No Evidence of Competition Between the Blacklegged Tick (Acari: Ixodidae) and American Dog Tick on the Rodent Host White-Footed Deermouse (Rodentia: Cricetidae) in Southwestern Tennessee.

Investigations that analyze interspecific associations of vectors on their hosts are important for understanding community structure and implementing ways to comprehend mechanisms of pathogen transmission. We assessed the interspecific association of two tick species (Ixodes scapularis Say (Ixodida: Ixodidae) and Dermacentor variabilis Say (Ixodida: Ixodidae)) on the rodent host Peromyscus leucopus Rafinesque (Rodentia: Cricetidae) at the Hobart Ames Research and Education Center in southwestern Tennessee. Of the rodents captured, 95 (63%) had neither species of tick, 6 (4%) had both tick species, 25 (16%) had I. scapularis only, and 26 (17%) had D. variabilis only. A coefficient of association (C7 = -0.08) was calculated, which suggested there was competition between the two species of ectoparasites, but this value was not significant, indicating that there was a neutral relationship between the tick species on P. leucopus. The co-occurrence of both tick species on their host at the same time suggested that the two tick species can occupy the same host and use the same resources without competing.

Small-mammal characteristics affect tick communities in southwestern Tennessee (USA).

Life histories can influence the degree of parasite infestations on a host. Pressures exerted on hosts based on age and sex convey varying degrees of parasite prevalence due to differences in host lifestyles, but it is not known how interactions between different host traits affect tick numbers. The objective of this study was to determine if host characteristics (e.g., age, sex, weight, and their interactions) affect the mean number of ticks found on small mammals regardless of host species or habitat. Sherman live traps were placed in forest and grass/forb habitats representative of the southeastern United States. After capture, host characteristics were recorded, and hosts were then searched for ticks. A total of 281 small mammals (148 Peromyscus leucopus, 34 P. maniculatus, 76 Sigmodon hispidus, 16 Microtus pinetorum, and 7 Ochrotomys nuttalli) and 610 ticks (488 Dermacentor variabilis, 114 Ixodes scapularis, 1 Amblyomma americanum, and 7 A. maculatum) were collected in this study. Host's age, sex, and weight affected the number of ticks collected from small mammals and significant interaction effects between host traits occurred (weight by sex, weight by age, and sex by age). For instance, female subadult rodents had significantly more ticks compared to female adults, male subadults had significantly fewer ticks compared to male adults, and the number of ticks on a host increased as host body mass increased. These results support the hypothesis that the number of ticks vary on rodent hosts based on life histories and trait interactions. Therefore, understanding the behavioral mechanisms of a host can aid in the management of parasites in the environment.

Macrophage migration inhibitory factor expression and protein localization in Amblyomma americanum (Ixodidae).

Amblyomma americanum (L.) ticks continue to emerge as disease vectors in many areas of the United States. Tick macrophage migration inhibitory factor (MIF) was first identified in A. americanum females and has been demonstrated to inhibit macrophage movement to the same extent as human MIF. This study was conducted to further characterize and elucidate the physiological role for MIF in tick feeding. A relative quantitative PCR assay was developed to determine the level of MIF gene expression during tick feeding. In addition, RNAi techniques were used to silence MIF prior to blood feeding. Physiological parameters of tick engorgement weight, length of feeding interval, and egg masses were observed to check for phenotypic manifestations of RNA silencing. Specific tick MIF antibody was used to localize MIF protein in frozen tick tissue sections. Tissue specific gene expression indicated that the midgut tissues were the most highly enriched for the MIF. Levels of gene expression did not parallel MIF protein pools seen in tissue sections. Of particular importance was the finding that unfed tick salivary glands appear to contain vesicles that are specific for MIF protein. This is the first demonstration of a pool of MIF that could be secreted during the first hours of tick feeding. While MIF silencing was demonstrated at the molecular level, no physiological phenotype was apparent. The MIF protein pools already available in the tissues may be sufficient to accomplish female tick feeding. Our studies show that the most prominent source of MIF during tick feeding is the midgut tissue. Future studies will address the role of MIF in blood feeding and nutrient digestion in the immature life stages of the tick.

Nitric oxide synthase and cGMP activity in the salivary glands of the American dog tick Dermacentor variabilis.

We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed.

Tick saliva: recent advances and implications for vector competence.

Secretions of the tick salivary glands are essential to the successful completion of the prolonged feeding of these ectoparasites as well as the conduit by which most tick-borne pathogens are transmitted to the host. In ixodid ticks the salivary glands are the organs of osmoregulation, and excess water from the bloodmeal is returned via saliva into the host. Host blood must continue to flow into the feeding lesion as well as remain fluid in the tick mouthparts and gut. The host's haemostatic mechanisms are thwarted by various anti-platelet aggregatory, anticoagulatory and anti-vasoconstrictory factors in tick saliva. Saliva components suppress the immune and inflammatory response of the host permitting the ticks to remain on the host for an extended period of time and, adventitiously, enhancing the transmission and establishment of tick-borne pathogens. Over the years much work has been done on the numerous enzyme and pharmacological activities found in the tick saliva. The present article reviews the most recent work on salivary gland secretions with special emphasis on how they favour pathogen transmission.

