Efficiency of ant-control agents in colony-level oral toxicity tests using Tetramorium tsushimae (Hymenoptera: Formicidae) for post-establishment control of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

The red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) causes serious damage worldwide as an invasive alien species. The species has expanded its range to the Pacific Rim since 2000s and Japan has faced its multiple introductions since 2017. While colony-level control methods are urgently needed, testing living colonies of the unestablished species is challenging especially due to various restrictions under the COVID-19 pandemic. Here, we proposed alternative long-term toxicity assays using artificial colonies of Tetramorium tsushimae Emery (Hymenoptera: Formicidae), a Japanese native species belonging to the same subfamily (Myrmicinae) as S. invicta. We conducted an acute toxicity test to determine if T. tsushimae is a suitable substitute for S. invicta using fipronil and found the LD50 value in T. tsushimae was close to that in S. invicta. Then, we conducted the long-term toxicity test with fipronil and two insect growth regulators (pyriproxyfen and etoxazole) using artificial colonies of T. tsushimae. All workers and larvae in the fipronil-treated colonies died within 3 days of treatment initiation. Emergence of new workers was observed after 18 days in the etoxazole-treated and control colonies, but not in the pyriproxyfen-treated colonies. We concluded that fipronil was the most promising insecticide for post-establishment control, and pyriproxyfen was effective as a toxic-bait agent for colony-level control. Supplementary Information: The online version contains supplementary material available at 10.1007/s13355-022-00800-x.

Unusual chromosome numbers and polyploidy in invasive fire ant populations.

Fire ants (Solenopsis invicta Buren in J Ga Entomol Soc 7:1-26, 1972), an invasive alien ant species, first spread from South America to the United States in the 1930s, the southern part of the United States by the end of the twentieth century, Oceania, Taiwan, and China in the twenty-first century, and finally to Japan and South Korea in 2017. As these ants have significant negative economic, human health, and environmental impacts, the purpose of this research was to accumulate cytogenetic information regarding fire ants and provide basic data for developing management strategies for their control. Fire ants were collected from invasive populations from Taiwan, Florida (USA), and Buenos Aires (Argentina), and a native population from Puerto Iguazu (Argentina), their point of origination, and analyzed with regard to chromosome number, morphology, and polyploidy, silver-stained nucleolar organizer regions (Ag-NORs), and 18S rDNA and telomere fluorescence in situ hybridization (FISH). The results showed that (1) fire ants from invaded populations differed in chromosome morphology compared to those from native populations; (2) the Florida and Taiwanese fire ant populations evinced greater variability in chromosome numbers and polyploidy variations; (3) the Taiwanese population exhibited significantly increased Ag-NOR signals in interphase cells, with signal number significantly positively correlating with distance from native populations; and (4) substantial diversity of signals was also apparent following 18S rDNA and telomere FISH analyses. Variation in these characteristics were hypothesized to be due to (1) the effect of hybridizations and interbreeding between closely related species or genetically distant populations, and (2) the potential effect of large amounts of insecticides sprayed for pest control.

Phylogeography of lethal male fighting in a social spider mite.

When males fight for access to females, such conflict rarely escalates into lethal fight because the risks and costs involved, that is, severe injury or death, are too high. The social spider mite, Stigmaeopsis miscanthi, does exhibit lethal male fights, and this male-male aggressiveness varies among populations. To understand the evolution of lethal fighting, we investigated aggressiveness in 42 populations and phylogenetic relationships in 47 populations along the Japanese archipelago. By analysis of the male weapon morph, a proxy for aggressiveness, we confirmed the existence of a mildly aggressive (ML) form, besides the low aggression (LW) and high aggression (HG) forms reported earlier. To evaluate demographic history of these three forms, we employed the approximate Bayesian computation approach using mtCOI sequences and taking into consideration the postlast glacial expansion history of the host plant, Miscanthus sinensis. As results, hierarchical split models are more likely to explain the observed genetic pattern than admixture models, and the ML form in the subtropical region was considered the ancestral group. The inferred demographic history was consistent with the one reconstructed for the host plant in a previous study. The LW form was split from the ML form during the last glacial period (20,000-40,000 years BP), and subsequently, the HG form was split from the ML form at the end of or after the last glacial period (5,494-10,988 years BP). The results also suggest that the mite invaded Japan more than once, resulting in the present parapatric distribution of LW and HG forms in eastern Japan.

Hatching synchrony is controlled by a two-step mechanism in the migratory locust Locusta migratoria (Acrididae: Orthoptera): Roles of vibrational stimuli.

