Massive horizontal gene transfer and the evolution of nematomorph-driven behavioral manipulation of mantids.

To complete their life cycle, a wide range of parasites must manipulate the behavior of their hosts.1 This manipulation is a well-known example of the "extended phenotype,2" where genes in one organism have phenotypic effects on another organism. Recent studies have explored the parasite genes responsible for such manipulation of host behavior, including the potential molecular mechanisms.3,4 However, little is known about how parasites have acquired the genes involved in manipulating phylogenetically distinct hosts.4 In a fascinating example of the extended phenotype, nematomorph parasites have evolved the ability to induce their terrestrial insect hosts to enter bodies of water, where the parasite then reproduces. Here, we comprehensively analyzed nematomorphs and their mantid hosts, focusing on the transcriptomic changes associated with host manipulations and sequence similarity between host and parasite genes to test molecular mimicry. The nematomorph's transcriptome changed during host manipulation, whereas no distinct changes were found in mantids. We then discovered numerous possible host-derived genes in nematomorphs, and these genes were frequently up-regulated during host manipulation. Our findings suggest a possible general role of horizontal gene transfer (HGT) in the molecular mechanisms of host manipulation, as well as in the genome evolution of manipulative parasites. The evidence of HGT between multicellular eukaryotes remains scarce but is increasing and, therefore, elucidating its mechanisms will advance our understanding of the enduring influence of HGT on the evolution of the web of life.

Visual learning in tethered bees modifies flight orientation and is impaired by epinastine.

Visual-orientation learning of a tethered flying bee was investigated using a flight simulator and a novel protocol in which orientation preference toward trained visual targets was assessed in tests performed before and after appetitive conditioning. Either a blue or a green rectangle (conditioned stimulus, CS) was associated with 30% sucrose solution (unconditioned stimulus, US), whereas the other rectangle was not paired with US. Bees were tested in a closed-looped flight simulator 5 min after ten pairings of the US and CS. Conditioned bees were preferentially oriented to the CS after such training. This increase in preference for CS was maintained for 24 h, indicating the presence of long-term memory. Because the total orienting time was not altered by conditioning, conditioning did not enhance orientation activity itself but increased the relative time for orientation to CS. When 0.4 or 4 mM epinastine (an antagonist of octopamine receptors) was injected into the bee's head 30 min prior to the experiment, both short- and long-term memory formation were significantly impaired, suggesting that octopamine, which is crucial for appetitive olfactory learning in insects, is also involved in visual orientation learning.

Possible Role of Polarized Light Information in Spatial Recognition in the Cricket Gryllus bimaculatus.

Many insects are able to use skylight e-vector patterns to deduce their heading direction. Crickets have been well known to orient themselves to certain e-vector orientations to keep their walking direction. However, it is still unknown if crickets are able to utilize polarized light information for spatial recognition. Using an experimental paradigm similar to the Morris water maze for rodents, here we examine the possibility that the cricket Gryllus bimaculatus can utilize polarized light information to find the target place. Crickets were placed in a round arena with a heated floor, a portion of which was cooled, and a cross-shaped e-vector pattern was presented from the top of the arena so that the cricket could find the cool spot by walking along the e-vector direction. When the arrangement of the e-vector pattern and the cool spot were fixed throughout the experiments, the time and the walking distance to find the cool spot were significantly decreased with increasing trials, but not when the e-vector pattern was rotated between each trial. Moreover, a model selection indicated that the visual stimulus contributed to the decrease in time and distance. To investigate the cricket's exploration patterns in the arena, a test trial in which the whole floor was uniformly heated was performed before and after the training trials. In the test trial, the crickets trained with the positionally fixed e-vector pattern showed wall-following behavior for a significantly longer time than those untrained and those trained with random e-vector patterns.

Enhanced polarotaxis can explain water-entry behaviour of mantids infected with nematomorph parasites.

