Evolution of horn length and lifting strength in the Japanese rhinoceros beetle Trypoxylus dichotomus.

What limits the size of nature's most extreme structures? For weapons like beetle horns, one possibility is a tradeoff associated with mechanical levers: as the output arm of the lever system-the beetle horn-gets longer, it also gets weaker. This "paradox of the weakening combatant" could offset reproductive advantages of additional increases in weapon size. However, in contemporary populations of most heavily weaponed species, males with the longest weapons also tend to be the strongest, presumably because selection drove the evolution of compensatory changes to these lever systems that ameliorated the force reductions of increased weapon size. Therefore, we test for biomechanical limits by reconstructing the stages of weapon evolution, exploring whether initial increases in weapon length first led to reductions in weapon force generation that were later ameliorated through the evolution of mechanisms of mechanical compensation. We describe phylogeographic relationships among populations of a rhinoceros beetle and show that the "pitchfork" shaped head horn likely increased in length independently in the northern and southern radiations of beetles. Both increases in horn length were associated with dramatic reductions to horn lifting strength-compelling evidence for the paradox of the weakening combatant-and these initial reductions to horn strength were later ameliorated in some populations through reductions to horn length or through increases in head height (the input arm for the horn lever system). Our results reveal an exciting geographic mosaic of weapon size, weapon force, and mechanical compensation, shedding light on larger questions pertaining to the evolution of extreme structures.

An Increase in Peroxiredoxin 6 Expression Induces Neurotoxic A1 Astrocytes in the Lumbar Spinal Cord of Amyotrophic Lateral Sclerosis Mice Model.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.

Fearless distasteful butterflies and timid mimetic butterflies: comparison of flight initiation distances in Papilioninae.

Prey are expected to flee from an approaching predator when the cost of remaining (i.e. being captured) exceeds the cost of fleeing. In species where individuals experience less predation pressure, delayed escape responses to predatory threats are predicted. Therefore, non-chalant behaviour should be observed in unpalatable and Batesian mimetic prey while rapid escape behaviour may be displayed in palatable and non-mimetic prey from closely related species. This study compared the flight initiation distances (FIDs; the distances at which a prey starts to flee during a standardized human approach) of several sympatric swallowtail butterflies (Papilionidae) in two areas with different temperate zones. As predicted, it was found that unpalatable species had significantly shorter FIDs than palatable species in both areas. By contrast, FIDs of Batesian mimic species were significantly longer than those of unpalatable model species and were not significantly different from those of palatable species. FIDs were not significantly different between mimetic and non-mimetic forms of the intraspecific polymorphic species Papilio polytes. The mimetic species or form may reduce the risk of revealing their identity (i.e. palatability) through early escape behaviour. These findings have implications for the selective forces that affect the escape tendency of prey.

Defense of bombardier beetles against avian predators.

Bombardier beetles (Coleoptera, Carabidae, Brachininae) possess a remarkable defense mechanism where a hot chemical spray is released from the tip of their abdomen, with an audible explosive sound. To date, the repellent properties of these chemicals have been tested against a limited number of taxa, such as amphibians and insects. To investigate the impact of bombardier beetle defenses on avian predators, feeding trials were conducted using the bombardier beetle (Pheropsophus jessoensis) and the Japanese quail (Coturnix japonica), a sympatric and generalist predator. All naïve, hand-reared quail attacked live beetles, indicating the absence of an innate aversion to them. However, most of the quail rejected consuming the beetles whether or not the beetles sprayed them with chemicals. Naïve quail also rejected dead P. jessoensis individuals. These results support the recent hypothesis that it is not essential for P. jessoensis to spray noxious chemicals to deter predators. We also found that some of the quail exposed to live P. jessoensis remembered to avoid them for up to 5 weeks. Our results provide the first evidence of the repelling effects of bombardier beetle defense mechanisms on avian predators.

Changes in the axo-glial junctions of the optic nerves of cuprizone-treated mice.