The eggs of the migratory locust, Locust migratoria, hatch in synchrony from their pod. In this study, we examined the mechanism controlling hatching synchrony. Two eggs obtained from the same pod hatched in synchrony when kept in contact with one another, whereas those separated by a few millimeters hatched less synchronously. When a screen separated the eggs, the hatching was even more sporadic, indicating that hatching synchrony might be controlled by a two-step mechanism. We hypothesize that in the first step the embryos shortly before hatching control the time to enter a standby stage using some signal from neighboring eggs. The eggs in the standby stage hatch promptly when an additional stimulus is received from neighboring eggs. Before this stage, eggs cannot respond to that stimulus by hatching but may spontaneously hatch later. Introduction of a newly hatched nymph to single eggs 1 or 2 days before hatching advanced hatching of these eggs, but hatching occurred only sporadically. Eggs kept in contact with other eggs that had been killed by freezing shortly before hatching hatched as if they had been kept singly in separate containers, providing no evidence for involvement of chemical stimuli in controlling hatching synchrony. By contrast, eggs separated by several millimeters hatched as synchronously as those kept in contact with one another when they were connected by a piece of wire. Furthermore, vibrational stimulation derived from music greatly advanced hatching of separately kept eggs; however, hatching synchrony was not achieved unless the music started shortly before hatching. These results are consistent with the two-step hypothesis and indicated that locust embryos used vibrational stimuli from neighboring eggs for synchronous hatching.

Patterns of reproductive isolation in a haplodiploid - strong post-mating, prezygotic barriers among three forms of a social spider mite.

In speciation research, much attention is paid to the evolution of reproductive barriers, preventing diverging groups from hybridizing back into one gene pool. The prevalent view is that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection and genetic drift in groups that are segregated spatially and/or temporally. Reproductive barriers, however, can also be reinforced by natural selection against maladaptive hybridization. These mutually compatible theories are both empirically supported by studies, analysing relationships between intensity of reproductive isolation and genetic distance in sympatric taxa and allopatric taxa. Here, we present the - to our knowledge - first comparative study in a haplodiploid organism, the social spider mite Stigmaeopsis miscanthi, by measuring premating and post-mating, pre- and post-zygotic components of reproductive isolation, using three recently diverged forms of the mite that partly overlap in home range. We carried out cross-experiments and measured genetic distances (mitochondrial DNA and nuclear DNA) among parapatric and allopatric populations of the three forms. Our results show that the three forms are reproductively isolated, despite the absence of premating barriers, and that the post-mating, prezygotic component contributes most to reproductive isolation. As expected, the strength of post-mating reproductive barriers positively correlated with genetic distance. We did not find a clear pattern of prezygotic barriers evolving faster in parapatry than in allopatry, although one form did show a trend in line with the ecological and behavioural relationships between the forms. Our study advocates the versatility of haplodiploid animals for investigating the evolution of reproductive barriers.

Host-ant specificity of endangered large blue butterflies (Phengaris spp., Lepidoptera: Lycaenidae) in Japan.

Large blue butterflies, Phengaris (Maculinea), are an important focus of endangered-species conservation in Eurasia. Later-instar Phengaris caterpillars live in Myrmica ant nests and exploit the ant colony's resources, and they are specialized to specific host-ant species. For example, local extinction of P. arion in the U. K. is thought to have been due to the replacement of its host-ant species with a less-suitable congener, as a result of changes in habitat. In Japan, Myrmica kotokui hosts P. teleius and P. arionides caterpillars. We recently showed, however, that the morphological species M. kotokui actually comprises four genetic clades. Therefore, to determine to which group of ants the hosts of these two Japanese Phengaris species belong, we used mitochondrial COI-barcoding of M. kotokui specimens from colonies in the habitats of P. teleius and P. arionides to identify the ant clade actually parasitized by the caterpillars of each species. We found that these two butterfly species parasitize different ant clades within M. kotokui.

Enhanced Sensitivity for Electrochemical Detection Using Screen-Printed Diamond Electrodes via the Random Microelectrode Array Effect.