A wide range of parasites manipulate the behaviours of their hosts in order to complete their life cycle1. Alteration of phototaxis is thought to be involved in host manipulation in many cases2,3. However, very little is known about what features of the light (intensity, spectrum, polarization) alter behaviour. Here we report that arboreal mantids (Hierodula patellifera) infected by nematomorph parasites (Chordodes sp.) are attracted to horizontally polarized light, which could induce the mantids to enter water, where the parasites can then emerge and reproduce. In a two-choice test, infected mantids were attracted to horizontally but not vertically polarized light. Uninfected mantids were not attracted to either. In a field experiment, 14 infected mantids entered a deep pool, where the water surface strongly reflected horizontally polarized light. By contrast, only two mantids entered a shallow pool, where the surface reflection had higher light intensity but weaker polarization. To our knowledge, this is the first study demonstrating that a manipulative parasite can take advantage of its hosts' ability to perceive polarized light stimuli to alter host behaviour. VIDEO ABSTRACT.

Orientation to polarized light in tethered flying honeybees.

Many insects exploit the partial plane polarization of skylight for visual compass orientation and/or navigation. In the present study, using a tethering system, we investigated how flying bees respond to polarized light stimuli. The behavioral responses of honeybees (Apis mellifera) to a zenithal polarized light stimulus were observed using a tethered animal in a flight simulator. Flight direction of the bee was recorded by monitoring the horizontal movement of its abdomen, which was strongly anti-correlated with its torque. When the e-vector orientation of the polarized light was rotated clockwise or counterclockwise, the bee responded with periodic right-and-left abdominal movements; however, the bee did not show any clear periodic movement under the static e-vector or depolarized stimulus. The steering frequency of the bee was well coordinated with the e-vector rotation frequency of the stimulus, indicating that the flying bee oriented itself to a certain e-vector orientation, i.e. exhibited polarotaxis. The percentage of bees exhibiting clear polarotaxis was much smaller under the fast stimulus (3.6 deg s-1) compared with that under a slow stimulus (0.9 or 1.8 deg s-1). Bees did not demonstrate any polarotactic behavior after the dorsal rim area of the eyes, which mediates insect polarization vision in general, was bilaterally covered with black paint. Preferred e-vector orientations under the clockwise stimulus varied among individuals and distributed throughout -90 to 90 deg. Some bees showed similar preferred e-vector orientations for clockwise and counterclockwise stimuli whereas others did not. Our results strongly suggest that flying honeybees utilize the e-vector information from the skylight to deduce their heading orientation for navigation.

Neuroethology of the Waggle Dance: How Followers Interact with the Waggle Dancer and Detect Spatial Information.

Since the honeybee possesses eusociality, advanced learning, memory ability, and information sharing through the use of various pheromones and sophisticated symbol communication (i.e., the "waggle dance"), this remarkable social animal has been one of the model symbolic animals for biological studies, animal ecology, ethology, and neuroethology. Karl von Frisch discovered the meanings of the waggle dance and called the communication a "dance language." Subsequent to this discovery, it has been extensively studied how effectively recruits translate the code in the dance to reach the advertised destination and how the waggle dance information conflicts with the information based on their own foraging experience. The dance followers, mostly foragers, detect and interact with the waggle dancer, and are finally recruited to the food source. In this review, we summarize the current state of knowledge on the neural processing underlying this fascinating behavior.

Host manipulation by parasites as a cryptic driver of energy flow through food webs.

Manipulative parasites alter predator-prey interactions, and thus may facilitate, shift or create energy flow pathways through food webs (referred to hereafter as manipulation-mediated energy flow, MMEF). The ecological significance of MMEF would be determined not only by the strength of host manipulation, but also ecological and epidemiological factors, including host biomass, parasite incidence, and trophic position of the host-parasite association in their food webs. While previous theory has predicted that strong manipulation will destabilize host-parasite dynamics, a recently proposed theoretical framework claims that a switching strategy (sequential manipulation from predation suppression to enhancement) should allow parasites to induce strong predation enhancement and thus large MMEF. We formally outline the current and future directions to better understand the causes and consequences of MMEF across biological hierarchies.

Antennal RNA-sequencing analysis reveals evolutionary aspects of chemosensory proteins in the carpenter ant, Camponotus japonicus.

Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants.

Aggressive behavior in the antennectomized male cricket Gryllus bimaculatus.

