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.

Bacillus predominates in the Ophiocordyceps pseudolloydii-infected ants, and it potentially improves protection and utilization of the host cadavers.

The bacterial communities that colonize the cadaver environment of insects infected and killed by parasitic fungi can be selected by the sympatric fungi and provide novel impacts. In this study, we found that Bacillus cereus/thuringiensis predominate the bacterial community in Dolichoderus thoracicus ant cadavers colonized by O. pseudolloydii. The most predominant bacterial strains in these ant cadavers were hemolytic and able to produce hydrolytic enzymes for digesting the ant tissue. A relatively intense lethal effect on the co-cultured nematode was displayed by a hemolytic strain. Moreover, the antagonistic effect against pathogenic fungi detected in the bacteria sympatric with O. pseudolloydii was reported here. Naphthoquinones have been shown to confer antibacterial activities and produced by the ant-pathogenic Ophiocordyceps fungi. However, our results did not show the naphthoquinone tolerance we expected to be detected in the bacteria from the ant infected by O. pseudolloydii. The bacterial diversity in the samples associated with O. pseudolloydii infected ants as revealed in this study will be a step forward to the understanding of the roles playing by the microbial community in the native habitats of O. pseudolloydii.

Anthropogenic Influence on the Distribution of the Longlegged Ant (Hymenoptera: Formicidae).

The longlegged ant Anoplolepis gracilipes (Smith) is a highly invasive tramp ant species known for its deleterious effects on native ecosystems. While tramp ants are associated with human activity, information on how different intensities of human activity affect their distribution is limited. This study investigated how anthropogenic activities affected the distribution of A. gracilipes in Penang, a tropical island in northern peninsular Malaysia. Three study sites (Youth Park, Sungai Ara, and Bukit Jambul/Relau) were selected, containing four sub-locations corresponding to different levels of human activity (low, moderate, high, and very high), determined by the average number of passersby observed over 30 min. Baited index cards were placed at each sub-location to evaluate ant abundance and distribution. The results demonstrated that A. gracilipes worker abundance was highest in areas of moderate human activity, as opposed to areas with low and higher human activity. The low abundance of A. gracilipes in comparatively undisturbed localities may be attributed to unsuitable microclimate, lack of propagule pressure, and diminished honeydew availability. In contrast, its exclusion from more urbanized localities could be explained by high interspecific competition with other tramp species and the absence of preferred nesting sites.

Searching Behavior in the Tropical Fire Ant Solenopsis geminata (Hymenoptera: Formicidae).

Social insects have evolved different search strategies to find target objects in unknown environments. In the present study, the searching behavior of the tropical fire ant Solenopsis geminata was investigated in a circular arena. The average time, search path, speed, and search patterns of worker ants in a circular arena were determined. The results showed that fire ant workers followed six major search patterns. The variation in the searching patterns of workers may explain the different levels of exploration. Most workers (56.8%) tended to search in small loops and progressively increase the search area size. These workers mostly turned in one direction, either clockwise or counterclockwise. More workers turned in a consistent pattern than in an inconsistent pattern. Moving speed was also higher in workers that maintained their turning directions than in those that changed directions. We thus propose that following search patterns consisting of loops of increasing size may be an effective strategy. The tropical fire ant S. geminata is a globally invasive species that was introduced to Taiwan 40 years ago and has continued to threaten residents. Based on behavioral studies of S. geminata, we may gain a better understanding of their exploratory behavior in the ecosystem in Taiwan.

Molecular identification of the broodsacs from Leucochloridium passeri (Digenea: Leucochloridiidae) with a review of Leucochloridium species records in Taiwan.

Leucochloridium spp. have been established in Taiwan since the 1930s, with five species listed: Leucochloridium turdi, Leucochloridium passeri, Leucochloridium muscularae, Leucochloridium sime, and Leucochloridium taiwanese listed until now. Because of the similar morphology among the adults of the Leucochloridiidae family, the taxonomic status of most of them should be reconsidered. Broodsacs are the most distinct characteristic shared by Leucochloridium flukes, and L. passeri is, by far, the only one whose broodsacs have been described. In this study, broodsacs collected from an infected Succinea sp. (amber snails) in Taiwan were morphologically compared to previous descriptions and sequenced using both nuclear and mitochondrial genetic markers for identification. Our samples resembled broodsacs of L. passeri previously described in northern Taiwan and those recently collected in Okinawa, Japan. The conspecific status of the broodsacs collected in Taiwan and Okinawa was supported by the identical DNA sequences identified in this study. Broodsacs have rarely been reported in tropical and subtropical regions, although some Leucochloridium spp. have been recorded. In such cases, genetic markers will be crucial to link Leucochloridium broodsacs in snails (intermediate host) with their corresponding adult stage in birds (definitive host).