The electrochemical properties of screen-printed diamond electrodes with various insulating polyester (PES) resin binder/boron-doped diamond powder (BDDP) ratios were investigated for high sensitivity electrochemical detection. For PES/BDDP weight ratios in the range of 0.3-0.5, the BDDP-printed electrodes exhibited cyclic voltammetry (CV) characteristics for Fe(CN)6(3-/4-) that are typical of a planar electrode, whereas microelectrode-like characteristics with sigmoidal CV curves were observed for PES/BDDP ratios of 1.0-2.0. Cu elemental mapping images of copper-electrodeposited BDDP-printed electrodes indicated the formation of island structures with conductive BDDP domains surrounded by an insulating PES matrix for large PES/BDDP ratios. The electrochemical detection of ascorbic acid (AA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was also investigated using polycrystalline BDD thin-film and BDDP-printed electrodes (PES/BDDP ratio = 0.3 and 1.0). As a result, the signal-to-background (S/B) ratios for the voltammetric detection of AA and 8-OHdG were in the order BDDP-printed electrode (PES/BDDP = 1.0) > BDDP-printed electrode (PES/BDDP = 0.3) > polycrystalline BDD thin film electrode, based on the large faradaic current with respect to the background current. Therefore, the BDDP-printed electrode with a large insulating binder/BDDP ratio has the potential for use as a disposable electrode for electrochemical detection. The electrode is cheaper, lighter and more sensitive than conventional BDD electrodes.

Discovery of a bird-parasitic fly, Carnus orientalis (Diptera: Carnidae), in Japan, with bionomic remarks and a key to Carnus species.

A bird-parasitic fly, Carnus orientalis Maa, 1968, is recorded for the first time from Japan, and it is taxonomically reexamined on the basis of specimens collected in Okinawa Prefecture. Adult flies were found from nestlings of Ryukyu scops owl (Otus elegans Cassin, 1852), which is a new host for C. orientalis. Bionomic remarks regarding C. orientalis are presented, and akey to the world species of Carnus is also provided.

Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

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.

Combined use of a synthetic trail pheromone and insecticidal bait provides effective control of an invasive ant.

BACKGROUND: Except for sex pheromones, use of pheromones in pest management has been largely unexplored. A high concentration of trail pheromone disrupts ant trail following and foraging, and thus synthetic trail pheromone can be a novel control agent for pest ants. In this study, a year-long treatment of small areas (100 m(2) plots of urban house gardens) with synthetic trail pheromone, insecticidal bait or both was conducted to develop a novel control method of the invasive Argentine ant Linepithema humile (Mayr). RESULTS: The ant population could be maintained lower than or similar to the initial level only by combined treatment with synthetic trail pheromone and insecticidal bait. Actually, the ant population was nearly always lowest in combination treatment plots. Throughout the study period, the ant population in plots treated with either one of synthetic trail pheromone or insecticidal bait remained similar to no-treatment plots. CONCLUSIONS: Combination treatment may be a more effective and environmentally friendly method for controlling invasive ants than conventional methods. Extermination of ants by insecticidal bait and inhibition of re-infestation by pheromone may be the mechanism of the combination effect. This is the first study to show a significant effect of synthetic trail pheromone on ant population.

Workers select mates for queens: a possible mechanism of gene flow restriction between supercolonies of the invasive Argentine ant.

Some invasive ants form large networks of mutually non-aggressive nests, i.e., supercolonies. The Argentine ant Linepithema humile forms much larger supercolonies in introduced ranges than in its native range. In both cases, it has been shown that little gene flow occurs between supercolonies of this species, though the mechanism of gene flow restriction is unknown. In this species, queens do not undertake nuptial flight, and males have to travel to foreign nests and cope with workers before gaining access to alien queens. In this study, we hypothesized that male Argentine ants receive interference from workers of alien supercolonies. To test this hypothesis, we conducted behavioral and chemical experiments using ants from two supercolonies in Japan. Workers attacked males from alien supercolonies but not those from their own supercolonies. The level of aggression against alien males was similar to that against alien workers. The frequency of severe aggression against alien males increased as the number of recipient workers increased. Cuticular hydrocarbon profiles, which serve as cues for nestmate recognition, of workers and males from the same supercolony were very similar. Workers are likely to distinguish alien males from males of their own supercolony using the profiles. It is predicted that males are subject to considerable aggression from workers when they intrude into the nests of alien supercolonies. This may be a mechanism underlying the restricted gene flow between supercolonies of Argentine ants. The Argentine ant may possess a distinctive reproductive system, where workers participate in selecting mates for their queens. We argue that the aggression of workers against alien males is a novel form of reproductive interference.

Male killing and incomplete inheritance of a novel spiroplasma in the moth Ostrinia zaguliaevi.

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.

Expression of a doublesex homologue is altered in sexual mosaics of Ostrinia scapulalis moths infected with Wolbachia.

