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Acari are arthropods with the conserved features of an exoskeleton that require the shedding of cuticle for growth. Steroid hormones, ecdysteroids, regulate the mechanisms necessary for growth and development. Acari lack juvenile hormones, so they utilize ecdysteroids as the solo lipophilic hormone throughout their life for molting as well as for reproduction. Although some research on ecdysteroidogenesis has been conducted in the Acari, two important questions remain unclear: the active form of ecdysteroid and sites for ecdysteroidogenesis. The active form of ecdysteroids in Acari is controversial. Analysis by mass spectrometry confirms that most Acari use 20-hydroxyecdysone (20E), but the spider mite Tetranychus urticae (Koch) (Acari: Tetranychidae) uses Ponasterone A (25-deoxy-20-hydroxyecdysone). Analyses of ecdysteoridogenic genes provide clear evidence that the ovary is the primary site of ecdysteroidogenesis in both immature and mature ticks. In this review, the known functions of ecdysteroids are summarized and recent progress on ecdysteroidogenesis is introduced.

In tritrophic interactions between cucumber plants, the cucumber moth Diaphania indica Saunders (Lepidoptera: Crambidae) and a larval parasitoid Apanteles taragamae Viereck (Hymenoptera: Braconidae), female A. taragamae may use herbivore-induced plant volatiles (HIPVs) to locate their host. However, the specific compound or blend of chemicals attracting A. taragamae remains unknown. In this study, differences in volatiles released from uninfested, mechanically damaged and host-infested cucumber plants were examined by the headspace volatile collection method. Responses of the larval parasitoid A. taragamae to the volatile extracts were examined in a four-arm olfactometer. We also investigated the attraction of female A. taragamae to a single compound identified as an HIPV from host-infested cucumber plants. Parasitoids discriminated between the volatiles from uninfested, host-infested and mechanically damaged plants. Chemical analysis of headspace volatiles from host-infested cucumber plants showe...

Cotesia kariyai Watanabe (Hymenoptera: Braconidae) is a specialist larval parasitoid of Mythimna separata Walker (Lepidoptera: Noctuidae). Cotesia kariyai wasps use herbivore-induced plant volatiles (HIPVs) to locate hosts. However, complex natural habitats are full of volatiles released by both herbivorous host- and non-host-infested plants at various levels of intensity. Therefore, the presence of non-hosts may affect parasitoid decisions while foraging. Here, the host-finding efficiency of naive C. kariyai from HIPVs influenced by host- and non-host-infested maize [Zea mays L. (Poaceae)] plants was investigated with a four-arm olfactometer. Ostrinia furnacalis Guenee (Lepidoptera: Crambidae) was selected as a non-host species. One unit (1 U) of host- or non-host-infested plant was prepared by infesting a potted plant with five host or seven non-host larvae. In two-choice bioassays, host-infested plants fed upon by different numbers of larvae, and various units of host- and non-host-infested plants (infestation units; 1 U, 2 U, and 3 U) were arranged to examine the effects of differences in volatile quantity and quality on the olfactory responses of C. kariyai with the assumption that volatile quantity and quality changes with differences in numbers of insects and plants. Cotesia kariyai was found to perceive quantitative differences in volatiles from host-infested plants, preferring larger quantities of volatiles from larger numbers of larvae or plants. Also, the parasitoids discriminated between healthy plants, host-infested plants, and non-host-infested plants by recognising volatiles released from those plants. Cotesia kariyai showed a reduced preference for host-induced volatiles, when larger numbers of non-host-infested plants were present. Therefore, quantitative and qualitative differences in volatiles from host- and non-host-infested plants appear to affect the decision of C. kariyai during host-habitat searching in multiple tritrophic systems.

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC–MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders.

Retinoid X receptors (RXR) exist broadly from invertebrates to vertebrates, and play essential roles in physiological processes of these organisms. In arthropods, RXRs form a complex with the ecdysteroid receptor (EcR) and ecdysteroids to mediate the regulation of ecdysis and reproduction. Compared to EcR, RXR and its homologue ultraspiracle (USP) are much less well understood. Therefore, we identified RXR of the soft tick Ornithodoros moubata (OmRXR) and used real-time PCR to examine the expression of OmRXR. This is the first report of RXR from a soft tick. OmRXR showed higher homology to hard tick, crustacean and vertebrate RXRs than insect RXRs and USPs. OmRXR expression was observed during molting in the last instar nymphs coinciding with EcR expression and increases in ecdysteroid titers. Tick vitellogenesis normally occurs soon after engorgement and OmRXR expression coinciding with EcR expression and ecdysteroid titers in engorged females occurred before vitellogenin (Vg) synthesis and egg maturation. The ecdysteroid/EcR/RXR complex appears to be important in the regulation of molting and vitellogenesis of soft ticks.