Exogenous Indole-3-Acetic Acid Induced Ethanol Tolerance in Phylogenetically Diverse Saccharomycetales Yeasts.

Indole-3-acetic acid (IAA) is an exogenous growth regulatory signal that is produced by plants and various microorganisms. Microorganisms have been suggested to cross-communicate with each other through IAA-mediated signaling mechanisms. The IAA-induced tolerance response has been reported in several microorganisms, but has not yet been described in Saccharomycetales yeasts. In the present study, three common stressors (heat, osmotic pressure, and ethanol) were examined in relation to the influence of a pretreatment with IAA on stress tolerance in 12 different lineages of Saccharomyces cerevisiae. The pretreatment with IAA had a significant effect on the induction of ethanol tolerance by reducing the doubling time of S. cerevisiae growth without the pretreatment. However, the pretreatment did not significantly affect the induction of thermo- or osmotolerance. The IAA pretreatment decreased the lethal effects of ethanol on S. cerevisiae cells. Although yeasts produce ethanol to outcompete sympatric microorganisms, IAA is not a byproduct of this process. Nevertheless, the accumulation of IAA indicates an increasing number of microorganisms, and, thus, greater competition for resources. Since the "wine trait" is shared by both phylogenetically related and distinct lineages in Saccharomycetales, we conclude that IAA-induced ethanol tolerance is not specific to S. cerevisiae; it may be widely detected in both pre-whole genome duplication (WGD) and post-WGD yeasts belonging to several genera of Saccharomycetales.

Possible impacts of the predominant Bacillus bacteria on the Ophiocordyceps unilateralis s. l. in its infected ant cadavers.

Animal hosts infected and killed by parasitoid fungi become nutrient-rich cadavers for saprophytes. Bacteria adapted to colonization of parasitoid fungi can be selected and can predominate in the cadavers, actions that consequently impact the fitness of the parasitoid fungi. In Taiwan, the zombie fungus, Ophiocordyceps unilateralis sensu lato (Clavicipitaceae: Hypocreales), was found to parasitize eight ant species, with preference for a principal host, Polyrhachis moesta. In this study, ant cadavers grew a fungal stroma that was predominated by Bacillus cereus/thuringiensis. The bacterial diversity in the principal ant host was found to be lower than the bacterial diversity in alternative hosts, a situation that might enhance the impact of B. cereus/thuringiensis on the sympatric fungus. The B. cereus/thuringiensis isolates from fungal stroma displayed higher resistance to a specific naphthoquinone (plumbagin) than sympatric bacteria from the environment. Naphthoquinones are known to be produced by O. unilateralis s. l., and hence the resistance displayed by B. cereus/thuringiensis isolates to these compounds suggests an advantage to B. cereus/thuringiensis to grow in the ant cadaver. Bacteria proliferating in the ant cadaver inevitably compete for resources with the fungus. However, the B. cereus/thuringiensis isolates displayed in vitro capabilities of hemolysis, production of hydrolytic enzymes, and antagonistic effects to co-cultured nematodes and entomopathogenic fungi. Thus, co-infection with B. cereus/thuringiensis offers potential benefits to the zombie fungus in killing the host under favorable conditions for reproduction, digesting the host tissue, and protecting the cadaver from being taken over by other consumers. With these potential benefits, the synergistic effect of B. cereus/thuringiensis on O. unilateralis infection is noteworthy given the competitive relationship of these two organisms sharing the same resource.

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.

The role of anthropogenic disturbance and invasion of yellow crazy ant in a recent decline of land crab population.

