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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...

The behavioural responses of Apanteles taragamae, a larval parasitoid of the cucumber moth Diaphania indica, to the volatiles of cucumber plants was investigated in a four-arm olfactometer. Females...

The exotic Bactrocera dorsalis and the native Ceratitis anonae infest citrus and constitute a threat to citrus production in Nigeria. The immature stages of the two species are difficult to differentiate morphologically and share the same fruit in the study area. A 658 bp portion of the COI gene was amplified and sequenced in 88 life stages of the species using LCO1490/HCO2198 primers. The enzymes RsaI, Sau3AI and Hsp92II were used to construct banding patterns distinguishing life stages of B. dorsalis from C. anonae. DNA barcoding confirmed the species to be B. invadens syn. n. B. dorsalis and C. anonae. Rsa I produced bands of 322 and 336 bp in B. dorsalis and 201 and 457 bp in C. anonae. Sau3AI produced bands of 103, 144 and 364 bp in B. dorsalis and 164 and 471 bp in C. anonae. Hsp92II produced fragment lengths of 137 and 521 bp in B. dorsalis and 110, 231 and 290 bp in C. anonae. The identification method was confirmed by DNA barcoding and molecular phylogenetic analysis. All the life stages within a...

Species from the genus Mayetiola are observed in the main cereal cultures of Tunisia. Some researchers have studied M. destructor that attacks wheat and M. hordei that attacks barley. However, a third important species observed in oat, M. avenae, has not been studied and is not well documented in Tunisia. A method to easily separate the species is needed to clarify the occurrences of these gall midge species. This study aimed to first distinguish between the three species of gall midges by molecular characterization and second to reveal the phylogenetic relationships within and between the three species of Mayetiola collected from 5 different regions of northern Tunisia. To achieve these purposes, two regions of the mitochondrial DNA, cytochrome oxidase subunit I gene, and the 16S rRNA gene were amplified by polymerase chain reaction and sequenced. For each marker, a set of 75 individuals were used for DNA analysis. Phylogenetic trees were created using the DNA sequences of all samples from the 3 species. Results showed significant separation of the three different species into dissimilar clades. Each clade contained only specimens from the same species. Differences were observed between DNA sequences of the same species. The differences within the same species were not representative of geographical variations but coexisted within a population Therefore, using the COI and 16S rRNA genes as markers can clearly separate M. avenae, M. destructor and M. hordei.

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.

We evaluated the utility of DNA barcodes for identifying Thaumatotibia leucotreta (Meyrick) infesting sweet oranges in Nigeria. Approximately 658 bp of the mitochondrial gene cytochrome oxidase subunit I (COI) was amplified, sequenced, and analyzed for 32 adult samples, eight from each of the four geopolitical zones of Nigeria. All of the samples had identical sequences except for two, both of which showed a transition from “G” to “A” at position 433 bp. DNA barcodes of specimens from Nigeria showed 100% similarity with sequences of T. leucotreta in the Barcode of Life Database (BOLD) from Kenya and South Africa; all the Nigerian samples cluster with T. leucotreta from BOLD with a bootstrap support of 90. DNA barcoding is consistent with identifications of the samples based on morphology.

The exotic tephritid Bactrocera invadens (syn. Bactrocera dorsalis) and the native Ceratitis species constitute serious pests and major impediment to Citrus production in Nigeria. Lack of accurate and reliable diagnostic methods independent of the life stages intercepted for both genera hampers implementation of appropriate early management programmes to avoid economic losses and delays in quarantine decision regarding these pests. We used a PCR amplification technique to successfully establish RFLP patterns of the COI coding gene to differentiate the exotic B. invadens from the native Ceratitis species infesting sweet oranges in Nigeria. The universal barcoding primer pair LCO1490/HCO2198 was used to amplify 658 bp-long fragment of the mitochondrial COI coding gene. The amplified fragment was analysed by automated direct sequencing and RFLP. Intraspecific variation in nucleotide sequences of B. invadens was very low with not more than 2 bp substitutions in four out of 31 individuals sequenced. Among the ...

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.