Molecular mechanisms of ecdysteroid regulation in development and reproduction have been thoroughly investigated in Diptera and Lepidoptera, but few studies report the molecular actions of ecdysteroids in hemimetabolous insects and more primitive arthropods. Ecdysteroids appear to be the main hormones regulating development and vitellogenesis in ticks. An ecdysteroid receptor that showed high homology with EcRs of other arthropods was isolated from Ornithodoros moubata (OmEcRA). OmEcR expression patterns coincided with ecdysteroid titres in the haemolymph during moulting and vitellogenesis and differed between mated and virgin females. Therefore, OmEcR appears to mediate the regulation of moulting and vitellogenesis by ecdysteroids in O. moubata females as seen in other arthropods.

A blood meal is required for reproduction in most argasid female ticks. The blood meal appears to stimulate an organ in the posterior end to produce a fat body stimulating factor (FSF), which is thought to be an ecdysteroid, to induce vitellogenin (Vg) synthesis. In this study, the relationship of vitellogenesis and ecdysteroids was investigated by measuring Vg and ecdysteroid titers while observing oocyte development and oviposition in mated and virgin females. Oviposition occurred from day 10 after engorgement in mated females and continued up to 40-50 days, whereas egg maturation and oviposition did not occur in virgin females. Vg titers in the hemolymph peaked on day 6 after engorgement and subsequently declined in mated females. Interestingly, Vg synthesis occurred and ovarian development progressed to the development of early vitellogenic oocytes in virgin females but oocyte maturation and oviposition did not occur. Topical application of ecdysteroids induced oviposition in fed virgin females indicating that ecdysteroids may induce oviposition. Concentrations of ecdysteroids for 20 days after engorgement revealed several peaks in mated female whole body extracts, but no peaks in virgin female extracts. In the hemolymph of only mated females, ecdysteroid titers showed two peaks that followed the early peak of ecdysteroids in the whole body on day 4 and 6 after engorgement. In addition, ecdysteroids in the reproductive tissues increased with the development of the ovary in mated females and this increase coincided with the latter peaks of the whole body. These observations indicate that physiological elevation of ecdysteroids accelerate Vg synthesis, and may induce egg maturation and stimulate oviposition in fed mated Ornithodoros moubata females.

Antibacterial peptide defensin isoform A was previously isolated from the midgut contents of Ornithodoros moubata blood-fed females. However, not only defensin A, but also three other defensin isoforms showed gene expression in the midgut, suggesting the possibility that these antibacterial peptides are secreted into the midgut lumen. To further understand tick immune mechanisms, the involvement of antibacterial peptides in midgut defense was investigated. Three antibacterial peptides with molecular masses near defensin isoforms B, C and D were detected in the midgut contents of blood-fed females. Enzyme-linked immunosorbent assay analysis revealed that the antibacterial peptides in the midgut contents cross-reacted with defensin A antibodies and increased as a response to blood feeding. Simultaneously, the antibacterial activity of the midgut contents was enhanced by blood feeding. Secretion of antibacterial peptides into the midgut lumen and an increase in the peptide concentration following blood feeding was also confirmed. These findings further support the hypothesis that antibacterial peptides play an important role in the midgut defense of ticks.

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.

Ticks have an efficient defense system for preventing microbial infection. The antimicrobial peptide defensin is one effective molecule in this system. Here we investigated immune competence and the involvement of defensin in the humoral defense of the soft tick, Ornithodoros moubata. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that gene expression of all four defensin isoforms was up-regulated by bacteria or bacterial components. Defensin gene up-regulation by hemocoelic inoculation of bacteria involves the midgut and granulocytes. In immunodetection analysis, immunization by bacterial injection increases the relative concentration of defensin-like material in the hemolymph plasma. Furthermore, elevated antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria was observed after immunization by a liquid growth inhibition assay. Therefore, enhanced anti-Gram-positive bacterial activity appears to be partially dependent on the release of defensin into the hemolymph. These findings demonstrate that defensin plays an important role in the up-regulated humoral response of O. moubata.

Midgut contents of Ornithodoros moubata showed strong antibacterial activity against Staphylococcus aureus. A combination of reversed-phase chromatography and mass spectrometric analysis was used to isolate two antibacterial peptides from the tick midgut lumen. Partial amino acid sequences by Edman degradation of these two peptides showed they are identical with the 1–11 and 3–19 portions of rabbit α hemoglobin. Host rabbit α hemoglobin appears to be cleaved between Met32 and Phe33 to produce these two antibacterial peptides. Isolation of a host bovine hemoglobin fragment with antimicrobial activity has been demonstrated in the Ixodid tick, Boophilus microplus (Fogaca et al. 1999). Similar immune mechanisms in the two major families of ticks, Ixodidae and Argasidae, appear to use the hemoglobin of the host as an antimicrobial agent in midgut defense.