Demyelination induced by cuprizone in mice has served a useful model system for the study of demyelinating diseases, such as multiple sclerosis. Severity of demyelination by cuprizone, however, varies across different regions of the central nervous system; the corpus callosum is sensitive, while the optic nerves are resistant. Here, we investigated the effects of cuprizone on optic nerves, focusing on the axo-glial junctions. Immunostaining for sodium channels, contactin-associated protein, neurofascins, and potassium channels revealed that there were no massive changes in the density and morphology of the axo-glial junctions in cuprizone-treated optic nerves. However, when we counted the number of incomplete junctional complexes, we observed increased numbers of isolated paranodes. These isolated paranodes were immunopositive for both axonal and glial membrane proteins, indicating that they were the contact sites between axons and glia. These were not associated with sodium channels or potassium channels, suggesting the absence of physiological functions. When teased axons from cuprizone-treated optic nerves were immunostained, the isolated paranodes were found at the internode region of the myelin. From these observations, we conclude that cuprizone induces new contacts between axons and myelins at the internode region.

Attraction to Carbon Dioxide from Feeding Resources and Conspecific Neighbours in Larvae of the Rhinoceros Beetle Trypoxylus dichotomus.

Saprophagous (feeding on decaying matter) insects often use carbon dioxide (CO2) as a cue for finding food. Humus-feeding larvae of the giant rhinoceros beetle Trypoxylus dichotomus exhibit a clumped distribution in natural microhabitats, but the mechanisms driving the distribution were unknown. Herein, I examined whether larvae use CO2 as a cue for fermented humus and aggregate in the vicinity of the food. I found that (i) larvae of T. dichotomus are strongly attracted to CO2, (ii) larvae orient toward highly fermented humus when given a choice between highly and poorly fermented humus, (iii) the highly fermented humus emits more CO2 than the poorly fermented humus, and (iv) larvae grow larger when fed highly fermented humus rather than poorly fermented humus. The clumped distribution of larvae is probably formed along the concentration gradient of CO2 induced by heterogeneity of fermented organic materials in soil. My laboratory experiments also revealed that larvae are chemically attracted to each other. Moreover, CO2 concentrations in soil were increased by the larval respiration, and small amounts of CO2 (much less than emitted during respiration by a single larva) were sufficient for larval attraction. These results suggest that not only response to fermented food resources, but also respiratory CO2 from conspecifics may lead to aggregation. Enhanced densities resulted in reduced weight gain under experimental conditions. However, exploiting a high-value resource at enhanced densities still led to greater body weight compared to individually exploiting a low-value resource. This demonstrates the adaptive value of the response to CO2 sources in this species.

Chemically mediated group formation in soil-dwelling larvae and pupae of the beetle Trypoxylus dichotomus.

Many insects form groups through interactions among individuals, and these are often mediated by chemical, acoustic, or visual cues and signals. In spite of the diversity of soil-dwelling insects, their aggregation behaviour has not been examined as extensively as that of aboveground species. We investigated the aggregation mechanisms of larvae of the Japanese rhinoceros beetle Trypoxylus dichotomus, which live in groups in humus soil. In two-choice laboratory tests, 2nd- and 3rd-instar larvae gathered at conspecific larvae irrespective of the kinship. The ablation of maxillae, which bear chemosensilla, abolished aggregation behaviour. Intact larvae also exhibited aggregation behaviour towards a larval homogenate. These results suggest that larval aggregation is mediated by chemical cues. We also demonstrated that the mature larvae of T. dichotomus built their pupal cells close to a mesh bag containing a conspecific pupal cell, which indicated that larvae utilize chemical cues emanating from these cells to select the pupation site. Thus, the larvae of T. dichotomus may use chemical cues from the conspecifics in two different contexts, i.e. larval aggregation and pupation site selection. Using conspecific cues, larvae may be able to choose suitable locations for foraging or building pupal cells. The results of the present study highlight the importance of chemical information in belowground ecology.

Rhinoceros beetles suffer male-biased predation by mammalian and avian predators.

