Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 µm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

Pine wilt disease causes cavitation around the resin canals and irrecoverable xylem conduit dysfunction.

Physiological mechanisms of irreversible hydraulic dysfunction in seedlings infected with pine wilt disease (PWD) are still unclear. We employed cryo-scanning electron microscopy (cryo-SEM) to investigate the temporal and spatial changes in water distribution within the xylem of the main stem of 2-year-old Japanese black pine seedlings infested by pine wood nematodes (PWNs). Our experiment was specifically designed to compare the water relations among seedlings subjected to the following water treatment and PWN combinations: (i) well-watered versus prolonged drought (no PWNs); and (ii) well-watered with PWNs versus water-stressed with PWNs (four treatments in total). Cryo-SEM imaging observations chronicled the development of patchy cavitations in the xylem tracheids of the seedlings influenced by PWD. With the progression of drought, many pit membranes of bordered pits in the xylem of the main stem were aspirated with the decrease in water potential without xylem cavitation, indicating that hydraulic segmentation may exist between tracheids. This is the first study to demonstrate conclusively that explosive and irreversible cavitations occurred around the hydraulically vulnerable resin canals with the progression of PWD. Our findings provide a more comprehensive understanding of stressors on plant-water relations that may eventually better protect trees from PWD and assist with the breeding of trees more tolerant to PWD.

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.

Social context-dependent modification of courtship behaviour in Drosophila prolongata.

Induction of alternative mating tactics by surrounding conditions, such as the presence of conspecific males, is observed in many animal species. Satellite behaviour is a remarkable example in which parasitic males exploit the reproductive investment by other males. Despite the abundance of parasitic mating tactics, however, few examples are known in which males alter courtship behaviour as a counter tactic against parasitic rivals. The fruit fly Drosophila prolongata shows prominent sexual dimorphism in the forelegs. When courting females, males of D. prolongata perform 'leg vibration', in which a male vibrates the female's body with his enlarged forelegs. In this study, we found that leg vibration increased female receptivity, but it also raised a risk of interception of the female by rival males. Consequently, in the presence of rivals, males of D. prolongata shifted their courtship behaviour from leg vibration to 'rubbing', which was less vulnerable to interference by rival males. These results demonstrated that the males of D. prolongata adjust their courtship behaviour to circumvent the social context-dependent risk of leg vibration.

Moth hearing and sound communication.

Active echolocation enables bats to orient and hunt the night sky for insects. As a counter-measure against the severe predation pressure many nocturnal insects have evolved ears sensitive to ultrasonic bat calls. In moths bat-detection was the principal purpose of hearing, as evidenced by comparable hearing physiology with best sensitivity in the bat echolocation range, 20-60 kHz, across moths in spite of diverse ear morphology. Some eared moths subsequently developed sound-producing organs to warn/startle/jam attacking bats and/or to communicate intraspecifically with sound. Not only the sounds for interaction with bats, but also mating signals are within the frequency range where bats echolocate, indicating that sound communication developed after hearing by "sensory exploitation". Recent findings on moth sound communication reveal that close-range (~ a few cm) communication with low-intensity ultrasounds "whispered" by males during courtship is not uncommon, contrary to the general notion of moths predominantly being silent. Sexual sound communication in moths may apply to many eared moths, perhaps even a majority. The low intensities and high frequencies explain that this was overlooked, revealing a bias towards what humans can sense, when studying (acoustic) communication in animals.

Echolocation of insects using intermittent frequency-modulated sounds.

Using echolocation influenced by Doppler shift, bats can capture flying insects in real three-dimensional space. On the basis of this principle, a model that estimates object locations using frequency modulated (FM) sound was proposed. However, no investigation was conducted to verify whether the model can localize flying insects from their echoes. This study applied the model to estimate the range and direction of flying insects by extracting temporal changes from the time-frequency pattern and interaural range difference, respectively. The results obtained confirm that a living insect's position can be estimated using this model with echoes measured while emitting intermittent FM sounds.

