Preprandial inhibition of re-mating in Ixodes ticks (Acari: Ixodidae)

Questing female blacklegged ticks, Ixodes scapularis, Say in Massachusetts rarely bear more than one endospermatophore in their reproductive tracts. We evaluated the cause of this nonrandom distribution by examining the stability of endospermatophore retention in females and the effect of prior insemination of females on the copulatory behavior of male ticks. Endospermatophores were retained without degradation in unfed female ticks for >1 yr at 5 degrees C and for at least 4 mo at 21 degrees C. Males were much more likely to abort preprandial (before feeding) copulations without inseminating females bearing endospermatophores. This remating inhibition activity persisted in unfed females for at least 2 mo after insemination. Perprandial (during feedng) copulations were less restrictive, particularly when females became partially engorged. Males were more likely to remain in copula with previously inseminated females that were engorged, but generally did not transfer spermatophores to them until their fifth day of attachment to a rabbit. Little inhibitory activity was evident during the final, rapid engorgement phase of feeding. Thus, remating inhibition appears to degrade as feeding progresses. In summary, an unknown factor associated with the previous insemination of females inhibits subsequent spermatophore transfer by causing mating pairs to interrupt copulation soon after initiation. Remating inhibition is strongest in unfed ticks and becomes less apparent as females become engorged.     

Interaction between ticks (Acari: Ixodidae) and pathogenic nematodes (Nematoda): susceptibility of tick species at various developmental stages

The virulence of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) to tick species under laboratory conditions is reported. The susceptibility of larval, nymphal, and adult stages of the ticks Hyalomma excavatum (Koch), Rhipicephalus bursa (Canestrini & Fanz), and R. sanguineus (Latereille) to 2 strains of Steinernema carpocapsae and 3 strains of Heterorhabditis bacteriophora were compared in laboratory assays. Preimaginal stages of ticks were found to be more resistant to the nematodes than were adult ticks which exhibited 80-100% mortality in a dish containing 5,000 infective juveniles of H. bacteriophora IS-3 or IS-5 strains isolated in Israel. These 2 strains were found to be much more virulent to unfed adult ticks than were the other isolates. No marked difference was found between engorged ticks and unfed adults of R. sanguineus or H. excavatum in terms of mortality, whereas engorged males and unfed females of R. bursa were significantly more susceptible than unfed males or engorged females.     

Histopathology and Ultrastructure Features of the Midgut of Adult Females of the Tick Amblyomma cajennense Fabricius, 1787 (Acari: Ixodidae) in Various Feeding Stages and Submitted to Three Infestations

ABSTRACT

The digestive tube of the tick Amblyomma cajennense is responsible for the digestion during feeding on the host. This study analyzed the midgut of unfed, partially engorged, and fully engorged fed females as well as three infestations in rabbits. In A. cajennense, the digestive tube is long and from the midgut, two pairs of diverticula ramify and lead to a blind end. In some midgut regions were observed for the first time in ticks, structures termed here “nodules.” The midgut of unfed females possesses a pseudostratified epithelium composed of digestive and generative cells. In partially engorged and engorged females at 1st infestation and partially engorged at 2nd infestation, the epithelium becomes stratified. In partially engorged females at 2nd infestation, the epithelium exhibits a third cell type: secretory cell. So the intestinal epithelium undergoes several changes during the feeding process in ticks at subsequent infestations. As infestations progress in the same host, the latter becomes more resistant and female ticks require more days to complete their feeding cycle, which in A. cajennense is 25 days.     

Stimulation of vitellogenesis by pyrethroids in mated and virgin female adults, male adults, and fourth instar females of Ornithodoros moubata (Acari: Argasidae)

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.     

Changes in the concentration of globulins in naive guinea pigs during feeding by the immature stages of Rhipicephalus evertsi evertsi (Acari: Ixodidae)

The weight of Rhipicephalus evertsi evertsi Neumann nymphs, which as larvae and nymphs completed the entire blood meal on the same individual guinea pig, was significantly lower than the weight of those that as larvae and nymphs fed on two separate naive hosts. Nymphs of the latter category spent 1 wk (from unfed larvae to unfed nymphs) on one animal before their transfer to the second host to complete the blood meal. The albumin concentration of the host blood decreased and was related to the time that the immature ticks spent on the hosts. The albumin/globulin ratio also decreased. The alpha 1 globulin fraction increased soon after the guinea pigs were infested with ticks. No further changes in the levels of the alpha 1 globulin fraction were observed with time. The concentration of serum beta globulins increased only in guinea pigs infested with immature ticks for the entire larval and nymphal feeding period. A second infestation of those guinea pigs with larvae of R. e. evertsi resulted in further increases in the levels of serum beta globulins. The relationships among serum beta globulin levels, the weight of engorged nymphs, and host immunity are discussed.     

