Neuroregulation of foraging behavior mediated by the olfactory co-receptor Orco in termites.

Olfaction is critical for survival because it allows animals to look for food and detect pheromonal cues. Neuropeptides modulate olfaction and behaviors in insects. While how the neuroregulation of olfactory recognition affects foraging behavior in termites is still unclear. Here, we analyzed the change after silencing the olfactory co-receptor gene (Orco) and the neuropeptide Y gene (NPY), and then investigated the impact of olfactory recognition on foraging behavior in Odontotermes formosanus under different predation pressures. The knockdown of Orco resulted in the reduced Orco protein expression in antennae and the decreased EAG response to trail pheromones. In addition, NPY silencing led to the damaged ability of olfactory response through downregulating Orco expression. Both dsOrco- and dsNPY-injected worker termites showed significantly reduced walking activity and foraging success. Additionally, we found that 0.1 pg/cm trail pheromone and nestmate soldiers could provide social buffering to relieve the adverse effect of predator ants on foraging behavior in worker termites with the normal ability of olfactory recognition. Our orthogonal experiments further verified that Orco/NPY genes are essential in manipulating termite olfactory recognition during foraging under different predation pressures, suggesting that the neuroregulation of olfactory recognition plays a crucial role in regulating termite foraging behavior.

Circadian Rhythms of Locomotor Activity Mediated by Cryptochrome 2 and Period 1 Genes in the Termites Reticulitermes chinensis and Odontotermes formosanus.

Locomotor activity rhythms are crucial for foraging, mating and predator avoidance in insects. Although the circadian rhythms of activity have been studied in several termite species, the molecular mechanisms of circadian rhythms in termites are still unclear. In this study, we found that two termite species, R. chinensis and O. formosanus, exhibited clear circadian rhythms of locomotor activity in constant darkness along with rhythmically expressed core clock genes, Cry2 and Per1. The knockdown of Cry2 or Per1 expression in the two termite species disrupted the circadian rhythms of locomotor activity and markedly reduced locomotor activity in constant darkness, which demonstrates that Cry2 and Per1 can mediate the circadian rhythms of locomotor activity in termites in constant darkness. We suggest that locomotor activity in subterranean termites is controlled by the circadian clock.

The cGMP-dependent protein kinase gene can regulate trail-following behaviour and locomotion in the termite Reticulitermes chinensis Snyder.

Social behaviours in termites are closely related to the chemical communication between individuals. It is well known that foraging worker termites can use trail pheromones to orient and locomote along trails so as to take food resources back to the nest. However, it is still unclear how termites recognize trail pheromones. Here, we cloned and sequenced the cGMP-dependent protein kinase (PKG) gene from the termite Reticulitermes chinensis Snyder, and then examined the response of termites to trail pheromones after silencing PKG through RNA interference. We found that PKG knockdown impaired termite ability to follow trail pheromones accurately and exhibited irregular behavioural trajectories in response to the trail pheromone in the termite R. chinensis. Our locomotion assays further showed that PKG knockdown significantly increased the turn angle and angular velocity in the termite R. chinensis. These findings help us better understanding the molecular regulatory mechanism of foraging communications in termites.

In addition to cryptochrome 2, magnetic particles with olfactory co-receptor are important for magnetic orientation in termites.

The volatile trail pheromone is an ephemeral chemical cue, whereas the geomagnetic field (GMF) provides a stable positional reference. However, it is unclear whether and how the cryptic termites perceive the GMF for orientation in light or darkness until now. Here, we found that the two termite species, Reticulitermes chinensis and Odontotermes formosanus, use the GMF for orientation. Our silencing cryptochrome 2 (Cry2) impaired magnetic orientation in white light but had no significant impact in complete darkness, suggesting that Cry2 can mediate magnetic orientation in termites only under light. Coincidentally, the presence of magnetic particles enabled the magnetic orientation of termites in darkness. When knock-downing the olfactory co-receptor (Orco) to exclude the effect of trail pheromone, unexpectedly, we found that the Orco participated in termite magnetic orientation under both light and darkness. Our findings revealed a novel magnetoreception model depending on the joint action of radical pair, magnetic particle, and olfactory co-receptor.

