varies widely among species, but our understanding of antennal development comes almost solely from studies of one species—the fruit fly, Drosophila melanogaster. Moreover, this knowledge applies mostly to adult structures, since Drosophila lacks external larval appendages. In contrast to Drosophila, the red flour beetle, Tribolium castaneum, has both larval and adult antennae, which are very different from one another in morphology. Thus, Tribolium provides an ideal system to compare modes of antennal development both within and between species. Here, we report that the Tribolium ortholog of spineless (Tc-ss) is required in both the larval and adult antennae. Knockdown of Tc-ss by RNAi during either larval or imaginal development causes transformation of the distal portion of the antennae to legs. Thus, the function of ss is conserved between Drosophila and Tribolium with respect to adult antennal specification and also between Tribolium larval and adult antennal development. The similarity of the Tc-ss RNAi phenotype to that of a classically described Tribolium mutation, antennapedia (ap) (of no relationship to the Drosophila Hox gene of the same name), led us to characterize the original ap mutation and two newly identified ap alleles. Our mapping and phenotypic data suggest that Tc-ss is the best candidate for the ap locus. These results represent a first step in characterizing larval and adult antennal patterning in Tribolium, which should provide important insights into the evolution of insect antennal development.     

Environmental and genetic control of cold tolerance in the Glanville fritillary butterfly

Thermal tolerance has a major effect on individual fitness and species distributions and can be determined by genetic variation and phenotypic plasticity. We investigate the effects of developmental and adult thermal conditions on cold tolerance, measured as chill coma recovery (CCR) time, during the early and late adult stage in the Glanville fritillary butterfly. We also investigate the genetic basis of cold tolerance by associating CCR variation with polymorphisms in candidate genes that have a known role in insect physiology. Our results demonstrate that a cooler developmental temperature leads to reduced cold tolerance in the early adult stage, whereas cooler conditions during the adult stage lead to increased cold tolerance. This suggests that adult acclimation, but not developmental plasticity, of adult cold tolerance is adaptive. This could be explained by the ecological conditions the Glanville fritillary experiences in the field, where temperature during early summer, but not spring, is predictive of thermal conditions during the butterfly's flight season. In addition, an amino acid polymorphism (Ala‐Glu) in the gene flightin, which has a known function in insect flight and locomotion, was associated with CCR. These amino acids have distinct biochemical properties and may thus affect protein function and/or structure. To our knowledge, our study is the first to link genetic variation in flightin to cold tolerance, or thermal adaptation in general.     

Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

Analysis of repetitive DNA distribution patterns in the <Emphasis Type="Italic">Tribolium castaneum</Emphasis> genome

Background  

Insect genomes vary widely in size, a large fraction of which is often devoted to repetitive DNA. Re-association kinetics indicate that up to 42% of the genome of the red flour beetle, Tribolium castaneum, is repetitive. Analysis of the abundance and distribution of repetitive DNA in the recently sequenced genome of T. castaneum is important for understanding the structure and function of its genome.     

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Genetic variance in fitness and its cross-sex covariance predict adaptation during experimental evolution

The additive genetic variation (V_A) of fitness in a population is of particular importance to quantify its adaptive potential and predict its response to rapid environmental change. Recent statistical advances in quantitative genetics and the use of new molecular tools have fostered great interest in estimating fitness V_A in wild populations. However, the value of V_A for fitness in predicting evolutionary changes over several generations remains mostly unknown. In our study, we addressed this question by combining classical quantitative genetics with experimental evolution in the model organism Tribolium castaneum (red flour beetle) in three new environmental conditions (Dry, Hot, Hot-Dry). We tested for potential constraints that might limit adaptation, including environmental and sex genetic antagonisms captured by negative genetic covariance between environments and female and male fitness, respectively. Observed fitness changes after 20 generations mainly matched our predictions. Given that body size is commonly used as a proxy for fitness, we also tested how this trait and its genetic variance (including nonadditive genetic variance) were impacted by environmental stress. In both traits, genetic variances were sex and condition dependent, but they differed in their variance composition, cross-sex and cross-environment genetic covariances, as well as in the environmental impact on V_A.     

Evolved variation in cold tolerance among populations of Eldana saccharina (Lepidoptera: Pyralidae) in South Africa

Among‐population variation in chill‐coma onset temperature (CT_min) is thought to reflect natural selection for local microclimatic conditions. However, few studies have investigated the evolutionary importance of cold tolerance limits in natural populations. Here, using a common‐environment approach, we show pronounced variation in CT_min (± 4 °C) across the geographic range of a nonoverwintering crop pest, Eldana saccharina. The outcomes of this study provide two notable results in the context of evolved chill‐coma variation: (1) CT_min differs significantly between geographic lines and is significantly positively correlated with local climates, and (2) there is a stable genetic architecture underlying CT_min trait variation, likely representing four key genes. Crosses between the most and least cold‐tolerant geographic lines confirmed a genetic component to CT_min trait variation. Slower developmental time in the most cold‐tolerant population suggests that local adaptation involves fitness costs; however, it confers fitness benefits in that environment. A significant reduction in phenotypic plasticity in the laboratory population suggests that plasticity of this trait is costly to maintain but also likely necessary for field survival. These results are significant for understanding field population adaption to novel environments, whereas further work is needed to dissect the underlying mechanism and gene(s) responsible.