Regulation of expression of the Borrelia burgdorferi beta(3)-chain integrin ligand,P66, in ticks and in culture

Borrelia burgdorferi is maintained in an infection cycle between mammalian and arthropod hosts. Appropriate gene expression by B. burgdorferi at different stages of this cycle is probably essential for transmission and establishment of infection. The B. burgdorferi beta(3) integrin ligand P66 is expressed by the bacteria in mammals, laboratory culture, and engorged but not unfed ticks. No in vitro culture conditions in which P66 expression reflected that in the unfed tick were found, suggesting that there are aspects of B. burgdorferi-tick interaction that remain unexplored.     

Arthropods as predators of ticks (Ixodoidea)

The existing information on arthropods as predators of ticks is based mainly on sporadic observations and their role in reducing tick populations and in most cases is still not clear. Some reports suggest that in certain ecological habitats arthropods play an important role in the control of the tick population. This publication reports on some 100 relevant publications that appeared between 1906 and 1999. Ants, beetles, and spiders seem to be the major arthropods preying on ticks. In general, engorged ticks are more often preyed upon by arthropods than are unfed or feeding ticks.     

Investigation of relationships between temperature and developmental rates of tick Ixodes scapularis (Acari: Ixodidae) in the laboratory and field

Relationships between temperature and preoviposition, preeclosion, and premolt developmental periods for the tick Ixodes scapularis Say were investigated by holding field-collected ticks in the laboratory at temperatures of 0 to 32 degrees C at constant daylength. The duration of these developmental periods decreased significantly with increasing temperature. Host of origin, prior storage at 4 degrees C, and season of collection of the ticks were also significantly associated with variations in the duration of the preoviposition period. For each developmental stage, the effect of temperature on development rate was best described as a power relationship. Laboratory-derived relationships were used to predict dates for molting, oviposition, and eclosion of engorged larvae and nymphs, engorged adult females and egg masses, respectively, placed in the field during 1989-1992. Predicted dates for oviposition by adult females, eclosion of eggs, and molting of engorged larvae were within 2 wk of the observed dates, and field-observed seasonal activity of questing larvae and nymphs also was predicted well by laboratory data. Molting of engorged nymphs and seasonal activity of questing adult ticks were, however, poorly predicted. Our findings suggest that duration of development in the field, of larvae from engorged adult females, and of nymphs from engorged larvae, may be explained largely by temperature effects alone, whereas emergence of adult I. scapularis from engorged nymphs may depend on temperature-independent diapause phenomena. The significance of these findings for understanding current and future distributions of I. scapularis, and of the pathogens it transmits, is discussed.     

Cloning and characterization of the autophagy-related gene 6 from the hard tick, <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis>

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.     

Replication and tissue tropisms of Crimean-Congo hemorrhagic fever virus in experimentally infected adult Hyalomma truncatum (Acari: Ixodidae).

Adult Hyalomma truncatum Koch ticks were inoculated intracoelomically with Crimean-Congo hemorrhagic fever (CCHF) virus to examine tissue tropisms of this virus in ticks. Virus was recovered from all 185 ticks examined between 2 and 42 d after inoculation with CCHF virus. Titers or virus recovered from unfed male and female ticks were essentially the same (means, 10(2.4) and 10(2.5) plaque-forming units [PFU] per tick, respectively). Infection rates and titers recovered per gland for both salivary glands and reproductive tissues (ovaries and testes) were positively associated with blood feeding; average titers were 10-fold greater in organs from fed compared with those from unfed individuals. In contrast, neither the tick's sex nor feeding status (unfed or engorged) affected the titer of virus recovered from hemolymph (mean, 10(1.6) PFU/microliters). Although virus was recovered from Malpighian tubules, midgut, muscle, and nervous tissues from nearly all of the ticks tested, viral titers were consistently low. The increase in viral titer associated with blood feeding by ticks appeared to be due primarily to proliferation of tissue (e.g., salivary gland and reproductive tissues), rather than to increased replication in tissue already present.     

