Hereditary nature of death-feigning frequency in a parasitoid wasp Heterospilus prosopidis using sib analysis.

Death feigning, a state of immobility observed in many animals in response to external stimuli, is an anti-predator behaviour. Although previous studies showed that death-feigning behaviours are quantitative genetic traits, the knowledge of the heritable basis of death-feigning behaviour is lacking. To investigate the heritable basis of death-feigning behaviour, we used three laboratory strains of a braconid parasitoid wasp, Heterospilus prosopidis. The heritable basis using half-sib analysis, and the effects of different geographical backgrounds, rearing conditions in the laboratory, and host age were evaluated. The results of the half-sib analysis showed that the frequency of death feigning varied among sires, suggesting a certain extent of additive genetic variance. Also, the frequency of death feigning varied between geographical backgrounds and among strains. Death-feigning frequency was not affected by the age of the host. Our findings highlight the importance of genetic factors underlying the basis of the death-feigning behaviour and provide support for the genetic alterations of traits from the perspective of evolution in various animal species.

Dominance and inheritance patterns of mobility and death feigning in beetle strains selected for moving activity.

Reciprocal crossing of different strains is a suitable method to investigate the dominance and inheritance of a focal trait. Herein, we performed reciprocal crossing among strains of Tribolium castaneum exhibiting a genetically high (H strain) and low (L strain) moving activity and investigated the related heritable factors in the F1 and F2 generations. We also evaluated death-feigning behavior, which negatively responded to artificial selection for moving activity in T. castaneum. The results obtained for the F1 generation suggest that low moving activity and short duration of death feigning were dominant. In the F2 generation, movement and death feigning exhibited continuous segregation. The distribution of each trait value in the F2 generation differed from that in the parental generation, and no individuals transgressing the distribution of trait values in the parental generation emerged in the F2 generation. These results suggest that the genetic correlation between movement and death-feigning behavior is controlled in a polygenic manner. Moreover, the examination of the proportions of both behaviors (high vs. low moving activity and long vs. short death-feigning duration) in the F1 generation revealed that the two behaviors may be controlled by the maternal genotype, suggesting that the gene(s) that control movement and death feigning are located on the sex chromosome in T. castaneum.

Latitudinal cline of death-feigning behaviour in a beetle (Tribolium castaneum).

Death-feigning behaviour is a phenomenon in which a prey is rendered motionless due to stimulation or threat from a predator. This anti-predator defence mechanism has been observed across numerous animal taxa and is considered adaptive in nature. However, longer durations of death feigning can result in decreased opportunities for feeding and reproduction, and therefore is often associated with fitness costs as compared to environments without predators. Differences have also been observed in the frequencies and durations of death feigning within populations, and these differences are thought to be influenced by the balance between survival and other fitness costs. Furthermore, this balance is predicted to vary in response to changes in environmental conditions. In this study, we examined the death feigning in 38 populations of the red flour beetle (Tribolium castaneum). Our results demonstrate that frequencies and durations of the death feigning in T. castaneum show geographical variations and a latitude cline, indicating that this behaviour is influenced by location as well as latitude. This study is the first to demonstrate the existence of a latitudinal cline in death feigning and suggests that death-feigning behaviour might have evolved in response to environmental factors that vary with latitude.

Responses to artificial selection for locomotor activity: A focus on death feigning in red flour beetle.

Whole-organism performance, including locomotor activity, is an important fitness trait in many animals. Locomotor activity is often classified into sprint speed and locomotor endurance and differences in sprint speed and locomotor endurance affect on other traits such as life-history traits. Previous studies found that locomotor endurance, sprint speed and brain dopamine (DA) levels are correlated with artificial selection for death feigning (an anti-predator behaviour that we refer to as 'death-feigning syndrome') in some insect species. Thus, if the syndrome has a genetic basis, death feigning, sprint speed and brain DA levels may be affected by artificial selection for locomotor endurance. We artificially selected for locomotor endurance over 10 generations in the red flour beetle Tribolium castaneum, and established higher (H) and lower activity (L) strains, then compared their death-feigning behaviour, sprint speed and brain DA levels. H-strain beetles exhibited significantly shorter duration of death-feigning, and significantly higher sprint speeds, suggesting variation in death-feigning syndrome. Surprisingly, although brain DA expression affects various animal behaviours, we found no significant differences in the brain DA expressions of H- and L-strain beetles. Thus, our results imply genetic correlations between locomotor endurance, sprint speed and death feigning, but not with brain DA expression, suggesting that differences in the biogenic amine results of our and previous studies may reflect differences in behavioural expression mechanisms.