Male crickets (Gryllus bimaculatus) exhibit intensively defensive aggressive behavior towards attacking males most often culminating in fighting. After the fight, the loser no longer exhibits aggressiveness in a second, separate encounter with another male; rather, the defeated male exhibits avoidance behavior. Here, we investigated the role of sensory input from the antennae in male defensive aggressive behavior. When we removed antennae from males (antennectomized males), we found that they showed little aggressiveness towards each other whereas they continued to exhibit typical fighting behavior towards an intact male. In addition, in a second encounter, antennectomized losers showed significantly higher aggressiveness towards another male than did intact losers. We further found that antennectomized crickets do not utilize visual or palpal sensory input to elicit aggressive behavior. In contrast, intact males showed aspects of aggressive behavior to male cuticular substances before and after winning a fight, and if they lost a fight they showed avoidance behavior. It thus appears that antennal sensory information is crucial in the mediation of aggressive and avoidance behaviors. However, sensory inputs from the antennae are not necessary to elicit defensive aggressive behavior but are necessary to discriminate conspecific males and initiate attacks against them.

Chemical discrimination and aggressiveness via cuticular hydrocarbons in a supercolony-forming ant, Formica yessensis.

BACKGROUND: Territorial boundaries between conspecific social insect colonies are maintained through nestmate recognition systems. However, in supercolony-forming ants, which have developed an extraordinary social organization style known as unicoloniality, a single supercolony extends across large geographic distance. The underlying mechanism is considered to involve less frequent occurrence of intraspecific aggressive behaviors, while maintaining interspecific competition. Thus, we examined whether the supercolony-forming species, Formica yessensis has a nestmate recognition system similar to that of the multicolonial species, Camponotus japonicus with respect to the cuticular hydrocarbon-sensitive sensillum (CHC sensillum), which responds only to non-nestmate CHCs. We further investigated whether the sensory system reflects on the apparent reduced aggression between non-nestmates typical to unicolonial species. METHODOLOGY/PRINCIPAL FINDINGS: F. yessensis constructs supercolonies comprising numerous nests and constitutes the largest supercolonies in Japan. We compared the within-colony or between-colonies' (1) similarity in CHC profiles, the nestmate recognition cues, (2) levels of the CHC sensillar response, (3) levels of aggression between workers, as correlated with geographic distances between nests, and (4) their genetic relatedness. Workers from nests within the supercolony revealed a greater similarity of CHC profiles compared to workers from colonies outside it. Total response of the active CHC sensilla stimulated with conspecific alien CHCs did not increase as much as in case of C. japonicus, suggesting that discrimination of conspecific workers at the peripheral system is limited. It was particularly limited among workers within a supercolony, but was fully expressed for allospecific workers. CONCLUSIONS/SIGNIFICANCE: We demonstrate that chemical discrimination between nestmates and non-nestmates in F. yessensis was not clear cut, probably because this species has only subtle intraspecific differences in the CHC pattern that typify within a supercolony. Such an incomplete chemical discrimination via the CHC sensilla is thus an important factor contributing to decreased occurrence of intraspecific aggressive behavior especially within a supercolony.

Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus.

Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.

Evidence for instantaneous e-vector detection in the honeybee using an associative learning paradigm.

Many insects use the polarization pattern of the sky for obtaining compass information during orientation or navigation. E-vector information is collected by a specialized area in the dorsal-most part of the compound eye, the dorsal rim area (DRA). We tested honeybees' capability of learning certain e-vector orientations by using a classical conditioning paradigm with the proboscis extension reflex. When one e-vector orientation (CS+) was associated with sugar water, while another orientation (CS-) was not rewarded, the honeybees could discriminate CS+ from CS-. Bees whose DRA was inactivated by painting did not learn CS+. When ultraviolet (UV) polarized light (350 nm) was used for CS, the bees discriminated CS+ from CS-, but no discrimination was observed in blue (442 nm) or green light (546 nm). Our data indicate that honeybees can learn and discriminate between different e-vector orientations, sensed by the UV receptors of the DRA, suggesting that bees can determine their flight direction from polarized UV skylight during foraging. Fixing the bees' heads during the experiments did not prevent learning, indicating that they use an 'instantaneous' algorithm of e-vector detection; that is, the bees do not need to actively scan the sky with their DRAs ('sequential' method) to determine e-vector orientation.