Comparison of methods for introducing and producing artificial infection of ixodid ticks (Acari: Ixodidae) with Ehrlichia chaffeensis.

Only 29.5 +/- 8.91% of engorged Amblyomma americanum (L.) nymphs that we inoculated with Ehrlichia chaffeensis molted successfully to adults compared with 75.8 +/- 7.46% of engorged nymphs that were not inoculated. However, 65.4 +/- 6.02% of unfed nymphs of this species were exposed for 2 h to E. chaffeensis suspension introduced to them through glass capillaries gained weight. These nymphs were placed on rabbits, and approximately 50% of them completed their feeding and molted successfully to adults. Weight gained was higher (71.8 +/- 17.33% and 69.8 +/- 23.26%) for unfed A. americanum females that fed from capillaries for 2 and 24, h respectively, than for nymphs. Similar values were recorded for Dermacentor variabilis (Say) (61.0 +/- 16.23%) and Rhipicephalus sanguineus (Latreille) (59.0 +/- 18.62%) females after 24 h of capillary feeding. The amount of E. chaffeensis suspension taken in by females of A. americanum, D. variabilis, and R. sanguineus during 24 h of feeding was 11.2 +/- 3.56, 10.9 +/- 4.29 and 6.3 +/- 2.35 microliters, respectively. This volume is equivalent to approximately 12,969, 12,622, and 7,295 infected cells ingested by the species mentioned above. Positive correlation between the volume taken in by the ticks and the weight gained by the females was found, but the initial weight of the unfed females did not effect the weight they gained. The pathogen was found in the females of all 3 species by polymerase chain reaction procedures for at least 7 d, indicating that the capillary feeding method can be successfully used for infecting unfed ticks. The potential use of this method is discussed.     

Laboratory-infected Ehrlichia chaffeensis female adult Amblyomma americanum salivary glands reveal differential gene expression

Ticks are efficient ectoparasites that are able to steal blood, a rich source of nutrients, from their vertebrate hosts. The nymphal developmental stage of ticks plays an important role for pathogen transmission to human and other animal hosts. In this article, we describe a bloodmeal-based sex differentiation tool to generate adult female ticks infected with Ehrlichia chaffeensis to investigate vector-pathogen interactions (functional genomics and gene expression studies). We demonstrate that there is a correlation between the uptake of blood during nymph attachment and the molting into male or female adult ticks. The data obtained from the bloodmeal experiments suggest that nymphs that molt into females presumably imbibe more blood than those that become male during the nymphal stage. The natural low E. chaffeensis infection rate in female adult Amblyomma americanum (L.) is a major limiting factor to investigate Ehrlichia-Amblyomma interactions. To generate Ehrlichia-infected female adult ticks, we inoculated obligate E. chaffeensis (Arkansas strain) infected DH82 cells into heavier engorged nymphs (> 12 mg) and allowed them to molt. Freshly molted adults were used to test the E. chaffeensis infection rate. E. chaffeensis genomic DNA was extracted from individual unfed and partially blood fed tick midgut and salivary gland tissues. The tissue samples were tested for the presence of E. chaffeensis using the nested polymerase chain reaction process. Polymerase chain reaction-amplified fragments were detected in unfed and partially fed tissues, demonstrating successful E. chaffeensis infection of tick tissues. This method was used to successfully show differential expression of selected tick genes in E. chaffeensis-infected midguts and salivary glands.     

Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis

The vectorial capacity of Rhipicephalus sanguineus in the transmission of canine visceral leishmaniasis has been evaluated through a laboratory-controlled experiment. One healthy Leishmania-free dog and two dogs naturally infected with Leishmania were infested with R. sanguineus in various stages of development. Engorged larvae, unfed nymphs, engorged nymphs, unfed adults, engorged female adults and fed male adults were collected from the experimental animals and examined for Leishmania infection by optical microscopy, polymerase chain reaction (PCR) and parasite culture. Leishmania forms were not detected in any of the 433 smears prepared from engorged colonies nor in any of the 118 smears prepared from unfed colonies. However, one flagellate structure was identified in one of the smears. All pools of R. sanguineus that had fed on the infected dogs tested PCR-positive for Leishmania DNA, with the single exception of the pool of engorged larvae. In contrast, all pools of ticks that had fed on the Leishmania-free dog were PCR-negative. Leishmania growth was not observed in any of the tick colonies following incubation on culture medium. Considering that no Leishmania forms were identified in any of the meticulously analysed smears derived from engorged colonies of R. sanguineus, it appears somewhat unlikely that the maintenance and multiplication of Leishmania occurs within the tick.     