Four enantiomeric 9-mer peptides named d-peptide A, B, C and D were designed and synthesized on the basis of 43-mer insect defensins from two beetles. The d-9-mer peptides maintained bacterial membrane disruptive activity similar to the original peptides and also showed various extents of growth inhibitory activity against different cancer cell lines. Of these peptides, d-peptide B exhibited the highest selective cancer cell cytotoxicity against the mouse myeloma cell line, P3-X63-Ag8.653. Flow cytometric and scanning electron microscopic analysis revealed d-peptide B disrupts mouse myeloma membrane construction, whereas no cytotoxic effect on normal leukocytes was observed. Moreover, a strong correlation between negatively charged phosphatidylserine (PS) density in cancer cell membrane surface and sensitivity to d-9-mer peptides were observed in various cancer cell lines. These results suggest that d-9-mer peptides have negative charge-dependent selective cancer cell cytotoxicity targeting PS in the cancer cell membrane. In addition, synergic growth inhibitory activity against mouse myeloma was observed in combinations of d-peptide B and dexamethasone. These results suggest d-9-mer peptides are promising candidates for novel anticancer drugs.

Four enantiomeric 9-mer peptides, D-peptides A (RLYLRIGRR-NH(2)), B (RLRLRIGRR-NH(2)), C (ALYLAIRRR-NH(2)), and D (RLLLRIGRR-NH(2)), were designed and synthesized on the basis of a beetle defensin antimicrobial peptide. These D-9-mer peptides have been reported to exhibit multiple functions, including antimicrobial and antiprotozoan activity, without cytotoxicity on normal fibroblasts and leukocyte cells. In this study, we found that the D-9-mer peptides inhibited telomerase activity (IC(100) = 40 microM). A new peptide, D-peptide C2 (ALYLAIRRRRRRRR-NH(2)), designed from D-peptide C to translocate into the cytoplasm by a penetrating sequence (octa-arginine), showed extremely strong telomerase inhibitory activity (IC(100) = 0.1 microM). D-Peptide C2 exhibited a great increase in cytotoxicity against various cancer cell lines (IC(50) = 3.4-26.4 microM). However, the immediate death of the cells suggested that the high cytotoxicity was not an effect of telomerase inhibitory activity. Mitochondrial swelling assay and microscopical observations of mitochondria indicated that the major target of the D-peptide C2 was the mitochondrial membrane.

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.

The effect of photoperiod on pupariation time and the ability to secrete ecdysteroids by the larval ring gland of the flesh fly, Boettcherisca peregrina, are compared among three tropical non-diapausing colonies, Bp (forest), Bp (Luwuk) and Bp (NC) and one temperate diapausing colony of Bp (Tokyo). Non-diapausing colonies do not enter diapause under any photoperiod at 20°C, whereas the Bp (Tokyo) diapausing colony enters diapause at the pupal stage under a short-day photoperiod. The times required for 50% pupariation of Bp (forest), Bp (Luwuk) and Bp (Tokyo) are 34 h, 36 h 20 min and 34 h 40 min under short days (SD), respectively. However, Bp (NC) pupariates in a much shorter time (26 h 10 min) compared with the others. The 50% pupariation times of Bp (NC), Bp (forest) and Bp (Tokyo) under long days (LD) are 27 h, 25 h 50 min and 26 h 40 min, respectively. However, it is much longer in Bp (Luwuk) at 53 h 20 min. The rates of secretion of ecdysteroids by the larval ring glands in vitro in Bp (Tokyo) and Bp (forest) are six- to ten-fold higher under SD (6.00 and 3.69 ng) than under LD (0.60 ng), whereas the ring glands of Bp (Luwuk) and Bp (NC) secrete 3.06 and 8.25 ng ecdysteroids under LD and 5.18 and 10.53 ng under SD, respectively. These results suggest there are at least three different physiological types among non-diapausing colonies of B.peregrina: Bp (forest), Bp (Luwuk) and Bp (NC).

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.

Ornithodoros moubataticks were fed on blood infected withBabesia equi. However, the parasites were quickly cleared as evidenced by the disappearance ofB. equi-specific ribosomal RNA from the ticks. We hypothesized that if theBabesiaparasite can escape midgut-associated barriers a non-vector tick can become infected withBabesia. To test this hypothesis,B. equiparasite-infected blood fromin vitroculture was injected into the haemocoel of ticks.B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs.Babesia-free dogs were infested withO. moubataticks that were infected by inoculation withB. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP ofB. gibsoniincreased gradually. These results indicate thatO. moubatamay be a useful vector model forBabesiaparasites and also a very important tool for studies on tick immunity againstBabesiaparasites and tick-Babesiainteractions.