The compartment structures of the antennal lobe in the ant, Aphaenogaster smythiesi japonica.

Pheromones are important cues for social insects such as ants. As a first step in elucidation of pheromonal information processing mechanisms in the myrmicine ant, we investigated the morphological structure of the antennal lobe. Using autofluorescence imaging, labeling of neuronal filamentous actin, and reduced silver impregnation staining, the antennal lobe was found to consist of five compartments that, each received input from a different antennal sensory tract. Two major tracts of projection neurons, the medial and lateral antenno-cerebral tract (m- and 1-ACT), originated from a different region of the antennal lobe. The m-ACT originated from the posterior part of the antennal lobe whereas the 1-ACT originated from the anterior part. These results demonstrate a spatial segregation of function within the antennal lobe.

Polarized skylight navigation in insects: model and electrophysiology of e-vector coding by neurons in the central complex.

Many insects exploit skylight polarization for visual compass orientation or course control. As found in crickets, the peripheral visual system (optic lobe) contains three types of polarization-sensitive neurons (POL neurons), which are tuned to different ( approximately 60 degrees diverging) e-vector orientations. Thus each e-vector orientation elicits a specific combination of activities among the POL neurons coding any e-vector orientation by just three neural signals. In this study, we hypothesize that in the presumed orientation center of the brain (central complex) e-vector orientation is population-coded by a set of "compass neurons." Using computer modeling, we present a neural network model transforming the signal triplet provided by the POL neurons to compass neuron activities coding e-vector orientation by a population code. Using intracellular electrophysiology and cell marking, we present evidence that neurons with the response profile of the presumed compass neurons do indeed exist in the insect brain: each of these compass neuron-like (CNL) cells is activated by a specific e-vector orientation only and otherwise remains silent. Morphologically, CNL cells are tangential neurons extending from the lateral accessory lobe to the lower division of the central body. Surpassing the modeled compass neurons in performance, CNL cells are insensitive to the degree of polarization of the stimulus between 99% and at least down to 18% polarization and thus largely disregard variations of skylight polarization due to changing solar elevations or atmospheric conditions. This suggests that the polarization vision system includes a gain control circuit keeping the output activity at a constant level.

De Novo synthesis of CREB in a presynaptic neuron is required for synaptic enhancement involved in memory consolidation.

Interaction between the activator type of cyclic AMP response element binding protein (CREB1) and the repressor type (CREB2) results in determining the emergence of long-lasting synaptic enhancement involved in memory consolidation. However, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time polymerase chain reaction (PCR) to CREB in the cerebral giant cells of Lymnaea. The cerebral giant cells play an important role in associative learning and employ a CREB cascade for the synaptic enhancement to neurons such as the B1 motoneurons. We injected the small interfering RNA (siRNA) of CREB1 or CREB2 into the cerebral giant cells and examined the changes in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation.

Contextual olfactory learning in cockroaches.

We investigated the capability of context-dependent olfactory learning in cockroaches. One group of cockroaches received training to associate peppermint odor (conditioning stimulus) with sucrose solution (appetitive unconditioned stimulus) and vanilla odor with saline solution under illumination and to associate peppermint with aversive unconditioned stimulus and vanilla with appetitive unconditioned stimulus in the dark. Another group received training with the opposite stimulus arrangement. Before training, both groups exhibited preference for vanilla over peppermint. After training, the former group preferred peppermint over vanilla under illumination but preferred vanilla over peppermint in the dark, and the latter group exhibited the opposite odor preference. We conclude that cockroaches are capable of disambiguating the meaning of conditioning stimuli according to visual context.

Impairment of olfactory discrimination by blockade of nitric oxide activity in the terrestrial slug Limax valentianus.

The terrestrial slug Limax readily associates an innately preferred food odor with the aversive taste of quinidine. We investigated slugs' olfactory discrimination capability among structurally similar alcohols and the effects of inhibition of nitric oxide (NO) synthesis to the olfactory discrimination behavior, using an olfactory discriminatory learning task. Limax could discriminate among the odor of 1-octanol (OT), 3-methylcyclohexanol (MC) and 1-hexanol (HX). OT was perceptually more similar to HX than was MC for them. When NO synthesis was inhibited by injecting N-nitro-L-arginine methyl ester (l-NAME) shortly before the discrimination test, slugs could not discriminate between OT and HX whereas the retrieval of olfactory memory and the discrimination between OT and MC remained intact. These results indicate that the NO cascade plays a crucial role for fine olfactory discrimination in Limax.