Immunization of guinea-pigs and cattle against adult Rhipicephalus appendiculatus ticks using semipurified nymphal homogenates and adult gut homogenate.

Guinea-pigs inoculated with crude homogenate of unfed nymphs of the tick Rhipicephalus appendiculatus and with three semipurified fractions of the homogenate obtained by gel permeation chromatography, acquired a significant degree of immunity to infestation with adults of this tick. Fraction 2 induced the highest reduction (66%) in mean weight of engorged females followed by crude homogenate and fractions 1 and 3. Calves immunized with crude homogenates of unfed nymphs, fraction 2 of nymphal homogenate, and gut homogenate of unfed females also acquired immunity against adults of R. appendiculatus. The mean weight of engorged females fed on calves inoculated with nymphal fraction 2 was the lowest of all five groups of calves on which females fed. The reduction in weight (38%) was not significantly different from that observed for females fed on calves inoculated with crude nymphal homogenate (31%) or females from third infestation of adult ticks. No differences in the weight and hatchability of egg batches produced by engorged females collected from the five groups of calves were observed. Analysis of sera collected from the five groups of calves showed that the concentration of albumin, alpha-1, alpha-2 and beta-globulins fluctuated and no significant differences between the treated groups were observed. The levels of gamma-globulin increased in treated groups including the group inoculated with adjuvant only, but unlike previous reports no increase in gamma-globulin or a correlation between the level of gamma-globulin and the degree of resistance acquired were observed in calves exposed to repeated tick infestations. However, the increase in the concentration of gamma-globulin in calves inoculated with fraction 2 or crude nymphal homogenate was higher than that observed in the other groups.     

Neutral lipid composition changes in the fat bodies of engorged females Rhipicephalus microplus ticks in response to fungal infections

The tick’s fat body plays an essential role in energy storage and utilization. This study aimed to analyze the fat body neutral lipid composition in Rhipicephalus microplus engorged females. In the first study (physiological profile of untreated ticks), the lipid analysis took place over the course of 4 days; the engorged females were incubated at optimal conditions and their fat bodies were dissected daily. Fat body lipid analysis after fungal infection with Metarhizium anisopliae sensu lato (s.l.) or Beauveria bassiana s.l. was performed with four groups: one without any treatment, one that was inoculated with a solution of 0.1 % Tween 80 in water, and two groups that were inoculated with M. anisopliae or B. bassiana conidial suspensions. The fat bodies were dissected 24 and 48 h after infection. Lipid analysis was conducted by thin-layer chromatography on a silica plate. The results of the physiological profile showed that the amounts of triacylglycerol (TAG) and free cholesterol (CHO) decreased with time, whereas cholesterol ester (CHOE) increased on the second and fourth days. Following M. anisopliae or B. bassiana infection, there was an increase in the amount of CHO after 24 h, whereas the other lipid classes were not altered. M. anisopliae caused an increase in CHOE and TAG and a reduction in CHO at 48 h after infection; however, B. bassiana infection did not cause significant alterations in the concentrations of these lipids. M. anisopliae and B. bassiana infection changed the fat body metabolism of engorged female R. microplus ticks. This study provides the first report of changes in the neutral lipid composition of the R. microplus fat body.     

Digestive enzymes in “hard ticks” (ixodoidea,ixodidae)

Summary The proteolytic activity of gut extracts prepared from Hyalomma excavatum female ticks was studied at different stages of blood feeding. Proteolytic enzyme activity was shown to be negligible in unfed ticks, increased from the onset of the tick feeding, reached a maximum at 60–70 hours post attachment and then declined to the initial low level. Proteolytic activity seems to be related to the presence of acidic protease(s) with an optimal pH of 2.9. It appears that a proteolytic enzyme inhibitor is present in the gut of the engorged tick which might be responsible for the low activity at the final stage of feeding.The possible bearing of the proteolytic phenomena on tick borne pathogens is discussed.     