Ticks transmit more different kinds of pathogenic organisms than any other hematophagous arthropod. This paper attempts to review the literature available on the cell-mediated and humoral responses of ticks. The cell-mediated defense responses by invertebrates are simpler than vertebrates, but are effective as a second line of defense against invasion by microorganisms. Hemocytes play a major role in this immune response through phagocytosis, nodule formation and encapsulation of invading foreign materials. These mechanisms appear to function in both soft and hard ticks. Humoral immune responses consist of three main types; humoral encapsulation, hemagglutination and production of antimicrobial proteins. Humoral encapsulation is elicited by the prophenoloxidase activating system and has only been shown to function in the soft tick Ornithodoros moubata. However, recent studies show the hard ticks also have prophenoloxidase activating cofactors indicating humoral encapsulation also functions in these ticks. Tick lectins function in hemagglutination of various microorganisms in both soft and hard ticks and also appear to function as recognition molecules of nonself and possibly in the transmission of pathogenic microorganisms. Ticks also have numerous antimicrobial proteins that can attack the invading microorganisms. Lysozymes have been shown to have bactericidal effects in several tick species and work in concert with other antimicrobial peptides to kill invaders. Cystatins, classic cysteine protease inhibitors, have recently been shown to function in innate immune responses of ticks. Hemoglobin fragments have antimicrobial activity in the midgut of ticks. Defensins show expression up-regulated by blood feeding and are effective antimicrobial agents with some differences between soft and hard ticks. Other novel antimicrobial peptides have been isolated from various species of soft and hard ticks, but their functions remain to be elucidated. Understanding these innate immune mechanisms in ticks is essential to develop strategies for reducing the transmission of diseases to man and animals.

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.

The ultrastructure and characteristics of hemocytes of argasid tick species, Ornithodoros moubata, during the ecdysdial phase are herein presented. Hemocyte classes/populations characterized based on their affinity with Giemsa stain and ultrastructural differences comprised the prohemocytes, nongranular cells (Nc), eosinophilic granular cells (Ec), basophilic granular cells (Bc), and unidentified cells. Significant changes/shift in the ratio of hemocyte classes/population was apparent in ticks before and after the ecdysial phase. The granule-scant basophilic granular cells (sBc) constituted the most abundant hemocyte population in the ecdysial phase. Nymphs in ecdysis showed increases in Nc and sBc and decrease in Ec, a phenomenon that was reversed in unengorged nymphs and adults ticks. The significant increase in total Bc population in ecdysis relative to nonengorged ticks clearly point to blastogenesis of Bc taking place during the ecdysial phase and Bc’s important role in the process of tissue...

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.

Linkage of an acetylcholinesterase (AChE) gene was detected in the house fly, Musca domestica L., by using the backcross method between a strain, aabys, that had a morphological multichromosomal marker on each of the five autosomes and a wild strain, LPR. Both strains were homozygous in this gene, and we used eight single nucleotide polymorphisms (SNPs) between them to distinguish the parental sequences in the backcrossed progeny, two of which resulted in the amino acid substitiutions common to the Drosophila and Aedes AChEs insensitive to organophosphates and carbamates. F, appeared to be a wild phenotype, and the AChE gene was heterozyous of aabys and LPR. In the backcross progeny, 32 (2(5)) phenotypes appeared, and 10 phenotypes with one wild or morphological marker were picked up for genotyping by the SNPs of AChE gene. A combination of the morphological markers and the SNPs revealed that the AChE structural gene is linked to autosome 2 in the house fly.

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.

Vitellogenins (Vgs) and vitellins (Vns) were purified from reproductive female adult hemolymph (HL) and newly laid eggs of the bean bug, Riptortus clavatus. They were separated into two (Vg-1 and 2) and three (Vn-1′, Vn-1, and Vn-2) sub-components, respectively, by ion exchange chromatography. Fused rocket immunoelectrophoresis using anti-(Vn-1) serum showed that they are distinguished clearly into two immunologically distinct groups, i.e., Vg-1/Vn-1/Vn-1′ and Vg-2/Vn-2. SDS-PAGE analysis showed Vn-1 (Vn-1′) and Vn-2 have the same and/or smaller subunit component polypeptides as Vg-1 and Vg-2, respectively. Vn-1′ has some lower molecular weight peptides than Vn-1. Quantitative rocket immunoelectrophoretic analysis showed that Vg-1 and Vn-1 (plus V-1′) were detected first on day 3 in the HL and day 4 in the ovary, respectively, after adult emergence, and increased by day 10 in non-diapause female adults. Vg-1 synthesis is induced in diapause female adults in 1 day after the treatment with juvenile hormone (JH) and JH analog (JHA) or in a few days after transfer from short day to long day conditions. © 1996 Wiley-Liss, Inc.