Polygene control and trait dominance in death-feigning syndrome in the red flour beetle Tribolium castaneum.

Death-feigning behavior is an anti-predatory technique used in several animal taxa and often correlates with inhibited movements (i.e., death-feigning syndrome). We performed a reciprocal crossing among strains exhibiting a genetically longer (L-strain) and shorter (S-strain) duration of death feigning. Then, we investigated related heritable factors in F1 and F2 populations. We also evaluated movement activities, which negatively responded to artificial selection for death feigning in T. castaneum. Our results indicated that death feigning occurred more frequently and for shorter periods in the F1 population. However, in the F2 population, death feigning and movement exhibited continuous segregation. Although the distribution of each trait value in the F2 generation differed from that of the parental generation, no individuals transgressing the distribution of trait values in the parental generation emerged from the F2 generation. Besides, chi-square analysis of the observed death feigning and movement of F1 and F2 progenies rejected the hypothesis of mono-major gene inheritance. These results suggested that the death-feigning syndrome was polygenically controlled, indicating the usefulness of reciprocal crossing experiments in assessing the quantitative inheritance of behavioral traits.

Death feigning as an adaptive anti-predator behaviour: Further evidence for its evolution from artificial selection and natural populations.

Death feigning is considered to be an adaptive antipredator behaviour. Previous studies on Tribolium castaneum have shown that prey which death feign have a fitness advantage over those that do not when using a jumping spider as the predator. Whether these effects are repeatable across species or whether they can be seen in nature is, however, unknown. Therefore, the present study involved two experiments: (a) divergent artificial selection for the duration of death feigning using a related species T. freemani as prey and a predatory bug as predator, demonstrating that previous results are repeatable across both prey and predator species, and (b) comparison of the death-feigning duration of T. castaneum populations collected from field sites with and without predatory bugs. In the first experiment, T. freemani adults from established selection regimes with longer durations of death feigning had higher survival rates and longer latency to being preyed on when they were placed with predatory bugs than the adults from regimes selected for shorter durations of death feigning. As a result, the adaptive significance of death-feigning behaviour was demonstrated in another prey-predator system. In the second experiment, wild T. castaneum beetles from populations with predators feigned death longer than wild beetles from predator-free populations. Combining the results from these two experiments with those from previous studies provided strong evidence that predators drive the evolution of longer death feigning.

Anti-predator behaviour depends on male weapon size.

Tonic immobility and escape are adaptive anti-predator tactics used by many animals. Escape requires movement, whereas tonic immobility does not. If anti-predator tactics relate to weapon size, males with larger weapons may adopt tonic immobility, whereas males with smaller weapons may adopt escape. However, no study has investigated the relationship between weapon size and anti-predator tactics. In this study, we investigated the relationship between male weapon size and tonic immobility in the beetle Gnathocerus cornutus. The results showed that tonic immobility was more frequent in males with larger weapons. Although most studies of tonic immobility in beetles have focused on the duration, rather than the frequency, tonic immobility duration was not affected by weapon size in G. cornutus. Therefore, this study is the first, to our knowledge, to suggest that the male weapon trait affects anti-predator tactics.

Arousal from Tonic Immobility by Vibration Stimulus.

