Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis.

1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter-receiver ecological communication in mushrooms.

Outcome of interspecific competition depends on genotype of conspecific neighbours.

The outcome of interspecific competition is affected by numerous abiotic and biotic factors. However, the effects of genetic relatedness of conspecific neighbours have not been elucidated. Here, we demonstrate that the genotype of a conspecific neighbour determines the outcome of interspecific competition. We investigated effects of genotype of neighbour sibling or non-sibling Plantago asiatica plants on competition with Trifolium repens plants. Pairs of P. asiatica, sibling or non-sibling, were grown in competition with T. repens. Sibling P. asiatica plants, but not non-sibling plants, increased leaf placement towards competitor T. repens plants, and that placement was correlated negatively with the biomass of T. repens and positively with seed production by P. asiatica. Seed production by P. asiatica plants competing with T. repens was greater in individuals paired with siblings than in those paired with non-siblings. Thus, our results show that the genotype of conspecific neighbours can alter the outcome of interspecific competition.

Seeds integrate biological information about conspecific and allospecific neighbours.

Numerous organisms integrate information from multiple sources and express adaptive behaviours, but how they do so at different developmental stages remains to be identified. Seeds, which are the embryonic stage of plants, need to make decisions about the timing of emergence in response to environmental cues related to survival. We investigated the timing of emergence of Plantago asiatica (Plantaginaceae) seed while manipulating the presence of Trifolium repens seed and the relatedness of neighbouring P. asiatica seed. The relatedness of neighbouring P. asiatica seed and the presence of seeds of T. repens did not on their own influence the timing of P. asiatica emergence. However, when encountering a T. repens seed, a P. asiatica seed emerged faster in the presence of a sibling seed than in the presence of a non-sibling seed. Water extracts of seeds gave the same result. We show that P. asiatica seeds integrate information about the relatedness of neighbouring P. asiatica seeds and the presence of seeds of a different species via water-soluble chemicals and adjust their emergence behaviour in response. These findings suggest the presence of kin-dependent interspecific interactions.

Chordotonal organs in hemipteran insects: unique peripheral structures but conserved central organization revealed by comparative neuroanatomy.

Hemipteran insects use sophisticated vibrational communications by striking body appendages on the substrate or by oscillating the abdominal tymbal. There has been, however, little investigation of sensory channels for processing vibrational signals. Using sensory nerve stainings and low invasive confocal analyses, we demonstrate the comprehensive neuronal mapping of putative vibration-responsive chordotonal organs (COs) in stink bugs (Pentatomidae and Cydinidae) and cicadas (Cicadidae). The femoral CO (FCO) in stink bugs consists of ventral and dorsal scoloparia, homologous to distal and proximal scoloparia in locusts, which are implicated in joint movement detection and vibration detection, respectively. The ligament of the dorsal scoloparium is distally attached to the accessory extensor muscle, whereas that of the ventral scoloparium is attached to a specialized tendon. Their afferents project to the dorso-lateral neuropil and the central region of the medial ventral association center (mVAC) in the ipsilateral neuromere, where presumed dorsal scoloparium afferents and subgenual organ afferents are largely intermingled. In contrast, FCOs in cicadas have decreased dorsal scoloparium neurons and lack projections to the mVAC. The tymbal CO of stink bugs contains four sensory neurons that are distally attached to fat body cells via a ligament. Their axons project intersegmentally to the dorsal region of mVACs in all neuromeres. Together with comparisons of COs in different insect groups, the results suggest that hemipteran COs have undergone structural modification for achieving faster signaling of resonating peripheral tissues. The conserved projection patterns of COs suggest functional importance of the FCO and subgenual organ for vibrational communications.

Defensive fungal symbiosis on insect hindlegs.

Tympanal organs as "insect ears" have evolved repeatedly. Dinidorid stinkbugs were reported to possess a conspicuous tympanal organ on female's hindlegs. Here we report an unexpected discovery that the stinkbug's "tympanal organ" is actually a novel symbiotic organ. The stinkbug's "tympanum" is not membranous but a porous cuticle, where each pore connects to glandular secretory cells. In reproductive females, the hindleg organ is covered with fungal hyphae growing out of the pores. Upon oviposition, the females skillfully transfer the fungi from the organ to the eggs. The eggs are quickly covered with hyphae and physically protected against wasp parasitism. The fungi are mostly benign Cordycipitaceae entomopathogens and show considerable diversity among insect individuals and populations, indicating environmental acquisition of specific fungal associates. These results uncover a novel external fungal symbiosis in which host's elaborate morphological, physiological and behavioral specializations underpin the selective recruitment of benign entomopathogens for a defensive purpose.

Trophic eggs compensate for poor offspring feeding capacity in a subsocial burrower bug.