Bifidobacteria were isolated from the intestinal tract of the Japanese honeybee, Apis cerana japonica, and investigated for potential application as a probiotic agent against Melissococcus plutonius, the causal agent of European foulbrood (EFB), based on the findings of in vitro inhibition assays. A total of 11 bifidobacteria strains (designated as AcjBF1-AcjBF11) were isolated using a culture-dependent method and their 16S rRNA gene sequences were analyzed. The AcjBF isolates belonged to three distinct bifidobacterial phylotypes that were similar to those found in the European honeybee, Apis mellifera. Although the Japanese and European honeybees are distinct species with different traits and habits, the observation that they share highly similar bifidobacterial phylotypes suggests that bifidobacteria are conserved among honeybee species. Despite having extremely high 16S rRNA gene sequence similarities, the AcjBF isolates had markedly different carbohydrate fermentation profiles. In addition, in vitro growth inhibition assays revealed that the cell-free supernatants of all AcjBF isolates exhibited antagonistic effects on M. plutonius growth. These results indicate that the bifidobacteria isolated from the gut of Japanese honeybee could potentially be employed as a new biological agent to control EFB.

Synthesis of the precursor yolk protein vitellogenin (Vg) occurs after engorgement in haematophagous arthropods. We identified the Vg cDNA of the soft tick Ornithodoros moubata (OmVg) and compared its expression in mated and virgin females. Both mated and virgin females showed increases in OmVg expression after engorgement but expression was higher in mated females than virgin females particularly as time advanced. Delayed mating in virgin females induced an increase in OmVg expression. OmVg expression was observed in the midgut and fat body by whole mount in situ hybridization, but enlarged fat body with high expression occurred in only mated females during the late phase of vitellogenesis. Therefore, engorgement initially induces OmVg expression but mating is necessary for continued Vg expression to produce mature eggs.

Vitellogenin (Vg) synthesis and oocyte development appear to be induced by ecdysteroids in the soft tick Ornithodoros moubata. Vg transcription in insects has been shown to be mediated by two nuclear receptors, an ecdysteroid receptor (EcR) and a retinoid X receptor (RXR). EcR and RXR expression in female ticks increase after engorgement. However, there is little information about EcR and RXR expression in tissues as related to reproduction. Therefore, the sites of EcR and RXR expression during vitellogenesis were determined. Expression levels of EcR and RXR were determined for salivary glands, midgut, ovary, anterior reproductive tract, fat body, muscle, and cuticle of mated females by reverse transcriptase RT-PCR and real-time PCR. Ovaries showed constant expression of EcR and RXR throughout the adult female stage. The fat body showed higher expression of EcR and RXR on one day after engorgement. The fat body of female ticks has been reported to be the organ of Vg synthesis. Therefore, increases in EcR and RXR of the fat body support the hypotheses that the site of Vg synthesis is the fat body and ecdysteroids regulate this synthesis in O. moubata. EcR and RXR in the ovary and anterior reproductive tract may also play roles in the regulation of oocyte development.

Vitellogenin (Vg) concentrations in the hemolymph and ovarian development were studied in Ornithodoros moubata after treatment with precocenes 1 (P1) and 2 (P2). Precocene was dissolved in acetone or DMSO and topically applied to the dorsal surface of ticks: (1) at adult ecdysis; (2) 24 h before engorgement; (3) immediately after engorgement; and (4) 24 h after engorgement. Subsequently, P1 and P2 were dissolved in olive oil and injected through the gonopore into the body cavity 24 h after engorgement. Vitellogenin concentration was measured on days 5 and 10 after engorgement and ovarian development was scored on day 10, 20 or 30. Oviposition was also recorded and the average weight of eggs laid by females was determined. No differences in concentration of Vg in the hemolymph occurred between the control ticks and ticks treated topically or by injection with P1 and P2. Precocene did not suppress the synthesis of Vg in O. moubata. However, oviposition was reduced in ticks that survived repeated treatment with high doses of P2 dissolved in acetone.

Chinzei, Y., D. Taylor, K. Miura and K. Ando, 1992. Vitellogenesis induction by synganglion factor in adult female tick, Ornithodoros moubata(Acari: Argasidae). J. Acarol. Soc. Jpn., 1(1): 15-26.Vitellogenesis, vitellogenin(Vg)induction in the hemolymph and ovarian development, was studied in unengorged female adults injected with synganglion(SyG)extract and treated with cypermethrin(CyM)in the tick, Ornithodoros moubata(Acari, Argasidae). CyM induced Vg in the hemolymph in the anterior parts of ligated females which contain the SyG, but did not induce vitellogenesis in the posterior part. This result suggests that the SyG is stimulated by treatment with CyM to release a factor for inducing vitellogenesis(vitellogenin inducing factor=VIF). We showed the injection of SyG extracts prepared from unengorged females induced a significant increase of Vg in the hemolymph and ovarian development, but that of SyG extracts from engorged females did not. This indicates that the unengorged female SyG contains the factor, VIF, which is released during or after engorgement. This SyG factor was sensitive to proteases, trypsin and chymotrypsin, indicating the factor is a peptide.