Tonic immobility (TI) is an effective anti-predator strategy. However, long immobility status on the ground increases the risk of being eaten by predators, and thus insects must rouse themselves when appropriate stimulation is provided. Here, the strength of vibration causing arousal from the state of TI was examined in strains artificially selected for longer duration of TI (L-strains: long sleeper) in a beetle. We provided different strengths of vibration stimuli to the long sleepers in Tribolium castaneum. Although immobilized beetles were never awakened by the stimuli from 0.01 to 0.12 mm in amplitude, almost of the beetles were aroused from immobilized status by the stimulus at 0.21 mm. There was a difference in sensitivity of individuals when the stimuli of 0.14 mm and 0.18 mm were provided. F2 individuals were also bred by crossing experiments of the strains selected for shorter and longer duration of TI. The arousal sensitivity to vibration was well separated in the F2 individuals. A positive relationship was observed between the duration of TI and the vibration amplitude, suggesting that immobilized beetles are difficult to arouse from a deep sleep, while light sleepers are easily aroused by even small vibrations. The results indicate a genetic basis for sensitivity to arousal from TI.

Costs of walking: differences in egg size and starvation resistance of females between strains of the red flour beetle (Tribolium castaneum) artificially selected for walking ability.

In many animals, movement often affects fitness components such as foraging or migration. On the other hand, individuals with higher mobility also often have fitness costs. This trade-off between movement and other traits may explain the maintenance of variation in moving ability in a population. However, few studies have focused on movement by walking, although many previous studies of insects have investigated the evolution of variations in mobility with wing polymorphism. In this study, we focused on the walking ability of the red flour beetle (Tribolium castaneum) and investigated whether females with higher than lower walking ability have fitness costs. The present results showed that females with genetically higher walking ability produced smaller eggs and had lower starvation resistance than females with lower walking ability. These results suggest that higher walking ability is costly for females, and this fitness cost may explain maintenance of variations of walking ability in a population.

Decoupling of Behavioral Trait Correlation Across Life Stages in Two Holometabolous Insects.

Many animal behaviors have a genetic base, and behavioral traits often correlate with one another. In this study, we tested for a behavioral correlation between tonic immobility and walking distance in the larval and adult stages independently of two holometabolous insects. We confirmed a negative correlation of traits between strains in adults of both the species; however, we did not find it in larvae of either species. This suggests that the negative correlation between tonic immobility and walking is decoupled across life stages from larva to adult. In contrast, previous studies have reported that phenotypic correlations between behavioral traits are maintained from larvae to adults in hemimetabolous insects. In addition, our present results differ from previous results with holometabolous insects. Therefore, our results suggest that metamorphosis can change trade-offs between behavioral traits.

Transcriptomic comparison between populations selected for higher and lower mobility in the red flour beetle Tribolium castaneum.

Movement is an important behavior observed in a wide range of taxa. Previous studies have examined genes controlling movement using wing polymorphic insects and genes controlling wing size. However, few studies have investigated genes controlling movement activity rather than morphological traits. In the present study, we conducted RNA sequencing using populations with higher (WL) and lower (WS) mobility established by artificial selection in the red flour beetle Tribolium castaneum and compared gene expression levels between selected populations with two replicate lines. As a result, we found significant differences between the selected populations in 677 genes expressed in one replicate line and 1198 genes expressed in another replicate line, of which 311 genes were common to the two replicate lines. Furthermore, quantitative PCR focusing on 6 of these genes revealed that neuropeptide F receptor gene (NpF) was significantly more highly expressed in the WL population than in the WS population, which was common to the two replicate lines. We discuss differences in genes controlling movement between walking activity and wing polymorphism.

Intralocus sexual conflict and offspring sex ratio.

Males and females frequently have different fitness optima for shared traits, and as a result, genotypes that are high fitness as males are low fitness as females, and vice versa. When this occurs, biasing of offspring sex-ratio to reduce the production of the lower-fitness sex would be advantageous, so that for example, broods produced by high-fitness females should contain fewer sons. We tested for offspring sex-ratio biasing consistent with these predictions in broad-horned flour beetles. We found that in both wild-type beetles and populations subject to artificial selection for high- and low-fitness males, offspring sex ratios were biased in the predicted direction: low-fitness females produced an excess of sons, whereas high-fitness females produced an excess of daughters. Thus, these beetles are able to adaptively bias sex ratio and recoup indirect fitness benefits of mate choice.

Juvenile hormone mediates developmental integration between exaggerated traits and supportive traits in the horned flour beetle Gnatocerus cornutus.