Distribution of dendrites of descending neurons and its implications for the basic organization of the cockroach brain.

To determine precisely the brain areas from which descending neurons (DNs) originate, we examined the distribution of somata and dendrites of DNs in the cockroach brain by retrogradely filling their axons from the cervical connective. At least 235 pairs of somata of DNs were stained, and most of these were grouped into 22 clusters. Their dendrites were distributed in most brain areas, including lateral and medial protocerebral, which are major termination areas of output neurons of the mushroom body, but not in the optic and antennal lobes, the mushroom body, the central complex, or the posteroventral part of the lateral horn. The last area is the termination area of major types of olfactory projection neurons from the antennal lobe, i.e., uni- and macroglomerular projection neurons, so these neurons have no direct connections with DNs. The distribution of axon terminals of ascending neurons overlaps with that of DN dendrites. We propose, based on these findings, that there are numerous parallel processing streams from cephalic sensory areas to thoracic locomotory centers, many of which are via premotor brain areas from which DNs originate. In addition, outputs from the mushroom body, central complex, and posteroventral part of the lateral horn converge on some of the premotor areas, presumably to modulate the activity of some sensorimotor pathways. We propose, based on our results and documented findings, that many parallel processing streams function in various forms of reflexive and relatively stereotyped behaviors, whereas indirect pathways govern some forms of experience-dependent modification of behavior.

Diurnal and circadian rhythm in compound eye of cricket (Gryllus bimaculatus): changes in structure and photon capture efficiency.

Day-night changes in rhabdom size of compound eyes were investigated in three groups of crickets (Gryllus bimaculatus): nymphs and adult males and females. In both adults and nymphs, the rhabdoms were larger at night than during a day. In adults, the mean rhabdom occupation ratios (RORs) of ommatidial retinulae at midnight were about two times greater than the values at midday. This change contributes to control of the photon capture efficiency (PCE) of the eye according to photic environment. The RORs of adult males at midnight were higher than those of both adult females and nymphs. This suggests that the PCE of the compound eye of adult males is the greatest of all groups. Under constant darkness, day-night changes in ROR were detected only in adult males, but neither in adult females nor in nymphs. On the other hand, no day-night changes were detected in any experimental group under constant light. These results suggest that the change in rhabdom size between day and night is an adaptation to the photic environment that is controlled mainly by the light-dark (day-night) cycle. However, the change in male adults is induced by an endogenous circadian clock.

Distribution of dendrites of descending neurons and its implications for the basic organization of the cockroach brain.

To determine precisely the brain areas from which descending neurons (DNs) originate, we examined the distribution of somata and dendrites of DNs in the cockroach brain by retrogradely filling their axons from the cervical connective. At least 235 pairs of somata of DNs were stained, and most of these were grouped into 22 clusters. Their dendrites were distributed in most brain areas, including lateral and medial protocerebra, which are major termination areas of output neurons of the mushroom body, but not in the optic and antennal lobes, the mushroom body, the central complex, or the posteroventral part of the lateral horn. The last area is the termination area of major types of olfactory projection neurons from the antennal lobe, i.e., uni- and macroglomerular projection neurons, so these neurons have no direct connections with DNs. The distribution of axon terminals of ascending neurons overlaps with that of DN dendrites. We propose, based on these findings, that there are numerous parallel processing streams from cephalic sensory areas to thoracic locomotory centers, many of which are via premotor brain areas from which DNs originate. In addition, outputs from the mushroom body, central complex, and posteroventral part of the lateral horn converge on some of the premotor areas, presumably to modulate the activity of some sensorimotor pathways. We propose, based on our results and documented findings, that many parallel processing streams function in various forms of reflexive and relatively stereotyped behaviors, whereas indirect pathways govern some forms of experience-dependent modification of behavior.