The yellow crazy ant, Anoplolepis gracilipes was first reported in Kenting National Park, Taiwan, in the 2000s, raising the concern of this invasive ant's potential effects on the native land crab populations. We investigated the distribution and abundance of A. gracilipes and its adverse effect on the land crab populations at four land crab hotspots within the park. Our survey results indicated that A. gracilipes was widespread and abundant at three of the study sites (Hsiangchiaowan, Shadao, and Natural Spring), while the distribution was much more confined at the fourth site (Houwan). Land crab populations experienced a notable decline at all the study sites except Houwan, indicating an inverse relationship between the population of land crabs and distribution/abundance of A. gracilipes. Combining the results of visual observations, the decline of land crab populations can be attributed, at least partially, to direct attacks by A. gracilipes on land crabs in their natural habitat and during the breeding migration season. Generalized additive model showed that worker abundance of this ant is associated with human disturbance levels, suggesting that anthropogenic disturbance may have contributed to the decline in land crab populations via promoting the abundance of A. gracilipes.

A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida).

Gordius chiashanus sp. nov., a newly described horsehair worm that parasitizes the Spirobolus millipede, is one of the three described horsehair worm species in Taiwan. It is morphologically similar to G. helveticus Schmidt-Rhaesa, 2010 because of the progressively broadening distribution of bristles concentrated on the male tail lobes, but it is distinguishable from G. helveticus because of the stout bristles on the mid-body. In addition, a vertical white stripe on the anterior ventral side and areoles on the inside wall of the cloacal opening are rarely mentioned in other Gordius species. Free-living adults emerged and mated on wet soil under the forest canopy in the winter (late November to early February) at medium altitudes (1100-1700 m). Mucus-like structure covering on the body surface, which creates a rainbow-like reflection, might endow the worm with high tolerance to dehydration. Although Gordius chiashanus sp. nov. seems to be more adaptive to the terrestrial environment than other horsehair worm species, cysts putatively identified as belonging to this hairworm species found in the aquatic paratenic host, Ephemera orientalis McLachlan, 1875, suggest the life cycle of Gordius chiashanus sp. nov. could involve water and land. The free-living adults emerged from the definitive hosts might reproduce in the terrestrial environment or enter an aquatic habitat by moving or being washed away by heavy rain instead of manipulating the behavior of their terrestrial definitive hosts.

Broad-Spectrum Activity of Volatile Organic Compounds from Three Yeast-like Fungi of the Galactomyces Genus Against Diverse Plant Pathogens.

The application of antagonistic fungi for plant protection has attracted considerable interest because they may potentially replace the use of chemical pesticides. Antipathogenic activities confirmed in volatile organic compounds (VOCs) from microorganisms have potential to serve as biocontrol agents against pre- and post-harvest diseases. In the present study, we investigated Galactomyces fungi isolated from rotten leaves and the rhizosphere of cherry tomato (Lycopersicon esculentum var. cerasiforme). VOCs produced by Galactomyces fungi negatively affected the growth of phytopathogenic fungi and the survival of nematodes. Mycelial growths of all nine examined phytopathogenic fungi were inhibited on agar plate, although the inhibition was more intense in Athelia rolfsii JYC2163 and Cladosporium cladosporioides JYC2144 and relatively moderate in Fusarium sp. JYC2145. VOCs also efficiently suppressed the spore germination and mycelial growth of A. rolfsii JYC2163 on tomatoes. The soil nematode Caenorhabditis elegans exhibited higher mortality in 24 h in the presence of VOCs. These results suggest the broad-spectrum activity of Galactomyces fungi against various plant pathogens and the potential to use VOCs from Galactomyces as biocontrol agents.

Carriers and cutters: size-dependent caste polyethism in the tropical fire ant (Solenopsis geminata).

Body size is an important life-history trait in eusocial insects which plays a key role in colony fitness. The division of labour, represented by caste polyethism, correlates with divergent morphological traits. Size polymorphism has been noted in the tropical fire ant, Solenopsis geminata; however, little is known regarding the differences in the size distributions of workers performing foraging tasks. In the present study, task partitioning was observed in the foraging activities of S. geminata. Two subgroups among foraging workers of S. geminata were discovered using the Gaussian mixture model: a large worker group (head width ≥ 0.924 mm) and a small worker group (head width < 0.924 mm). The foraging worker population comprised two distinct groups - 25.64% were large workers and 74.36% were small workers. Larger workers delivered heavier seeds faster than smaller workers, but this difference became less apparent when lighter seeds were being carried. When large prey such as crickets was encountered during foraging, S. geminata partitioned their tasks into cutting and transportation. The large workers were observed to cut cricket prey into fragments with their longer mandibles, and the small workers then transported these fragments back to the nest. These results present evidence of task partitioning among tropical fire ants, with different tasks being performed by ants of different castes.

