α-Acaridial [2(E)-(4-methyl-3-pentenyl)butenedial] is a novel monoterpene secreted from the house dust mites. Because of its molecular nature of a highly reactive, small lipidic compound, we addressed whether α-acaridial might function as a haptenic allergen that induced allergic contact dermatitis. Mice sensitized with α-acaridial were challenged by the same antigen on the ear skin. After 2 days, significant ear swelling with a prominent infiltration of CD4+ T lymphocytes was observed. In vitro, α-acaridial exhibited an outstanding ability to quickly interact with and chemically modify a reference protein. Virtually all cysteine residues and a sizable fraction of lysine residues were found to be selectively modified, suggesting that α-acaridial could potentially interact with any proteins. Previously, numerous mite-derived proteinaceous allergens have been associated with contact dermatitis. Our study now emphasizes that small lipidic compounds released from mites comprise a new class of mite allergens, and therefore, is of significant medical implications.

In the preceding symposium, we presented the result of the phylogenetic analysis of Stigmaeopsis spider mites (Acari: Tetranychidae) in Japan using sequence data of the Cytochrome Oxidase subunit I (COI) region of mitochondria DNA. We analyzed haplotypes representing S. longus, S. celarius, S. takahashii and S. saharai, which inhabit bamboo species of the genera Sasa and Phyllostachys (Poaceae; Bambusoideae), and two forms of S. miscanthi (HG and LW) living on Miscanthus sinensis (Poaceae; Panicoideae). Both neighborjoining and maximum-likelihood methods revealed that Stigmaeopsis consists of seven monophyletic groups, in which two forms of S. miscanthi form two distinct monophyletic groups, whereas four species of bamboo-inhabiting species comprise a polyphyletic assembly. These results imply a mismatch between diagnostic morphological characters (i.e. the length of dorsal setae) and their phylogenetic lineages for bamboo-inhabiting species, requiring re-evaluation of the use of these characters. In addition, our results indicate that plural lineages coexist in a single habitat at various regions, suggesting that differentiated lineages secondarily encounter around southwestern Japan. Interestingly, the result of phylogenetic analysis on mitochondrial COI and nuclear ITS1 region was, in part, inconsistent in internal branches. In this symposium, I re-analyzed more these data by phylogenetic network analysis. Based on the results, I make some inference about the process of habitat expansion of these mites.

Blood feeding tightly regulates the reproductive cycles in ticks. Vitellogenesis and nutritional signaling are a key event in the tick reproductive cycle. Here we report the identification of a Haemaphysalis longicornis GATA factor, (HlGATA), which is synthesized after a blood meal and acts as a transcriptional activator of vitellogenin (Vg). HlGATA shares structural similarity with other GATA factors of invertebrates and vertebrates. Tick GATA mRNA accumulated in the fat body and midgut prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in engorged females. RNA interference (RNAi)-mediated knock down of GATA transcript resulted in a significant inhibition of Vg expression and effectively disrupts egg development after blood meal in engorged tick. In addition, HlGATA translation was inhibited by RNAi-mediated knock down of S6 kinase. These experiments have revealed that the GATA factor, which is the specific transcriptional activator of Vg gene, represents important molecule for tick reproduction.

Antimicrobial peptides represent an essential alternative first line of defense. Naturally occurring molecules associated with the innate immune system in disease-bearing vectors such as mosquito, tick could be the target for searching more potent and effective agents to combat against the pathogens resistant to conventionally used antibiotics. Recently, we explored expression of a defensinlike peptide, longicin from the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analog (P4) displayed antimicrobial/fungicidal/parasiticidal activity and, therefore, proposed to be a chemotherapeutic compounds against tick-borne disease organisms. Structural characterization of antimicrobial peptides is very important to understand the peptide activity. In addition, harmful side effects such as lysis of red blood cells or cytotoxicity towards mammalian host cells commonly associated with antimicrobial peptides as potential therapeutic agent should also be elucidated. In this study, we analyzed some structural features using bioinformatics tool, CD Spectroscopy, and also determined cytolytic activity of P4 peptide. According to the chemicophysical characteristics, the P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of β-sheet which was also observed in modelled tertiary structure. CD spectroscopy results also revealed the existence of a β-sheet and changes of helical content in the presence of membrane-mimic condition. These structural observations on P4 suggest that the antimicrobial activity could be due to the well developed β-sheet. In addition, sequence homology search showed that antimicrobial molecule identified in other ticks and in organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved antimicrobial domain. The peptide P4 also showed less cytolytic activity against various cell lines or erythrocytes of various species. The data presented here strongly suggests that the peptide P4 could be developed as future therapeutic agent against tick-borne microorganisms.