Ecdysteroidogenesis is essential for arthropod development and reproduction. Although the importance of ecdysteroids has been demonstrated, there is little information on the sites and enzymes for synthesis of ecdysteroids from Chelicerates. Ecdysteroid functions have been well studied in the soft tick Ornithodoros moubata, making this species an excellent candidate for elucidating ecdysteroidogenesis in Chelicerates. Results showed that O. moubata has at least two ecdysteroidogenic enzymes, Spook (OmSpo) and Shade (OmShd). RNAi showed both enzymes were required for ecdysteroidogenesis. Enzymatic assays demonstrated OmShd has the conserved functions of ecdysone 20-hydroxylase. OmSpo showed specific expression in the ovaries of final nymphal and adult stages, indicating O. moubata utilizes the ovary as an ecdysteroidogenic tissue instead of specific tissues as seen in other arthropods. On the other hand, OmShd expression was observed in various tissues including the midgut, indicating functional ecdysteroids can be produced in these tissues. In nymphal stages, expression of both OmSpo and OmShd peaked before molting corresponding with high ecdysteroid titers in the hemolymph. In fed adult females, OmSpo expression peaked at 8–10 days after engorgement, while OmShd expression peaked immediately after engorgement. Mated females showed more frequent surges of OmShd than virgin females. These results indicate that the regulation of synthesis of ecdysteroids differs in nymphs and adult females, and mating modifies adult female ecdysteroidogenesis. This is the first report to focus on synthesis of ecdysteroids in ticks and provides essential knowledge for understanding the evolution of ecdysteroidogenesis in arthropods.

Two defensin genes A and B were previously demonstrated to be up-regulated by blood feeding in the soft tick, Ornithodoros moubata [Nakajima et al. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31: 747-751]. In this study, two defensin isoforms C and D similar to defensins A and B were newly cloned. A total of four defensins have been identified in O. moubata. All four Ornithodoros defensins are coded as prepro-defensins. Ornithodoros defensin genes consist of four exons and three introns, an organization reported in mussel defensins but not insect defensins. Ornithodoros defensin C and D genes are predominantly expressed in the midgut and up-regulated in response to blood feeding. The mature peptide of the previously cloned Ornithodoros defensin A was purified from the midgut lumen, indicating defensin is secreted into the midgut. These findings confirm the involvement of Ornithodoros defensin in midgut immunity.

Animals are constantly threatened by pathogenic microorganisms and have developed cellular and humoral immune responses to combat these infections. Invertebrates possess only an innate non-specific immune response. Antimicrobial substances are major components of innate immunity not only in invertebrates but also in vertebrates. Despite the importance of ticks as vectors of disease very little is known about their immune system. Our recent studies have revealed that four defensin isoforms are present in Ornithodoros moubata. These four isoforms are constitutively expressed in the midgut and up-regulated in response to blood feeding. Moreover, a mature peptide of defensin isoform A has been isolated from the tick midgut lumen. These findings indicate Ornithodoros defensins are involved in tick midgut defense against potentially harmful invasive microbes.

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.

European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee, Apis cerana japonica F. (Hymenoptera: Apidae) , were characterized and evaluated for their potential to inhibit the growth of Melissococcus plutonius corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae) , the causative agent of European foulbrood. Analysis of 16S rRNA gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to Bacillus, Staphylococcus, and Pantoea. The isolates were screened against the pathogenic bacterium M. plutonius by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus Bacillus exhibited inhibitory activity against M. plutonius. In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with M plutonius, suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.

Effects of juvenile hormones (JH) and JH analogs on the release of vitellogenin (Vg) into the hemolymph and ovarian development in unfed adult female ticks, Ornithodoros moubata (Murray), were investigated. Topical application of acetone solvent and injection of acetone or oils showed some increase in Vg titer in the hemolymph. Topical application of JH (JH I, JH II, JH III) and JH analogs (methoprene, S21149, S21150, and S31183) dissolved in acetone to unengorged adult females elevated Vg titer in the hemolymph but only to the same level as the acetone controls. These effects were independent of dose. Injection of JH (JH I, JH II, JH III) and methoprene dissolved in mineral oil also did not significantly increase the Vg titer in the hemolymph compared with the controls (mineral oil injection). Electrophoretic analysis of hemolymph from females 5 d after treatment topically or by injection with JH and JH analogs showed faint Vg bands which comigrated with Vg's (Vg-1 and Vg-2) of normal, engorged female hemolymph, but Vg bands were detected more clearly in the hemolymph samples that were collected greater than 2 wk after treatment. However, the same level of Vg also was detected in the hemolymph of females treated with acetone or mineral oil. Vg synthesis in females treated with JH and JH analogs was analyzed by in vivo labeling and fluorography, which showed that Vg synthesis was not induced by application of JH to unengorged females.

Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30-60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.

A two-step HPLC purification procedure resulted in a factor from the stable fly that depolarizes the lumen-negative transepithelial voltage (Vt) of the adult stable fly Malpighian tubule. When applied to tubules of the female mosquito, Aedes aegypti, this factor partially mimics the electrophysiological actions of the mosquito natriuretic factor (MNF). It also selectively increases active transepithelial Na transport by the mosquito Malpighian tubule. The blood meal causes a transient increase in hemolymph Na and Cl contents and hemolymph volume during the course of the 24-h post-feeding period. The level of a factor that is immunologically cross-reactive with the human atrial natriuretic peptide (ANP) increases more than 6-fold within 6 h following a blood meal by the stable fly. The temporal pattern of the levels of the ANP-immunoreactive factor closely parallels the blood meal-induced rise and subsequent fall in hemolymph NaCl content and hemolymph volume, suggesting a functional correlation between the ANP-immunoreactive factor and the rate of NaCl and fluid loss from the hemolymph.

A cDNA encoding tick chitinase was cloned from a cDNA library of mRNA from Haemaphysalis longicornis eggs and designated as CHT1 cDNA. The CHT1 cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced amino acid sequence shows a 31% amino acid homology to Aedes aegypti chitinase and a multidomain structure containing one chitin binding peritrophin A domain and two glycosyl hydrolase family 18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass of 108 kDa with a pI of 5.0. A recombinant baculovirus AcMNPV.CHT1-expressed rCHT1 is glycosylated and able to degrade chitin. Chitin degradation was ablated by allosamidin in a dose-dependent manner. The optimal temperature and pH for activity of the purified chitinase were 45 degrees C and pH 5-7. The CHT1 cDNA has an ELR motif for chemokine-mediated angiogenesis and appears to be a chitinase of the chemokine family. Localization analysis using mouse anti-rCHT1 serum revealed that native chitinase is highly expressed in the epidermis and midgut of the tick. AcMNPV.CHT1 topically applied to H. longicornis ticks exhibited replication. This is the first report of insect baculovirus infection of ticks. The importance of AcMNPV.CHT1 as a novel bio-acaricide for tick control is discussed.

In the present study, morphological observations on the extracellular structures found on the apical surface of the midgut epithelium, known as the peritrophic membrane (PM) or glycocalyx, are described in Haemaphysalis longicornis females and larvae. These structures have been hypothesized to provide protection to the microvilli of epithelial cells of the digestive tract. Our aim was to determine whether the extracellular structures are important in the digestion of the blood meal and/or as a protection against infection or injury. The PM was detectable in the midgut of engorged larvae by electron microscopy, but not in engorged females. However, a PM-like structure, stainable with toluidine blue, was observed in females by light microscopy. From the results of confocal laser scanning and electron microscopic observations with wheat germ agglutinin (WGA lectin) staining for chitin of the PM, however, the structure was clearly recognized. The structure in the female is likely to be PM because staining with WGA lectin in the presence of GlcNAc indicates the presence of chitin and various morphologies of PM have been reported in insects and ticks. These results show morphologically that different types of PM-like structure are formed in larvae and females of H. longicornis.

Ticks and mites respond to a limited spectrum of stimuli in their search for hosts and mates. Airborne chemical signals include carbon dioxide, ammonia, organic acids, terpenoids, 2,6-dichlorophenol, and other phenolic compounds. These are detected primarily by sensilla in and adjacent to Haller's organ. Most ixodid species examined have one or more multiporose sensilla that detect such volatiles. These olfactoreceptors enable the ticks to respond to remote volatile chemicals from hosts and from the other ticks, e.g., sex pheromones. Other sensilla, probably mechanogustatory in function, also occur on the tarsi. Gustatory sensilla on the palps detect assembly pheromones that enable ticks and mites to respond to conspecific or heterospecific chemical stimuli in their environment. Responses to those stimuli in ticks result in clustering, i.e., arrestant behavior. Arrestant behavior also occurs in certain mites. Finally, cheliceral chemosensilla enable ticks to recognize specific phagostimulants in host blood, e.g., ATP and glutathione, that stimulate feeding. InDennacentor variabilis andD. andersoni, these same cheliceral chemosensilla recognize species-specific genital sex pheromones in the vulvae of conspecific mates, without which they do not copulate.