The Association between Virus Prevalence and Intercolonial Aggression Levels in the Yellow Crazy Ant, Anoplolepis Gracilipes (Jerdon).

The recent discovery of multiple viruses in ants, along with the widespread infection of their hosts across geographic ranges, provides an excellent opportunity to test whether viral prevalence in the field is associated with the complexity of social interactions in the ant population. In this study, we examined whether the association exists between the field prevalence of a virus and the intercolonial aggression of its ant host, using the yellow crazy ant (Anoplolepis gracilipes) and its natural viral pathogen (TR44839 virus) as a model system. We delimitated the colony boundary and composition of A. gracilipes in a total of 12 study sites in Japan (Okinawa), Taiwan, and Malaysia (Penang), through intercolonial aggression assay. The spatial distribution and prevalence level of the virus was then mapped for each site. The virus occurred at a high prevalence in the surveyed colonies of Okinawa and Taiwan (100% infection rate across all sites), whereas virus prevalence was variable (30%-100%) or none (0%) at the sites in Penang. Coincidentally, colonies in Okinawa and Taiwan displayed a weak intercolonial boundary, as aggression between colonies is generally low or moderate. Contrastingly, sites in Penang were found to harbor a high proportion of mutually aggressive colonies, a pattern potentially indicative of complex colony composition. Our statistical analyses further confirmed the observed correlation, implying that intercolonial interactions likely contribute as one of the effective facilitators of/barriers to virus prevalence in the field population of this ant species.

Apoptosis as a primary defense mechanism in response to viral infection in invasive fire ant Solenopsis invicta.

Apoptosis is considered an innate defense mechanism of insect hosts at the early stage of pathogen infection. The present study attempts to determine whether apoptosis is involved in defending the fire ant, Solenopsis invicta from a natural viral pathogen Solenopsis invicta virus 1 (SINV-1). Results of TEM examination and TUNEL assay both revealed the signature of apoptosis in the midgut epithelium of SINV-1-infected fire ant larvae. A time-course study was conducted to monitor changes in the dynamics of SINV-1 viral titers and apoptosis levels in the midgut epithelium of SINV-1-infected larvae. We found that the viral titer significantly decreases as apoptosis level increases, suggesting that the apoptotic epithelium constitutes a barrier against dissemination of SINV-1. These findings serve as the very first empirical evidence for the virus-induced apoptosis in ants and also help explain some previously observed mortality patterns and behavioral alterations associated with SINV-1 in fire ants.

Viral infections in fire ants lead to reduced foraging activity and dietary changes.

Despite the presence of conserved innate immune function, many insects have evolved a variety of mechanical, chemical, and behavioral defensive responses to pathogens. Illness-induced anorexia and dietary changes are two behavioral defensive strategies found in some solitary insects, but little is known regarding the role of such behaviors in social insects, especially in ants. In the present study we examined if such reduced foraging activity exists for a social insect, the invasive fire ant Solenopsis invicta, and its viral pathogen, Solenopsis invicta virus 1 (SINV-1). Virus-free fire ant colonies were split into two colony fragments, one of which subsequently was inoculated with SINV-1. Four food resources with different macronutrient ratios were presented to both colony fragments. SINV-1-inoculated colony fragments consistently displayed reduced foraging performance (e.g., foraging intensity and recruitment efficiency), a decline in lipid intake, and a shift in dietary preference to carbohydrate-rich foods compared with virus-free fragments. These findings provide the first evidence for virus-induced behavioral responses and dietary shifts in shaping the host-pathogen interactions in fire ants. The findings also suggest a possible mechanism for how fire ant colonies respond to viral epidemics. Potential implications of these behavioral differences for current management strategies are discussed.

A new orthopteran-parasitizing horsehair worm, Acutogordius taiwanensis sp. n., with a redescription of Chordodes formosanus and novel host records from Taiwan (Nematomorpha, Gordiida).