Background Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. Methodology/Principal Findings We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. Conclusions/Significance Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods.

Neutering of part-fed females eliminated copulatory behavior inDermacentor variabilis (Say) andD. andersoni Stiles males. Extracts from the anterior reproductive tracts of part-fed (7 days) females partly restored the male copulatory behavior in conspecific neutered females. Similar extracts from unfed females did not restore the behavior, suggesting that the pheromone was produced during feeding. Perception of the genital sex pheromone by sensillae on the male cheliceral digits was confirmed by electrophysiological techniques.

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.

The effects of several pyrethroids on vitellogenesis, i.e., vitellogenin (Vg) synthesis and ovarian development in unfed mated female adults of Ornithodoros moubata were investigated. Survival of ticks treated with pyrethrin, resmethrin, and etophenprox was very low. Ticks treated with cypermethrin (CyM), fenvalerate (Fev), and flucythrinate (Flu) survived even when high concentrations were used. Vg titer in the hemolymph of unfed mated females was the same level on day 5 and about three times higher on day 10 after treatment with CyM, Fev, and Flu, as that of engorged adult females. CyM was the most effective in inducing vitellogenesis and was further evaluated in unfed virgin females, males, and fourth instars. CyM was shown to stimulate vitellogenesis in unfed virgin females adults. Oviposition did not occur but ovaries were well developed. CyM also was shown to stimulate Vg in the hemolymph of unfed and fed adult males; however, the Vg levels were very low compared to that of engorged adult females. Extremely high concentrations of Vg were observed in the hemolymph of female nymphs (fourth instar), particularly engorged nymphs, treated with CyM (10 micrograms). No ovarian development was observed in these nymphs. Vg stimulated by CyM was shown to be immunologically and electrophoretically the same as that of normal engorged females.

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri . Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [ 35 S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.

In the present study, monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established. Afterward, artificial feeding was performed to assess the tickcidal effect of fetal bovine serum meal containing each mAb. As a result, Om21 showed the strongest tickcidal effect on adult female O. moubata. The reactivity of various tick cells and organs, including the hemocyte, midgut, trachea, ovary, fat body, and muscle, to Om21 was then examined by an indirect immunofluorescent antibody test and by immunoelectron microscopy. Om21 reacted with not only hemocytes but also with fat body cells, epidermis, cuticle of the trachea, connective tissue of the muscle, and the basement membrane of the midgut, trachea, fat body, oocyte, and epidermis. These results suggest that Om21 passing through the midgut epithelium induced a tickcidal effect on hemocytes or various organs. However, the target of Om21 could not be identified in the present study. The antihemocyte mAb produced in this study, Om21, may be useful for the immunological control of ticks.

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.

Many species of ticks and mites must obtain a blood meal from a vertebrate or invertebrate host for development and reproduction to occur. This blood meal results in the transmission of various disease agents from infected animals to uninfected animals. Ticks are second only to mosquitoes as vectors of human diseases. Ticks and/or mites transmit a wide range of microorganisms: viruses, rickettsia, bacteria, and spirochetes, and also protozoa and nematoda.

Chemical analysis and male bioassay responses were used to investigate possible components of the genital sex pheromone of D. variabilis and D. andersoni . Bioassays with D. variabilis indicated greatest responses to myristic, palmitic, stearic, arachidic, and behenic fatty acids as well as myristic and stearic methyl esters. Responses of D. variabilis decreased with changes in chain length, different functional groups and unsaturation. Bioassays with D. andersoni indicated greatest responses to palmitic acid as well as moderate responses to other fatty acids and methyl esters. Chemical analysis revealed the presence of myristic, palmitic, palmitoleic, stearic, oleic and linoleic acids and several unknown compounds in the vaginal lumen and on the external gonopore area. Total free fatty acids in vaginal extracts were 24.8 ng/female equivalent in D. variabilis and 297.2 ng/female equivalent in D. andersoni . Saturated free fatty acids ranging in chain length from C14 to C22 appear to serve as components of the genital sex pheromone of D. variabilis and D. andersoni . Most of these components are present in the lumen of the vestibular vagina and on the external gonopore surface.