Alteration of Termite Locomotion and Allogrooming in Response to Infection by Pathogenic Fungi.

Termites, being vulnerable to parasitic or pathogenic infections due to large number of individuals living together in colonies, have evolved various behavioral and physiological tactics to resist the infections by those pathogens. Locomotion can help termites collect information on parasites and accordingly exhibit hygienic behaviors. Termites inevitably encounter entomopathogenic fungi during nesting and foraging. However, how these fungal pathogens influence locomotion of termites and how hygienic behavior benefits their survival remains unknown. Here, we examined locomotion alteration of the termite Reticulitermes chinensis (Isoptera: Rhinotermitidae) after infections with different concentrations of the entomopathogenic fungus Metarhizium anisopliae (Hypocreales: Clavicipitaceae). When R. chinensis was isolated, the low concentration (5 × 103 conidia/ml) significantly increased termite locomotion after 6, 12, and 24 h compared with control. However, the high concentrations (5 × 107, 5 × 109 conidia/ml) significantly decreased termite locomotion after 48 h, and termite survival was also significantly lower at 5 × 107 and 5 × 109 conidia/ml compared with the low concentrations and the control. When R. chinensis was in group, however, the locomotion significantly increased 24 h after exposure to 5 × 103 and 5 × 109 conidia/ml but was normalized after 48 h of exposure compared with the control. Allogrooming was significantly higher at 5 × 103 and 5 × 109 conidia/ml compared with the control. The fungal infection did not result in significantly higher mortality of the group termites probably owing to their allogrooming. These findings enhance our understanding on how a termite species copes with biotic stress (i.e., fungal infections) via adaptive behaviors.

Silencing Orco Impaired the Ability to Perceive Trail Pheromones and Affected Locomotion Behavior in Two Termite Species.

Sophisticated social behaviors in termite colonies are mainly regulated via chemical communication of a wide range of pheromones. Trail pheromones play important roles in foraging behavior and building tunnels and nests in termites. However, it is almost unclear how termites perceive trail pheromones. Here, we cloned and sequenced of olfactory co-receptor (Orco) genes from the two termites Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae) and Odontotermes formosanus (Shiraki) (Isoptera: Termitidae), and then examined their responses to trail pheromones after silencing Orco through RNA interference (RNAi). We found that Orco knockdown impaired their ability to perceive trail pheromones and resulted in the disability of following pheromone trails in the two termite species. Our locomotion behavior assays further showed that Orco knockdown significantly decreased the distance and velocity in the two termite species, but significantly increased the angular velocity and turn angle in the termite R. chinensis. These findings strongly demonstrated that Orco is essential for termites to perceive their trail pheromones, which provides a potential way to control termite pests by damaging olfactory system.

Bioactivity of diatomaceous earth against the subterranean termite Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae).

The effects of diatomaceous earth (DE) on the penetrating behavior, tunneling behavior, mortality, and body surface characteristics of the subterranean termite Reticulitermes chinensis were investigated in this study. Our results show that the workers of R. chinensis were able to penetrate 1- and 2-mm layers of dry DE but not 3-mm layers. After treatment with dry DE for 6 h, the mortality of termites reached 100%, which was significantly higher than in the treatment with DE with a 10 and 25% moisture content and treatment with sand of three different moisture contents. The tunneling distances of workers in DE with 10, 25, and 50% moisture contents were all significantly shorter than those in sand with the same moisture contents (10, 25, and 50%), indicating that DE has a good suppressing effect on the tunneling behavior of workers. After treatment with dry DE for different times (1, 3, and 6 h), many DE particles adhered to the bodies of workers, whereas no particles adhered to the body of workers in the case of treatment with dry sand. The treatment with dry DE for 6 h resulted in the death of all workers, which presented conspicuous abdominal shrinkage, whereas workers treated with sand had no significant mortality and no obvious abdominal shrinkage. In summary, we suggest that dry DE has ideal insecticidal activity against the subterranean termite R. chinensis and can be further exploited for controlling termites inside houses.