A description of a new species of horsehair worm, Acutogordius taiwanensissp. n., a redescription of Chordodes formosanus, and novel host records for the latter are provided. Acutogordius taiwanensissp. n. is morphologically similar to A. protectus with moderately flat areoles on its tail tips, but is distinguishable by small mid-body ornamentations. Despite the distinct differences in the post-cloacal crescents between 14 male samples, their conspecific status, along with that of nine female samples, was upheld by a phylogenetic comparison of partial cytochrome oxidase subunit I (COI) sequences. Chordodes formosanus is another common horsehair worm species in Taiwan, which was previously believed to specifically parasitize Hierodula mantids. However, in this study, five C. formosanus were observed emerging from an Acromantis mantid, and two long-horned grasshopper hosts (Leptoteratura sp. and Holochlora japonica). These five worms showed high degrees of similarity in COI sequences and morphology, but one of these individuals bore abnormal crowned areoles, which has never been observed in C. formosanus, and may be attributed to the incomplete development of this particular individual.

Annual Survey of Horsehair Worm Cysts in Northern Taiwan, with Notes on a Single Seasonal Infection Peak in Chironomid Larvae (Diptera: Chironomidae).

The life cycle of the freshwater horsehair worm typically includes a free-living phase (adult, egg, larva) and a multiple-host parasitic phase (aquatic paratenic host, terrestrial definitive host). Such a life cycle involving water and land can improve energy flow in riparian ecosystems; however, its temporal dynamics in nature have rarely been investigated. This study examined seasonal infection with cysts in larval Chironominae (Diptera: Chironomidae) in northern Taiwan. In the larval chironomids, cysts of 3 horsehair worm species were identified. The cysts of the dominant species were morphologically similar to those of Chordodes formosanus. Infection with these cysts increased suddenly and peaked 2 mo after the reproductive season of the adult horsehair worms. Although adult C. formosanus emerged several times in a year, only 1 distinct infection peak was detected in September in the chironomid larvae. Compared with the subfamily Chironominae, samples from the subfamilies Tanypodinae and Orthocladiinae were less parasitized. This indicates that the feeding behavior of the chironomid host likely affects horsehair worm cyst infections; however, bioconcentration in predatory chironomids was not detected.

Morphological allometry and intersexuality in horsehair-worm-infected mantids, Hierodula formosana (Mantodea: Mantidae).

Parasitic castration is a strategy used by parasites to minimize damage to the host by consuming its reproductive system, which results in the morphological alteration of the host. We determined that the forewing shape and density of the antennal sensilla of field-collected adult male mantids (Hierodula formosana), infected by horsehair worms (Chordodes formosanus) was partially feminized (intersexuality), and both male and female mantids infected by horsehair worms exhibited allometric changes in their wings and walking legs. In addition, the testes of most infected male adults disappeared or reduced in size, whereas the number of ovarioles in infected female adults was unaffected. The infection mainly influenced the structures related to host reproduction and locomotion, suggesting unbalanced energy exploitation and the reduction of parasitic virulence. In addition, the intersexuality of infected male adults indicated that sexual differentiation in insects, which researchers have considered to be an autonomous process, was influenced by the infection. The similarity of the antennae of infected male adults with those of last-instar female nymphs suggested that parasitic juvenilization may cause such feminization, but the mechanism of parasitic influence on insect sex characteristics should be studied further.

A new horsehair worm, Chordodes formosanus sp. n. (Nematomorpha, Gordiida) from Hierodula mantids of Taiwan and Japan with redescription of a closely related species, Chordodes japonensis.

A new species of horsehair worm, Chordodes formosanussp. n., is described and compared to a closely related species, Chordodes japonensis. Although both species possess the same six cuticular structures of areoles on the surface, the significantly longer filaments on the female crowned areoles can be used as diagnostic characters for the new species. The different taxonomic status of these two species was also confirmed after analyzing the partial cytochrome oxidase subunit I sequence, and the mantid hosts, which are respectively limited to the genus Tenodera for Chordodes japonensis and Hierodula for Chordodes formosanussp. n. In addition, the immature stages of eggs and larvae of the new species are also described and discussed in detail.