Caenorhabditis elegans transfers across a gap under an electric field as dispersal behavior.

Interactions between different animal species are a critical determinant of each species' evolution and range expansion. Chemical, visual, and mechanical interactions have been abundantly reported, but the importance of electric interactions is not well understood. Here, we report the discovery that the nematode Caenorhabditis elegans transfers across electric fields to achieve phoretic attachment to insects. First, we found that dauer larvae of C. elegans nictating on a substrate in a Petri dish moved directly to the lid through the air due to the electrostatic force from the lid. To more systematically investigate the transfer behavior, we constructed an assay system with well-controlled electric fields: the worms flew up regardless of whether a positive or negative electric field was applied, suggesting that an induced charge within the worm is related to this transfer. The mean take-off speed is 0.86 m/s, and the worm flies up under an electric field exceeding 200 kV/m. This worm transfer occurs even when the worms form a nictation column composed of up to 100 worms; we term this behavior "multiworm transfer." These observations led us to conclude that C. elegans can transfer and attach to the bumblebee Bombus terrestris, which was charged by rubbing with flower pollen in the lab. The charge on the bumblebee was measured with a coulomb-meter to be 806 pC, which was within the range of bumblebee charges and of the same order of flying insect charges observed in nature, suggesting that electrical interactions occur among different species.

Nanoscale Mechanical Stimulation Method for Quantifying C. elegans Mechanosensory Behavior and Memory.

Withdrawal escape response of C. elegans to nonlocalized vibration is a useful behavioral paradigm to examine mechanisms underlying mechanosensory behavior and its memory-dependent change. However, there are very few methods for investigating the degree of vibration frequency, amplitude and duration needed to induce behavior and memory. Here, we establish a new system to quantify C. elegans mechanosensory behavior and memory using a piezoelectric sheet speaker. In the system, we can flexibly change the vibration properties at a nanoscale displacement level and quantify behavioral responses under each vibration property. This system is an economic setup and easily replicated in other laboratories. By using the system, we clearly detected withdrawal escape responses and confirmed habituation memory. This system will facilitate the understanding of physiological aspects of C. elegans mechanosensory behavior in the future.

Host orientation using volatiles in the phoretic nematode Caenorhabditis japonica.

Host orientation is the most important step in host-searching nematodes; however, information on direct cues from hosts to evoke this behaviour is limited. Caenorhabditis japonica establishes a species-specific phoresy with Parastrachia japonensis. Dauer larvae (DL), the non-feeding and phoretic stage of C. japonica, are predominantly found on female phoretic hosts, but the mechanisms underlying the establishment of this phoresy remain unknown. To determine whether C. japonica DL are able to recognize and orient themselves to a host using a volatile cue from the host, we developed a Y-tube olfactory assay system in which C. japonica DL were significantly attracted to the air from P. japonensis but not to the air from three other insects or to CO2. These results demonstrated that C. japonica DL utilize volatiles for host recognition and orientation and that the presence of a specific volatile kairomone released by the host attracts C. japonica DL.

Propagation of Caenorhabditis japonica in the nest of its carrier insect, Parastrachia japonensis.

We demonstrated the disembarkation of the bacterial-feeding nematode Caenorhabditis japonica dauer larvae (DL) from adult Parastrachia japonensis female insects and observed the propagation of nematodes in artificial insect nests. Our results clarify the process of propagation in this nematode species and provide insights into the nematode-insect relationship. Quiescent C. japonica DL resumed their mobility only at > 99.9% relative humidity (RH) at 25°C in the presence or absence of the carrier insect. In artificial nests with > 99.9% RH, DL resumed their mobility and the number of DL on female insects decreased gradually after oviposition, although numerous DL remained on the insects. Very few DL were detected on mother insects after hatching. Nematode propagation was observed on the egg mass after hatching and on nymphal carcasses; the total number of nematodes in the nest increased dramatically after this point. These results indicate that humidity is an important factor for disembarkation of C. japonica DL and that C. japonica propagates in the nest of P. japonensis where it feeds on the remains of eggs and nymph carcasses, indicating that C. japonica and P. japonensis have a unique phoretic and necromenic association.

Species-specific and female host-biased ectophoresy in the roundworm Caenorhabditis japonica.