Various animals produce inviable eggs or egg-like structures called trophic eggs, which are presumed to be an extended maternal investment for the offspring. However, there is little knowledge about the ecological or physiological constraints associated with their evolutionary origin. Trophic eggs of the seminivorous subsocial burrower bug (Canthophorus niveimarginatus) have some unique characteristics. Trophic eggs are obligate for nymphal survival, and first-instar nymphs die without them. To identify the cause of nymphal death, we hypothesized that first-instar nymphs starve to death because they cannot feed on anything but trophic eggs. Although first-instar nymphs fed on artificially exposed endosperm did survive, nymphs that were provided with intact seed were not able to penetrate the seed vessel and starved to death. Another hypothesis that trophic eggs play a role in transferring the midgut symbiont, essential for survival in heteropteran bugs, from mother to offspring was rejected because almost all nymphs had retained the symbiont without feeding on trophic eggs. These results suggest that poor feeding capacity of the offspring is the cause of nymphal death, and the important constraint that promotes the evolution of the curious trophic egg system in C. niveimarginatus.

Locomotive Syndrome is a Risk Factor for the Dropout of Continuous Positive Airway Pressure Treatment in Patients with Obstructive Sleep Apnea Syndrome.

Objective: The purpose of this study was to investigate the risk factors linked to patient dropout from continuous positive airway pressure (CPAP) therapy for the treatment of obstructive sleep apnea syndrome (OSAS). Methods: This study included 1191 patients with OSAS at baseline assessment, who were followed for 3 years. We evaluated clinical parameters, indicators related to OSAS treatment, exercise habits and the presence of locomotive syndrome (LS). LS was evaluated by a 'loco-check', as established by the Japanese Orthopedic Association. The OSAS patients were categorized at baseline as belonging to an 'LS group' or a 'non-LS group', and clinical parameters were compared. Results: Eighty-six patients (7.2%) dropped out of CPAP therapy during the 3 year follow-up period. The dropout rate of the LS group was significantly higher than that of the non-LS group. Using a Cox-proportional hazard model, the LS, old age and poor compliance were determined to be significant risk factors for dropping out of CPAP therapy. The hazard ratios (95% CI) of LS, elderly people and poor CPAP compliance were 2.11 (1.31-3.48), 1.80 (1.11-2.94) and 1.61 (1.04-2.47), respectively. Conclusion: LS may be the critical risk factor for dropping out of CPAP therapy among patients with OSAS.

Egg-Cracking Vibration as a Cue for Stink Bug Siblings to Synchronize Hatching.

Egg clutches of many animals hatch synchronously due to parental control [1, 2] or environmental stimulation [3, 4]. In contrast, in some animals, embryos actively synchronize their hatching timing with their siblings to facilitate adaptive behavior in sibling groups, such as mass migration [5, 6]. These embryos require synchronization cues that are detectable from eggs and indicative of when the siblings hatch, such as pre-hatching vocalizations in birds and crocodiles [7, 8]. Previous studies, using methods including artificial presentation of non-specific mechanical stimuli, demonstrated that vibrations or other mechanical forces caused by sibling movements are cues used by some turtles and insects [9-13]. However, there is no evidence about which movements of tiny embryos or hatchlings, among multiple possibilities, can generate mechanical cues actually detectable through eggs. Here, we show that embryos of the brown marmorated stink bug, Halyomorpha halys, synchronize hatching by responding to single pulsed vibrations generated when siblings crack open their eggshells. An egg-cracking vibration seems to be transmitted to distant eggs within a clutch while still maintaining its function as a cue, thus leading to the highly synchronized hatching pattern previously reported [14]. In this species, it is possible that embryos attempt to hatch with short lags after earlier-hatched siblings to avoid egg cannibalism by them [14]. The present study illustrates the diversity of social-information use by animal embryos for success in the sibling group.

Maternal hatching synchronization in a subsocial burrower bug mitigates the risk of future sibling cannibalism.

Sibling cannibalism-the killing and consumption of conspecifics within broods-carries a high risk of direct and inclusive fitness loss for parents and offspring. We reported previously that a unique vibrational behavior shown by the mother of the subsocial burrower bug, Adomerus rotundus (Heteroptera: Cydnidae), induced synchronous hatching. Maternal regulation may be one of the most effective mechanisms for preventing or limiting sibling cannibalism. Here, we tested the hypothesis that synchronous hatching induced by maternal vibration in A. rotundus prevents sibling cannibalism. Mothers and their mature egg masses were allocated to three groups: synchronous hatching by maternal vibration (SHmv), synchronous hatching by artificial vibration (SHav), and asynchronous hatching (AH). We then investigated the influence of each hatching strategy on the occurrence of sibling cannibalism of eggs and early-instar nymphs in the laboratory. No difference in the proportion of eggs cannibalized was observed among the three groups. However, the proportion of nymphs cannibalized was higher in the AH group than in the SHmv group. The difference in the number of days to first molting within clutch was significantly higher in the AH group than in the SHmv group. Junior nymphs were sometimes eaten by senior nymphs. However, immediately after molting, senior nymphs were at a high risk of being eaten by junior nymphs. Our results indicate that synchronous hatching of A. rotundus is necessary to mitigate the risk of sibling cannibalism.