Sexually selected exaggerated traits are often coupled with modifications in other nontarget traits. In insects with weapons, enlargements of nontarget characters that functionally support the weapon often occur (i.e. supportive traits). The support of sexual traits requires developmental coordination among functionally related multiple traits-an explicit example of morphological integration. The genetic theory predicts that developmental integration among different body modules, for which development is regulated via different sets of genes, is likely to be coordinated by pleiotropic factors. However, the developmental backgrounds of morphological integrations are largely unknown. We tested the hypothesis that the juvenile hormone (JH), as a pleiotropic factor, mediates the integration between exaggerated and supportive traits in an armed beetle Gnatocerus cornutus. During combat, males of this beetle use exaggerated mandibles to lift up their opponents with the supportive traits, that is, the head and prothoracic body parts. Application of methoprene, a JH analog (JHA), during the larval to prepupal period, induced the formation of large mandibles relative to the body sizes in males. Morphometric examination of nontarget traits elucidated an increase in the relative sizes of supportive traits, including the head and prothoracic body parts. In addition, reductions in the hind wing area and elytra length, which correspond to flight and reproductive abilities, were detected. Our findings are consistent with the genetic theory and support the idea that JH is a key pleiotropic factor that coordinates the developmental integration of exaggerated traits and supportive characters, as well as resource allocation trade-offs.

Freezing or death feigning? Beetles selected for long death feigning showed different tactics against different predators.

Prey evolve antipredator strategies against multiple enemies in nature. We examined how a prey species adopts different predation avoidance tactics against pursuit or sit-and-wait predators. As prey, we used three strains of Tribolium beetles artificially selected for short (short strain) or long (long strain) duration of death feigning, and a stock culture (base population). Death feigning is known to be effective for evading a jumping spider in the case of the long strains, while the present study showed that the long-strain beetles used freezing against a sit-and-wait type predator, Amphibolus venator, in this study. The short- strain beetles were more easily oriented toward predators. The time to predation was also shorter in the short strains compared to the long strains. The results showed that, as prey, the short strains displayed the same behavior, escaping, against both types of predators. Traditionally, death feigning has been thought to be the last resort in a series of antipredator avoidance behaviors. However, our results showed that freezing and death feigning were not parts of a series of behaviors, but independent strategies against different predators, at least for long-strain beetles. We also examined the relationship between a predator's starvation level and its predatory behavior. In addition, the orientation behavior toward and predation rate on the prey were observed to determine how often the predatory insect attacked the beetles.

Male aggressive behavior and exaggerated hindlegs of the bean bug Riptortus pedestris.

Males of the bean bug species Riptortus pedestris possess larger hindlegs than females. Observations of male-male interactions showed that the enlarged hindlegs are used as weapons in male fights, and that males with larger hindlegs win fights more frequently. Morphological analysis based on the positive allometry test showed that the femora of larger males are relatively bigger than those of smaller males, but femora of larger females are not relatively larger than those of smaller females. These results suggest that sexual selection in R. pedestris favors larger hindlegs for male fighting. In addition, the thorax and abdomen lengths were larger in the male than in the female. The males often lift their abdomen with their back to the opponent for displays against an opponent. As a result, abdominal size may be under stronger selection in the male than in the female, as for the exaggerated hindlegs.

An empirical test of the bet-hedging polyandry hypothesis: Female red flour beetles avoid extinction via multiple mating.

Bet-hedging via polyandry (spreading the extinction risk of the female's lineage over multiple males) may explain the evolution of female multiple mating, which is found in a wide range of animal and plant taxa. This hypothesis posits that females can increase their fitness via polyandrous mating when "unsuitable" males (i.e., males causing reproductive failure for various reasons) are frequent in the population and females cannot discriminate such unsuitable mates. Although recent theoretical studies have shown that polyandry can operate as a bet-hedging strategy, empirical tests are scarce. In the present study, we tested the bet-hedging polyandry hypothesis by using the red flour beetle Tribolium castaneum. We compared female reproductive success between monandry and polyandry treatments when females mated with males randomly collected from an experimental population, including 20% irradiated (infertile) males. In addition, we evaluated geometric mean fitness across multiple generations as the index of adaptability of bet-hedging traits. Polyandrous females showed a significantly higher egg hatching rate and higher geometric mean fitness than monandrous females. These results strongly support the bet-hedging polyandry hypothesis.