Caenorhabditis japonica is a bacteriophagous nematode species that was discovered on the semi-social burrower bug, Parastrachia japonensis, which demonstrates egg-guarding and provisioning behaviors. To understand the life history of C. japonica in relation to P. japonensis, we demonstrated the specificity of this association and fluctuations in nematode number on the insect throughout the year. C. japonica dauer larvae (DL), larvae in a nonfeeding diapause stage, were predominantly found as clumps on the adult female insects but rarely found on the male insects in all populations examined. This female-biased association was consistent throughout the year, but after the nymphs hatched, nematodes were not detected on the mother insects showing provisioning behavior. DL appeared on the nymphs, and the number of DL on the newly emerged female insects gradually increased thereafter. C. japonica has never been detected on other invertebrates collected from the P. japonensis habitat thus far. Our data suggest that the life cycles of C. japonica and P. japonensis are synchronized.

Negative gravitactic behavior of Caenorhabditis japonica dauer larvae.

Gravity on Earth is a constant stimulus and many organisms are able to perceive and respond to it. However, there is no clear evidence that nematodes respond to gravity. In this study, we demonstrated negative gravitaxis in a nematode using dauer larvae (DL) of Caenorhabditis japonica, which form an association with their carrier insect Parastrachia japonensis. Caenorhabditis japonica DL demonstrating nictation, a typical host-finding behavior, had a negative gravitactic behavior, whereas non-nictating C. japonica and C. elegans DL did not. The negative gravitactic index of nictating DL collected from younger nematode cultures was higher than that from older cultures. After a 24 h incubation in M9 buffer, nictating DL did not alter their negative gravitactic behavior, but a longer incubation resulted in less pronounced negative gravitaxis. These results are indicative of negative gravitaxis in nictating C. japonica DL, which is maintained once initiated, seems to be affected by the age of DL and does not appear to be a simple passive mechanism.

Species-specific recognition of the carrier insect by dauer larvae of the nematode Caenorhabditis japonica.

Host recognition is crucial during the phoretic stage of nematodes because it facilitates their association with hosts. However, limited information is available on the direct cues used for host recognition and host specificity in nematodes. Caenorhabditis japonica forms an intimate association with the burrower bug Parastrachia japonensis. Caenorhabditis japonica dauer larvae (DL), the phoretic stage of the nematode, are mainly found on adult P. japonensis females but no other species. To understand the mechanisms of species-specific and female carrier-biased ectophoresy in C. japonica, we investigated whether C. japonica DL could recognize their hosts using nematode loading and chemoattraction experiments. During the loading experiments, up to 300 C. japonica DL embarked on male and female P. japonensis, whereas none or very few utilized the other shield bugs Erthesina fullo and Macroscytus japonensis or the terrestrial isopod Armadillidium vulgare. In the chemoattraction experiments, hexane extracts containing the body surface components of nymphs and both adult P. japonensis sexes attracted C. japonica DL, whereas those of other shield bugs did not. Parastrachia japonensis extracts also arrested the dispersal of C. japonica DL released at a site where hexane extracts were spotted on an agar plate; i.e. >50% of DL remained at the site even 60 min after nematode inoculation whereas M. japonensis extracts or hexane alone did not have the same effect. These results suggest that C. japonica DL recognize their host species using direct chemical attractants from their specific host to maintain their association.

Low survivorship of dauer larva in the nematode Caenorhabditis japonica, a potential comparative system for a model organism, C. elegans.

The nematode dauer larva (DL) is a non-aging diapause stage. The DL of the model nematode Caenorhabditis elegans has been studied as a model system for aging and longevity. However, information on DL in other nematode species is limited. In this study, the survivorship, storage, energy consumption, and oxidative stress tolerance of Caenorhabditis japonica DL were examined. C. japonica is a close relative of C. elegans, but has species-specific phoretic associations with the shield bug Parastrachia japonensis. Also, its DL has a much longer lifespan than C. elegans in a biological setting. However, when C. japonica DLs were detached from their phoretic host, they did not survive more than 10 days while more than 80% of C. elegans survived under the same conditions. Also, C. japonica DL showed more active movement (swimming) and lower tolerance to oxidative stress than C. elegans DL. Because the concentration of triacylglycerol (TAG), the energy source of nematodes, did not decrease significantly during the experiment, exhaustion of the energy reservoir did not cause the low survivorship of C. japonica. Instead, low tolerance to oxidizing stress and increased production of reactive oxygen species in C. japonica were the main causes of the reduced survivorship. The fact that C. japonica DL cannot survive away from its insect host indicates that its longevity is increased by unknown factors derived from the host. Despite these significant differences between C. japonica and C. elegans, these two species are phylogenetically closely related (they are derived from a common ancestor). Therefore, C. japonica could be a good comparative system for C. elegans, and further physiological and molecular analyses of C. japonica DL may provide important information about the internal and external factors affecting the longevity of nematodes in general.