The life cycle of ticks is characterized by alternate off-host and on-host conditions. The life span is estimated at several years and most ixodid ticks spend more than 95% of their life off the host. They seem to have a unique strategy to endure the off-host state for a long period. By electron microscopy, isolation membrane-, autophagosome- and autolysosome-like structures were found in the midgut epithelial cells of unfed ticks. Therefore, we focused on autophagy which is well-conserved from yeast to higher eukaryotes and induced by starvation. We have identified homologues of autophagy-related (ATG) genes (ATG3, ATG4 and ATG8) from cDNA libraries of the 3-host tick Haemaphysalis longicornis. Each expression profile of H. longicornis ATG (HlATG) genes and HlAtg proteins at the stages of nymph and adult were examined by real-time PCR and immunoblotting. Moreover, autophagy is known to be induced by inactivation of target of rapamycin (TOR), a phosphatidylinositol kinase. To examine the effect of TOR function on the expression of HlAtg protein(s), rapamycin, a specific inhibitor of the signal transduction mediated by TOR, was injected to unfed adults. It was revealed that the expression of HlAtg protein(s) was enhanced in response to the rapamycin. This result indicates that tick have the nutrient-sensitive TOR signaling pathway which regulate autophagy.

Gamasida (Acari, Parasitiformes) is one the most diverse group in mites, and they inhabit in various environments, for example soil, tree canopy, tree trunk, leaves, animal, inter-tidal zone. About 10,000 species have been described in the world, half of them are predatory mites in soil and litter, and they prey on nematodes, small insects and mites. Although many predatory gamasid mites have been recorded from soil and litter in Japan, difference of diversity and difference of species composition of gamasid mites in different soil environments have not surveyed and discussed yet. In the present study, we surveyed soil gamasid mites in urban environment in Sapporo City, Hokkaido, Japan, and compared with gamasid mites in secondary forests (mixed forest, deciduous broad-leaved forest) in and near Sapporo. Our study focused on the following points: 1) difference of diversity in each environment; 2) characteristic taxa in each envrironment; and 3) availability of some taxa as bioindicator to evaluate environment. Number of species and index of diversity were higher in the fauna of secondary forests. Diversity of the families Parasitidae and Laelapidae were higher in urban environment fauna, while diversity of Parholaspidae and Veigaiidae were higher in the forest one. In the forest, mites of the family Zerconidae appeared exclusively, whereas one species of the family Parholaspidae and Parasitidae were dominant in urban mite fauna. Family Zerconidae and some species of Parholaspidae and Parasitidae may be available as bioindicator to evaluate soil environment.

Terrestrial animals are widely considered to be well protected from damage caused by ambient UV radiation, and thus the direct effects of solar UV-B radiation on arthropods have not attracted a great deal of attention. However, if plant-dwelling arthropods have evolved behavioral adaptations to solar UV-B radiation, but not morphological or physiological adaptations, the resources available to them would be strongly limited. Tetranychus urticae and their dominant natural enemies, phytoseiid mites, usually stay on the lower leaf surfaces. Because of the accumulation of UV-B-absorbing compounds such as flavonoids in the leaves, the upper and lower leaf surfaces provide different UV environments. We tested whether UV irradiation affects survival and reproduction of T. urticae and, if so, whether staying on the lower leaf surfaces is beneficial for their performance in solar UV radiation. Consequently, we observed lethal effects of artificial UV-B irradiation and solar UV radiation on T. urticae. However, by remaining on lower leaf surfaces, the mites could avoid such deleterious effects. These suggest that staying on the lower leaf surfaces is a behavioral adaptation of T. urticae to avoiding harmful solar UV-B radiation. Lethal effects of UV-B irradiation also exerted strong deterrent effects on the hatchability of phytoseiid mites. Additionally, preliminary experiments suggested that phytoseiid mites avoided and quickly escaped from a place irradiated by solar UV. Therefore, solar UV-B radiation may strongly affect the diurnal and spatial patterns of distribution or migration of mite communities on host plant canopies.

Population density of the citrus red mite, Panonychus citri (McGregor), in Japanese pear orchards remained low until mid-August, even after inoculation of pear leaves with a considerable number of adult female P. citri from May onwards. This raised the possibility that pear leaves contain a natural compound that suppresses an increase of P. citri populations. The rate of development from larva to adult was significantly lower on leaves collected in July than on leaves collected earlier or later, in several years. The population suppression was caused by molting inhibition and ovicidal activity, according to our close observation in the laboratory [Gotoh and Kubota (1997) Exp. Appl. Acarol. 21: 343-356]. To clarify whether a natural pear compound caused this molting inhibition, a methanol crude extract of pear leaves was isolated and added to a newly developed artificial diet, consisting of sodium caseinate, sucrose, levulose, glucose and inositol. The compound extracted from pear leaves resulted in the molting inhibition as observed on pear leaves. Based on infrared and NMR spectral analysis, the compound extracted from pear leaves closely resembled the synthetic acaricide hexythiazox. Furthermore, the LC50-values of the compound extracted from pear leaves for ovicidal activity of P. citri eggs and for inhibition of molting to protonymphs were similar to those of hexythiazox. These results strongly suggest that the molting deterrent extracted from pear leaves was in fact hexythiazox, an acaricide in use on pear trees, rather than a natural product. This suggestion becomes even stronger, considering that the molting inhibition was observed in a hexythiazox-spray year, but not in a non-spray year.