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.

Substrate-borne vibrations induce behavioral responses in the leaf-dwelling cerambycid, Paraglenea fortunei.

Many insects utilize substrate-borne vibrations as a source of information for recognizing mates or predators. Among various substrates, plant leaves are commonly used for transmitting and receiving vibrational information. However, little is known about the utilization of vibrations by leaf-dwelling insects, especially coleopteran beetles. We conducted two experiments to examine the response of the leaf-dwelling cerambycid beetle, Paraglenea fortunei, to substrate-borne vibrations. We recorded and analyzed vibrations of host plant leaves from four different sources: wind (0.5 m/s), a beetle during landing, a walking beetle, and a beetle walking in the wind (0.5 m/s). We then measured the behavioral thresholds, the lowest amplitudes that induce behavioral responses, from beetles walking and resting on horizontal and vertical substrates with pulsed vibrations ranging from 20 Hz to 1 kHz. The vibrational characteristics of biotic and abiotic stimuli clearly differed. Beetle-generated vibrations (landing, walking, and walking in the wind) were broadly high in the low-frequency components above ∼30 Hz, while wind-generated vibrations showed a dominant peak at ∼30 Hz and a steep decrease thereafter. Among four situations, beetles walking on horizontal substrates showed lowest thresholds to vibrations of 75-500 Hz, which are characteristic of beetle-generated vibrations. Given that P. fortunei beetles are found on horizontal leaf surfaces of the host plant, vibrations transmitted though horizontal substrates may induce a strong freeze response in walking beetles to detect conspecifics or heterospecifics. Our findings provide evidence that leaf-dwelling beetles can discriminate among biotic and abiotic factors via differences in vibrational characteristics.

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.

Bridging biotremology and chemical ecology: a new terminology.

Living organisms use both chemical and mechanical stimuli to survive in their environment. Substrate-borne vibrations play a significant role in mediating behaviors in animals and inducing physiological responses in plants, leading to the emergence of the discipline of biotremology. Biotremology is experiencing rapid growth both in fundamental research and in applications like pest control, drawing attention from diverse audiences. As parallels with concepts and approaches in chemical ecology emerge, there is a pressing need for a shared standardized vocabulary in the area of overlap for mutual understanding. In this article, we propose an updated set of terms in biotremology rooted in chemical ecology, using the suffix '-done' derived from the classic Greek word 'δονέω' (pronounced 'doneo'), meaning 'to shake'.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Shape classification technology of pollinated tomato flowers for robotic implementation.

Three pollination methods are commonly used in the greenhouse cultivation of tomato. These are pollination using insects, artificial pollination (by manually vibrating flowers), and plant growth regulators. Insect pollination is the preferred natural technique. We propose a new pollination method, using flower classification technology with Artificial Intelligence (AI) administered by drones or robots. To pollinate tomato flowers, drones or robots must recognize and classify flowers that are ready to be pollinated. Therefore, we created an AI image classification system using a machine learning convolutional neural network (CNN). A challenge is to successfully classify flowers while the drone or robot is constantly moving. For example, when the plant is shaking due to wind or vibration caused by the drones or robots. The AI classifier was based on an image analysis algorithm for pollination flower shape. The experiment was performed in a tomato greenhouse and aimed for an accuracy rate of at least 70% for sufficient pollination. The most suitable flower shape was confirmed by the fruiting rate. Tomato fruit with the best shape were formed by this method. Although we targeted tomatoes, the AI image classification technology is adaptable for cultivating other species for a smart agricultural future.

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.

Electrophysiological and Morphological Characterization of Contact Chemosensilla in Adults and Larvae of the Butterfly, Atrophaneura alcinous.