Male sexually-selected traits often impose an increased risk of predation on their bearers, causing male-biased predation. We investigated whether males of the sap-feeding Japanese rhinoceros beetle Trypoxylus dichotomus were more susceptible to predation than females by comparing the morphology of beetles caught in bait traps with the remains of beetles found on the ground. The males of this species are larger than the females and have a horn on the head. We found that predation pressure was greater for males than for females, and that larger individuals of both sexes were more vulnerable to predation. We identified two predators, the raccoon dog Nyctereutes procyonoides and jungle crow Corvus macrorhynchos, by monitoring sap-site trees with infrared video cameras. Raccoon dogs visited sap-site trees at night, while crows came after daybreak. The highest frequency of visits by both predators was observed in the first half of August, which matches the peak season of T. dichotomus. Raccoon dogs often left bite marks on the remains of prey, whereas crows did not. Bite marks were found on most of the remains collected at two distant localities, which suggested that predation by raccoon dogs is common. Size- and sex-dependent differences in the conspicuousness and active period of T. dichotomus probably explain these biased predation patterns. Our results suggest that having a large horn/body is costly in terms of the increased risk of predation. Predation cost may act as a stabilizing selection pressure against the further exaggeration of male sexual traits.

Pupal vibratory signals of a group-living beetle that deter larvae: Are they mimics of predator cues?

Pupae of some insects produce sounds or vibrations, but the function of the sounds/vibrations has not been clarified in most cases. Recently, we found vibratory communication between pupae and larvae of a group-living beetle Trypoxylus dichotoma, which live in humus soil. The vibratory signals produced by pupae were shown to deter approaching larvae, thereby protecting themselves. In the present study, we tested our hypothesis that pupal signals are mimics of vibratory noises associated with foraging of moles, the most common predators of T. dichotoma. Mole vibrations played back in laboratory experiments deterred larval approaches in the same way as pupal signals. These findings suggest that to deter conspecific larvae, pupae of T. dichotoma may have exploited a preexisting response of larvae to predator vibrations by emitting deceptive signals.

Deceptive vibratory communication: pupae of a beetle exploit the freeze response of larvae to protect themselves.

It is argued that animal signals may have evolved so as to manipulate the response of receivers in a way that increases the fitness of the signallers. In deceptive communication, receivers incur costs by responding to false signals. Recently, we reported that pupae of the soil-inhabiting Japanese rhinoceros beetle Trypoxylus dichotoma produce vibratory signals to deter burrowing larvae, thereby protecting themselves. In the present study, monitoring of vibrations associated with larval movement revealed that T. dichotoma larvae remained motionless for ca 10 min when pupal vibratory signals were played back transiently (freeze response). Furthermore, pupal signals of T. dichotoma elicited a freeze response in three other scarabaeid species, whose pupae do not produce vibratory signals. This indicates that the freeze response to certain types of vibration evolved before the divergence of these species and has been evolutionarily conserved, presumably because of the fitness advantage in avoiding predators. Pupae of T. dichotoma have probably exploited pre-existing anti-predator responses of conspecific larvae to protect themselves by emitting deceptive vibratory signals.

Physiological adaptation of the Asian corn borer Ostrinia furnacalis to chemical defenses of its host plant, maize.

A number of gramineous plants such as maize contain cyclic hydroxamic acids (cHx) that are toxic to many herbivores. Among the Ostrinia species found in Japan, the Asian corn borer Ostrinia furnacalis is the only one that utilizes maize, a gramineous plant. We used O. furnacalis and two congeners, Ostrinia scapulalis and Ostrinia latipennis, to obtain insights into the physiological adaptation of O. furnacalis to cHx. When an artificial diet containing a low concentration (0.3mg/g diet) of cHx was fed to the larvae of O. furnacalis and O. scapulalis, larval growth and survival were significantly less affected in O. furnacalis than O. scapulalis. An artificial diet containing a high level (0.7mg/g diet) of cHx was found to severely retard the growth of both species, albeit to different degrees. In an assay in vitro, homogenate of the digestive tract of O. furnacalis larvae degraded cHx more rapidly than that of O. scapulalis or O. latipennis. The degradation was found to be enzymatic and dependent on a cofactor, UDP-glucose, suggesting that UDP-glucosyltransferase or other UDP-glucose-dependent enzymes were involved. This enzymatic adaptation probably has enabled O. furnacalis to utilize plants containing cHx.