Adaptive Advantage of Myrmecochory in the Ant-Dispersed Herb Lamium amplexicaule (Lamiaceae): Predation Avoidance through the Deterrence of Post-Dispersal Seed Predators.

Seed dispersal by ants (myrmecochory) is found worldwide, but the benefits that plants obtain from this mutualism remain uncertain. In the present study, we conducted laboratory experiments to demonstrate seed predator avoidance as a benefit of myrmecochory using the annual ant-dispersed herb Lamium amplexicaule, the disperser ant Tetramorium tsushimae, and the seed predatory burrower bug Adomerus rotundus. We compared the predation intensity of Lamium amplexicaule seeds by Adomerus rotundus under the presence or absence of Tetramorium tsushimae. Both the number of seeds sucked by Adomerus rotundus adults and the feeding duration of sucked seeds by nymphs were significantly reduced in the presence of ants. This effect was most likely due to the behavioral alteration of Adomerus rotundus in response to the ant presence, because ants seldom predated Adomerus rotundus during the experiment. Our results demonstrated that the presence of ants decreases post-dispersal seed predation, even when the ants do not bury the seeds. The present study thus suggests that the non-consumptive effects of ants on seed predators benefit myrmecochorous plants.

Maternal vibration: an important cue for embryo hatching in a subsocial shield bug.

Hatching care has been reported for many taxonomic groups, from invertebrates to vertebrates. The sophisticated care that occurs around hatching time is expected to have an adaptive function supporting the feeble young. However, details of the characteristics of the adaptive function of hatching care remain unclear. This study investigated the hatching care of the subsocial shield bug, Parastrachia japonensis (Heteroptera: Parastrachiidae) to verify its function. Results show that the P. japonensis mothers vibrated the egg mass intermittently while maintaining an egg-guarding posture. Then embryos started to emerge from their shells synchronously. Unlike such behaviors of closely related species, this vibrating behavior was faint, but lasted more than 6 h. To investigate the effect of this behavior on hatching synchrony and hatching success, we observed the hatching pattern and the hatching rate in control, mother-removed, and two artificial vibration groups. Control broods experienced continuous guarding from the mother. Intermittent artificial vibration broods were exposed to vibrations that matched the temporal pattern of maternal vibration produced by a motor. They showed synchronous hatching patterns and high hatching rates. However, for mother-removed broods, which were isolated from the mother, and when we provided continuous artificial vibration that did not match the temporal pattern of the maternal vibration, embryo hatching was not only asynchronous: some embryos failed to emerge from their shells. These results lead us to infer that hatching care in P. japonensis has two functions: hatching regulation and hatching assistance. Nevertheless, several points of observational and circumstantial evidence clearly contraindicate hatching assistance. A reduction in the hatching rate might result from dependence on maternal hatching care as a strong cue in P. japonensis. We conclude that the hatching care of P. japonensis regulates the hatching pattern and serves as an important cue to induce embryo hatching.

Diverse strategies for vertical symbiont transmission among subsocial stinkbugs.

Sociality may affect symbiosis and vice versa. Many plant-sucking stinkbugs harbor mutualistic bacterial symbionts in the midgut. In the superfamily Pentatomoidea, adult females excrete symbiont-containing materials from the anus, which their offspring ingest orally and establish vertical symbiont transmission. In many stinkbug families whose members are mostly non-social, females excrete symbiont-containing materials onto/beside eggs upon oviposition. However, exceptional cases have been reported from two subsocial species representing the closely related families Cydnidae and Parastrachiidae, wherein females remain nearby eggs for maternal care after oviposition, and provide their offspring with symbiont-containing secretions at later stages, either just before or after hatching. These observations suggested that sociality of the host stinkbugs may be correlated with their symbiont transmission strategies. However, we found that cydnid stinkbugs of the genus Adomerus, which are associated with gammaproteobacterial gut symbionts and exhibit elaborate maternal care over their offspring, smear symbiont-containing secretions onto eggs upon oviposition as many non-social stinkbugs do. Surface sterilization of the eggs resulted in aposymbiotic insects of slower growth, smaller size and abnormal body coloration, indicating vertical symbiont transmission via egg surface contamination and presumable beneficial nature of the symbiosis. The Adomerus symbionts exhibited AT-biased nucleotide compositions, accelerated molecular evolutionary rates and reduced genome size, while these degenerative genomic traits were less severe than those in the symbiont of a subsocial parastrachiid. These results suggest that not only sociality but also other ecological and evolutionary aspects of the host stinkbugs, including the host-symbiont co-evolutionary history, may have substantially affected their symbiont transmission strategies.