A homologue of the sex-determining gene doublesex, Osdsx, was identified in the adzuki bean borer Ostrinia scapulalis. Three isoforms of the Osdsx transcript (Osdsx(M), Osdsx(FL) and Osdsx(FS)) differing in length were found. Osdsx(M) was specifically found in males, and contained an 852-bp ORF encoding a protein of 284 amino acids. Osdsx(FL) and Osdsx(FS) were found in females, and had the same 813-bp ORF encoding a protein of 271 amino acids. The Osdsx gene was inferred to have six exons and five introns. The variation in the transcript could be explained by the alternative splicing of Osdsx: Osdsx(M) was formed by the splicing of exons 1, 2, 5 and 6, Osdsx(FS) by the splicing of exons 1-4 and 6, and Osdsx(FL) by the splicing of exons 1-6. RT-PCR analysis indicated that Osdsx was transcribed in a sex-specific manner in all somatic tissues examined, regardless of developmental stage. In Wolbachia-induced sexual mosaics of O. scapulalis, which are genetically male, the female-specific isoform of Osdsx (Osdsx(FL)) was shown to be expressed in addition to the male-specific isoform (Osdsx(M)). This finding provides the first evidence that Wolbachia manipulates the sex of its host by interfering either with the sex-specific splicing of Osdsx itself or with another upstream sex determination process.

Heat treatment of the Adzuki bean borer, Ostrinia scapulalis infected with wolbachia gives rise to sexually mosaic offspring.

A maternally inherited intracellular bacterium, Wolbachia, causes reproductive alterations in its arthropod hosts. In the adzuki bean borer, Ostrinia scapulalis (Walker) (Lepidoptera: Crambidae), naturally-occurring Wolbachia selectively kills male progeny. This Wolbachia strain appears to have a feminizing effect, since antibiotic treatment of infected female moths gives rise to male progeny with sexually mosaic phenotypes. It is proposed that male-specific death occurs through the feminizing effect, and sexual mosaics are produced when this effect is incompletely exerted. Here we examined whether the treatment of infected female moths with high temperatures (34 degrees C, 36 degrees C, or 38 degrees C), which is likely to suppress the activity of Wolbachia, induces sexually mosaic progeny. It was found that eggs laid within 24 h after treatment of Wolbachia-infected mothers at 36 degrees C gave rise to seven sexual mosaics along with 54 normal females. The time lag between treatment and the appearance of mosaic progeny was much shorter with heat treatment than antibiotic treatment, suggesting that heat treatment is more useful for spotting developmental timing when Wolbachia exerts its feminizing effect on O. scapulalis embryos.

Sex-specific death in the Asian corn borer moth (Ostrinia furnacalis) infected with Wolbachia occurs across larval development.

Maternally inherited endosymbiotic bacteria of the genus Wolbachia induce various kinds of reproductive alterations in their arthropod hosts. In a Wolbachia-infected strain of the adzuki bean borer moth, Ostrinia scapulalis (Lepidoptera: Crambidae), males selectively die during larval development, while females selectively die when Wolbachia are eliminated by antibiotic treatment. We found that naturally occurring Wolbachia in the congener O. furnacalis caused sex-specific lethality similar to that in O. scapulalis. Cytogenetic analyses throughout the entire larval development clarified that the death of males (when infected) and females (when cured) took place mainly during early larval stages. However, some individuals also died after complete formation of larval bodies but before egg hatching, or at late larval stages, even in the penultimate instar. Although the specific timing was highly variable, death of males and females occurred before pupation without exception. The potential association of sex-specific lethality with the sex determination mechanism was also examined and is discussed.

Transinfection reveals the crucial importance of Wolbachia genotypes in determining the type of reproductive alteration in the host.

Wolbachia , a group of endosymbiotic bacteria in arthropods, alter the reproduction of their hosts in various ways. A Wolbachia strain (wSca) naturally infecting the adzuki bean borer moth Ostrinia scapulalis induces male killing, while another strain (wKue) infecting the Mediterranean flour moth Ephestia kuehniella induces cytoplasmic incompatibility (CI) in the resident host. Transinfection of Wolbachia can be a powerful tool to elucidate the relative importance of Wolbachia and the host in determining the type of reproductive alterations. Recently, male killing was shown to occur in E. kuehniella transinfected with w Sca. In the present study, we transferred w Kue to O. scapulalis by embryonic microinjection. In the O. scapulalis transinfected with wKue, CI, but not male killing occurred. Thus, in addition to wSca, wKue was shown to induce the same type of alteration in a foreign host as in its natural host. These results demonstrate the crucial role of the Wolbachia genotype in determining the type of reproductive alteration. However, the present study also revealed the involvement of host factors. First, the degree of incompatibility induced by wKue in O. scapulalis was stronger than that in E. kuehniella , indicating that host factors can affect the level of CI. Second, the vertical transmission rate of wKue in O. scapulalis was generally low, suggesting that the host affects the dynamics of Wolbachia transmission.