Responses to relaxed and reverse selection in strains artificially selected for duration of death-feigning behavior in the red flour beetle, Tribolium castaneum

Divergent lines selected artificially for many generations make it possible to answer two questions: (1) whether genetic variation still exists within the selected population; and (2) whether the selection itself is costly for the selected strain. In previous studies, the red flour beetle Tribolium castaneum was divergently selected artificially for duration of death-feigning, and strains selected for longer (L-strain) and shorter (S-strain) durations of death-feigning have been established (Miyatake et al. 2004, 2008). Because the selection experiments have been conducted for more than 27 generations, genetic variation may be eroded. Furthermore, because another previous study reported physiological costs to L-strains, the L-strains selected artificially for longer duration of death-feigning may have suffered more costs than the S-strains. In the present study, therefore, we relaxed the selection pressure after the 27th or 30th generation of S- and L-strains. We also carried out reverse selection during the most recent eight generations of S- and L-strains. The results showed that each strain clearly responded to relaxation of selection and reverse selection, suggesting that (1) additive genetic variation still existed in both strains after long-term selection, and (2) selection for shorter and longer duration of death-feigning was costly. These results suggest that anti-predator behavior is controlled by many loci, and longer or shorter duration of death-feigning is costly in a laboratory without predators

Lines selected for different durations of tonic immobility have different leg lengths in the red flour beetle Tribolium castaneum

Tonic immobility is an adaptive anti-predator behaviour observed in many species. This anti-predator behaviour is often correlated with a species' movement motivation, so a relationship between the duration of tonic immobility and morphological traits supporting movement would be expected. Using the red flour beetle, Tribolium castaneum, we carried out two-way artificial selection for the duration of tonic immobility over more than 43 generations, establishing populations with longer (L) and shorter (S) tonic immobility durations compared to those of a non-selected control (C) population. Here, we investigated differences in walking motivation and leg length between the selected populations. Walking motivation was significantly higher in beetles from the S population than that in those from the L population. Moreover, S-population beetles of both sexes had significantly longer legs than those from L and C populations. The present results suggest the evolution of longer legs in response to selection pressure for a shorter duration of tonic immobility in T. castaneum

Aggregation pheromone interrupts death feigning in the red flour beetle Tribolium castaneum

Death feigning is a behavior in which a prey is rendered motionless due to stimulation or threat by a predator. This anti-predator defense mechanism has been observed across a wide range of animal taxa and is considered adaptive. However, long durations of death feigning can decrease opportunities for feeding and reproduction, and therefore is a fitness cost as compared to environments without predators. Because death feigning is thought to be affected by the balance between survival and other fitness costs, selection pressure may drive individuals who are capable of plastic changes in the intensity of death feigning. Pheromones, which are important semiochemicals that affect foraging and reproductive success, may be one of the factors influencing the intensity of death-feigning behavior. In this study, we investigated the effect of an aggregation pheromone on the death-feigning behavior of the red flour beetle Tribolium castaneum. We found that beetles exposed to the pheromone showed a significantly shorter duration of death feigning than beetles that were not exposed to the pheromone. Therefore, our results suggest that an aggregation pheromone can plasticly alter the death-feigning behavior in T. castaneum

Effects of temperature during successive generations on life-history traits in a seed beetle Callosobruchus chinensis (Chrysomelidae: Coleoptera)

Temperature is an important environmental factor for life-history traits in poikilothermic animals. Many of experiments on evolution have been conducted using Drosophila species, and effects on life-history traits vary depending on the study. On the other hand, few studies have been conducted on the effects of temperature on life-history traits in the other insect species. In the present study, we reared adzuki bean beetles under two different temperatures, high and low, for 2 years (20 generations), and compared life-history traits including body size of females, fecundity, egg size, rate of egg hatching, emergence rate, development time, and wing length. No differences in responses were found in these traits between selection strains, except the rate of egg hatching. That is, the rates of egg hatching in high-temperature (32 °C) selection strains were significantly higher than those in low-temperature (24 °C) selection strains. We discuss the cause of change in egg hatchability during successive generations under different temperature treatments from the following viewpoints including evolutionary adaptation to high temperature and the experimental protocol

Spatio-temporal distribution of adults and eggs of the West Indian sweetpotato weevil Euscepes postfasciatus (Coleoptera: Curculionidae) on sweet potato stems

The West Indian sweetpotato weevil, Euscepes postfasciatus, a serious pest of sweet potatoes, is being eradicated by sterile insect technique (SIT) in the south-western islands of Japan. Information on the diurnal movement of the target pests on host plants and where mating and egg-laying behavior occurs on the host is important for the application of SIT, which eradicates the target pest through mating of released sterile males and wild females. However, little such information is available on this species. In this study, male and female adults were released on host plants to examine the diurnal distribution on seedlings according to sex, as well as the sites where mounting behavior and egg laying occurs. The results showed that females left the host plant more frequently at night, whereas males were more likely to remain on the host plant at night. Both males and females stayed on the nodes of the host plant during the daytime. Mounting behavior also tended to occur more often at nodes. Furthermore, compared to unmated females, mated females stayed at the vertical top of the seedlings. However, it was found that eggs were often laid close to the roots rather than at the top of the vertical stems, even when the seedlings were placed upside down. The results of previous studies and this study will be discussed from the perspective of the application of SIT against E. postfasciatus

Effects of caffeine on mating behavior and sperm precedence in Tribolium castaneum

Biogenic amines such as dopamine are physiologically neuroactive substances that affect behavioral and physiological traits in invertebrates, and it has long been known that these substances affect mating behavior in insects. Caffeine is a dopamine activator and thus enhances dopamine receptor activity. However, the effects of caffeine intake on insect mating behavior have been largely unexplored. Therefore, we examined the effect of caffeine on mating behavior in the red flour beetle Tribolium castaneum. Caffeine, which activates dopamine, affected the mating behavior of T. castaneum males. Males who orally ingested caffeine courted faster than males who did not, resulting in faster mounting of females and less time to a male's external aedeagus protrusion. However, the present results showed no difference in sperm precedence measured as a P2 value between males fed caffeine and males not fed caffeine. We discuss the effects of caffeine on insect mating and the possibility that caffeine consumption may cause males to mate with more females in the laboratory

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

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

Artificial selection on walking distance suggests a mobility-sperm competitiveness trade-off

Securing matings is a key determinant of fitness, and in many species, males are the sex that engages in mate searching. Searching for mates is often associated with increased mobility. This elevated investment in movement is predicted to trade-off with sperm competitiveness, but few studies have directly tested whether this trade-off occurs. Here, we assessed whether artificial selection on mobility affected sperm competitiveness and mating behavior, and if increased mobility was due to increased leg length in red flour beetles (Tribolium castaneum). We found that, in general, males selected for decreased mobility copulated for longer, stimulated females more during mating, and tended to be better sperm competitors. Surprisingly, they also had longer legs. However, how well males performed in sperm competition depended on females. Males with reduced mobility always copulated for longer than males with high mobility, but this only translated into greater fertilization success in females from control populations and not the selection populations (i.e. treatment females). These results are consistent with a mate-searching/mating-duration trade-off and broadly support a trade-off between mobility and sperm competitiveness

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