Amplitude of circadian rhythms becomes weaken in the north, but there is no cline in the period of rhythm in a beetle.

Many species show rhythmicity in activity, from the timing of flowering in plants to that of foraging behavior in animals. The free-running periods and amplitude (sometimes called strength or power) of circadian rhythms are often used as indicators of biological clocks. Many reports have shown that these traits are highly geographically variable, and interestingly, they often show latitudinal or longitudinal clines. In many cases, the higher the latitude is, the longer the free-running circadian period (i.e., period of rhythm) in insects and plants. However, reports of positive correlations between latitude or longitude and circadian rhythm traits, including free-running periods, the power of the rhythm and locomotor activity, are limited to certain taxonomic groups. Therefore, we collected a cosmopolitan stored-product pest species, the red flour beetle Tribolium castaneum, in various parts of Japan and examined its rhythm traits, including the power and period of the rhythm, which were calculated from locomotor activity. The analysis revealed that the power was significantly lower for beetles collected in northern areas than southern areas in Japan. However, it is worth noting that the period of circadian rhythm did not show any clines; specifically, it did not vary among the sampling sites, despite the very large sample size (n = 1585). We discuss why these cline trends were observed in T. castaneum.

Genomic characterization between strains selected for death-feigning duration for avoiding attack of a beetle.

Predator avoidance is an important behavior that affects the degree of adaptation of organisms. We compared the DNA variation of one of the predator-avoidance behaviors, the recently extensively studied "death-feigning behavior", between the long strain bred for feigning death for a long time and the short strain bred for feigning death for a short time. To clarify how the difference in DNA sequences between the long and short strains corresponds to the physiological characteristics of the death-feigning duration at the transcriptome level, we performed comprehensive and comparative analyses of gene variants in Tribolium castaneum strains using DNA-resequencing. The duration of death feigning involves many gene pathways, including caffeine metabolism, tyrosine metabolism, tryptophan metabolism, metabolism of xenobiotics by cytochrome P450, longevity regulating pathways, and circadian rhythm. Artificial selection based on the duration of death feigning results in the preservation of variants of genes in these pathways in the long strain. This study suggests that many metabolic pathways and related genes may be involved in the decision-making process of anti-predator animal behavior by forming a network in addition to the tyrosine metabolic system, including dopamine, revealed in previous studies.

Cross-species behavior analysis with attention-based domain-adversarial deep neural networks.

Since the variables inherent to various diseases cannot be controlled directly in humans, behavioral dysfunctions have been examined in model organisms, leading to better understanding their underlying mechanisms. However, because the spatial and temporal scales of animal locomotion vary widely among species, conventional statistical analyses cannot be used to discover knowledge from the locomotion data. We propose a procedure to automatically discover locomotion features shared among animal species by means of domain-adversarial deep neural networks. Our neural network is equipped with a function which explains the meaning of segments of locomotion where the cross-species features are hidden by incorporating an attention mechanism into the neural network, regarded as a black box. It enables us to formulate a human-interpretable rule about the cross-species locomotion feature and validate it using statistical tests. We demonstrate the versatility of this procedure by identifying locomotion features shared across different species with dopamine deficiency, namely humans, mice, and worms, despite their evolutionary differences.

Dispersal and ejaculatory strategies associated with exaggeration of weapon in an armed beetle.

Weapons used in male fighting can be costly to males and are often reported to trade off with other characters such as wings or spermatogenic investment. This study investigated whether increased investment into weapons can generate evolutionary changes in mating strategy for armed males. Male flour beetles, Gnatocerus cornutus, have enlarged mandibles that are used in male-male competition. We subjected these weapons to 12 generations of bidirectional selection and found trade-offs between weapons and two other male characters: wing and testis size. In addition, probably as a consequence of the observed changes in investment, dispersal ability and ejaculatory volume differ significantly between the lines. This indicates that the exaggeration of a weapon can be associated with dispersal and ejaculatory strategies. Thus, altered investment into weapons can lead to correlated changes in life-history traits.