Polymerase chain reaction detection efficiency of the human granulocytic ehrlichiosis agent (Rickettsiaceae: Ehrlichieae) in ticks (Acari: Ixodidae) is dependent on the DNA extraction method

Several methods of extracting DNA from ticks were examined to improve the efficiency of polymerase chain reaction (PCR) detection of the human granulocytic ehrlichiosis (HGE) agent. DNA was extracted from laboratory-reared uninfected and HGE-infected ticks using 3 separate methods. In one treatment, unfed nymphs and engorged larvae of Ixodes scapularis Say, either individually or in pools of 3, were homogenized in 40 microliters of 1x PCR buffer and boiled for 30 min. A 2nd group of ticks was extracted using the QiaAmp Tissue kit, a silica column separation method. A 3rd group was extracted with DNA-STAT, a guanidinium thiocynate method. Five microliters of each extract was used for PCR amplification. Pathogen-free tick DNA samples did not amplify a product. Laboratory-infected ticks extracted either with the QiaAmp kit or those homogenized and boiled in PCR buffer amplified product in 37.5% and 87.5% of the samples, respectively. Infected ticks extracted with DNA STAT-60 amplified a product in 100% of samples. No differences were observed in detection efficiency between ticks tested singly or in pools.     

Immune responses of interferon gamma (IFN-gamma) knock out mice to repeated Haemaphysalis longicornis (Acari: Ixodidae) nymph infestations

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-gamma) deficient mice (IFN-gamma mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemoaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-gamma+/+ mice. However, the engorged body weight of the ticks from IFN-gamma +/+ mice were considerably lower than those from IFN-gamma-/- mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-gamma-/- mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-gamma-/- mice than in IFN-gamma+/+ mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-gamma-/- mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.     

Immunization of guinea-pigs against Rhipicephalus appendiculatus adult ticks using homogenates from unfed immature ticks.

Guinea-pigs immunized with homogenates of unfed larvae and nymphs of the tick Rhipicephalus appendiculatus developed significant levels of protective immunity to infestation with adults of this species. The mean engorged weight of female ticks feeding on immunized animals (181.96 +/- 05.63 mg and 170.11 +/- 11.54 mg) was reduced by an average of 46% and 51%, respectively, compared to that of female ticks feeding on control guinea-pigs, although in some individual animals the reduction was as high as 86%; the mean egg mass weight was also significantly reduced. Electrophoretic separation of the homogenates followed by immunostaining with post-infestation sera revealed several antigen bands common to all stages. Two bands of 36,500 and 23,000 molecular weight (MW) were recognized in all homogenates by post-adult infestation serum, but not by post-larval or post-nymphal infestation sera, suggesting that these may be antigens specifically involved in feeding by adult ticks, and are either not presented to the host's immune system or presented only in minimal amounts during feeding by immature stages. Sera from animals immunized with the homogenates did not recognize either of these antigens. Post-immunization sera did, however, stain two bands of 84,000 and 60,000 MW in the homogenates which were not recognized by post-infestation sera.     

Molecular evidence for potential transovarial transmission of Jingmen tick virus in Haemaphysalis longicornis fed on cattle from Yunnan Province,China

Jingmen tick virus (JMTV) is a novel tick-borne virus first identified from Jingmen city, Hubei Province of China in 2010. It has been proved that JMTV can cause human diseases and is widely distributed both inside and outside of China. However, the survival mode and transmission characteristics of JMTV still need further research, particularly in terms of transovarial transmission. In this study, an investigation was conducted to explore the presence of JMTV from engorged female ticks to their offspring. All engorged female adult ticks were collected from domestic cattle and allowed to lay eggs in appropriate humidity and temperature conditions. Maternal ticks, eggs and larvae were screened for JMTV RNA through real-time polymerase chain reaction (RT-PCR) and nested PCR methods. The results revealed the positive rate of 10.53% (10/95) in engorged ticks, 9.09% (2/22) in eggs and 8% (4/50) in larvae pools, respectively. Phylogenetic analysis confirmed that sequences from eggs and larvae had closer relationship with those isolates from maternal engorged ticks with more than 99.7% homology and JMTV manifested with evolutional conservatism. Our study has identified for the first time that JMTV could be transmitted from mother generation to offspring of Haemaphysalis Longicornis. Nonetheless, the efficiency of transovarial transmission in JMTV and the significance of ticks as amplification hosts still need to be further illustrated.