Distribution and electrophysiological responses of contact chemosensilla were examined in the Aristolochiaceae-feeding butterfly Atrophaneuraalcinous. In adult butterflies, tarsal contact chemosensilla of the foreleg were classified into two groups based on length: long- and short-type sensilla. Long-type sensilla were distributed much more widely in females than in males, whereas short-type sensilla were found at the edge of the tarsi in a similar manner in both sexes. Taste responses of the long- and short-type sensilla to methanol extracts of Aristolochia debilis and Citrus spp. were recorded. Aristolochia debilis extracts evoked spikes with different amplitudes, whereas Citrus spp. extracts evoked spikes with a single amplitude in the long-type sensilla. Short-type sensilla did not respond to either extract. Moreover, we recorded responses to different concentrations of sucrose and NaCl. Results suggest that adult butterflies can discriminate the taste of host plant components from other chemicals using long-type sensilla during oviposition and may recognize diets containing sugar and salts during feeding using short-type sensilla. In the larval mouthparts, there were lateral and medial styloconic sensilla on the maxillary galea and epipharyngeal sensillum on the epipharynx. Electrophysiological responses of these sensilla suggest that larvae can discriminate between host plant compounds.

Moths produce extremely quiet ultrasonic courtship songs by rubbing specialized scales.

Insects have evolved a marked diversity of mechanisms to produce loud conspicuous sounds for efficient communication. However, the risk of eavesdropping by competitors and predators is high. Here, we describe a mechanism for producing extremely low-intensity ultrasonic songs (46 dB sound pressure level at 1 cm) adapted for private sexual communication in the Asian corn borer moth, Ostrinia furnacalis. During courtship, the male rubs specialized scales on the wing against those on the thorax to produce the songs, with the wing membrane underlying the scales possibly acting as a sound resonator. The male's song suppresses the escape behavior of the female, thereby increasing his mating success. Our discovery of extremely low-intensity ultrasonic communication may point to a whole undiscovered world of private communication, using "quiet" ultrasound.

To females of a noctuid moth, male courtship songs are nothing more than bat echolocation calls.

It has been proposed that intraspecific ultrasonic communication observed in some moths evolved, through sexual selection, subsequent to the development of ears sensitive to echolocation calls of insectivorous bats. Given this scenario, the receiver bias model of signal evolution argues that acoustic communication in moths should have evolved through the exploitation of receivers' sensory bias towards bat ultrasound. We tested this model using a noctuid moth Spodoptera litura, males of which were recently found to produce courtship ultrasound. We first investigated the mechanism of sound production in the male moth, and subsequently the role of the sound with reference to the female's ability to discriminate male courtship songs from bat calls. We found that males have sex-specific tymbals for ultrasound emission, and that the broadcast of either male songs or simulated bat calls equally increased the acceptance of muted males by the female. It was concluded that females of this moth do not distinguish between male songs and bat calls, supporting the idea that acoustic communication in this moth evolved through a sensory exploitation process.

Inhibitory substances contained in calcium carbonate wettable powder on the oviposition of the peach fruit moth, Carposina sasakii.

Spraying a calcium carbonate suspension "White Coat" on the fruit of apples significantly suppresses the oviposition of the peach fruit moth, Carposina sasakii. In gas chromatography (GC) with an electroantennographic detector analysis, adult female antennae showed responses to three compounds that were identified as 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) and its two mono-hydrolyzed analogs, texanols (1- and 3-isobutyrates), all added as a plasticizer to the agents. An oviposition-choice test using adult moths revealed that TXIB has clear deterrent properties when applied to young apple fruits. Video recording analysis showed that female moths spent longer on self-grooming and searching around TXIB-treated fruits. In the same assay, pure calcium carbonate treatment prevented the moths from climbing up or landing on the fruits, while such was not the case with White Coat-treated fruits. TXIB, an adjuvant aimed to provide rain/wind resistance, weakened the slipperiness of the calcium carbonate coating but, coincidentally, maintained the oviposition inhibitory activity of the White Coat by its deterrent odorant.

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.