Variabilin, a novel RGD-containing antagonist of glycoprotein IIb-IIIa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis.

A novel inhibitor of human platelet aggregation, named variabilin, was isolated from salivary glands of the hard tick Dermacentor variabilis using a combination of gel filtration and high pressure liquid chromatography. Variabilin was a potent antagonist of the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa; alphaIIbbeta3) and the vitronectin receptor alphavbeta3. Amino acid sequence analysis by Edman degradation revealed that it has 47 residues, with a molecular weight of 4968.5. Like many other naturally occurring antagonists of GPIIb-IIIa, variabilin contains the RGD (Arg-Gly-Asp) motif. However, unlike the RGD-containing antagonists of GPIIb-IIIa, the RGD sequence of variabilin is not positioned in a loop bracketed by cysteine residues. It has little sequence homology to the other known naturally occurring antagonists of GPIIb-IIIa, including the disintegrins from snakes, decorsin and ornatin from leeches, and disagregin from soft ticks. Variabilin is the first RGD-containing antagonist isolated from ticks.

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Fluid secretion by isolated tick salivary glands dependent on an intact cytoskeleton.

Isolated salivary glands from female Dermacentor variabilis (Say) were pre-treated with either cytochalasin D or nocodazol, followed by stimulation with dopamine. Glands pre-treated with 1 microM-cytochalasin D did not secrete fluid; pre-treatment with 1 nM-cytochalasin D did not significantly reduce fluid secretion. Glands pre-treated with 1 microM-nocodazol had a significant reduction in fluid secretion (P = 0.003); 1 nM-nocodazol did not significantly affect fluid secretion. Ligation of the main salivary duct and pre-treatment with 1 microM-cytochalasin D significantly increased gland weight compared to the dopamine stimulated controls (P = 0.0412). Cytochalasin D-treated type III acini had a significantly larger mean diameter compared to the dopamine control (P = 0.0047). Glands treated with 1 mM-verapamil plus 10 microM-dopamine exhibited a significant decrease in fluid secretion (P = 0.017), and when ligated, had a significantly decreased weight compared to the controls (P = 0.0028).

The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis.

Polyclonal antibodies directed against D. variabilis vitellin were utilized for immunocytochemistry at the ultrastructural level. We localized vitellogenin (Vg) in rough endoplasmic reticulum cisternae, secretory granules and secreted products of fat body trophocytes and midgut vitellogenic cells from feeding and ovipositing females. Vg was localized in the oocyte Golgi bodies and in the yolk bodies of both feeding and ovipositing females. Uptake of exogenous Vg was indicated by the presence of immunospecific gold probe in coated pits and coated vesicles at the apical plasma membrane of oocytes from females in rapid engorgement and oviposition. In unmated females little detectable evidence of Vg uptake by developing oocytes suggests that mating and host detachment signal the beginning of vitellogenesis. We conclude that fat body trophocytes, midgut vitellogenic cells and oocytes are involved in the synthesis and/or processing of Vg and that feeding is the signal associated with the initiation of Vg synthesis and/or processing.

Tick (Acari: Ixodidae) attachment cement and salivary gland cells contain similar immunoreactive polypeptides.

A specific antiserum (12C) raised to a 90-kDa immunogenic component of salivary glands of the tick Rhipicephalus appendiculatus recognized similar 90-kDa polypeptides from salivary glands of the American dog tick, Dermacentor variabilis, and the lone star tick, Amblyomma americanum, as well as 70-kDa polypeptides in the cement of D. variabilis, A. americanum, and R. sanguineus (brown dog tick). The reduction in size of the polypeptide for these ticks suggests that it is modified in some way during or after secretion. Immunostaining of salivary glands of unfed- and partially-fed female D. variabilis localized an immunoreactive protein in the d- and e-cells of the type III acini. The quantity of label in granules of glands from unfed ticks was visibly greater than in the granules of glands from partially fed ticks, suggesting that this component is secreted within the first 2 d of feeding. Collectively, these data support the conclusion that a 90-kDa polypeptide of saliva is conserved among ixodid tick genera and is a component of the attachment cement.

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.

Determination of vitellogenin titer in the hemolymph of Dermacentor variabilis (Acarina: Ixodidae) using an indirect enzyme-linked immunosorbent assay.

Using polyclonal antibodies against Dermacentor variabilis (Say) egg vitellin, we developed an indirect, competitive enzyme-linked immunosorbent assay (ELISA). Vitellogenin in the hemolymph of females was quantitatively monitored through feeding (slow feeding and rapid engorgement feeding periods) and postfeeding (preoviposition and oviposition) until death. Vitellogenin concentrations in hemolymph ranged from 1 microgram/ml on the first day of feeding to a maximum of 430 micrograms/ml on the third day of oviposition. The temporal pattern of vitellogenin production in this tick shows a gradual increase in the hemolymph vitellogenin titer throughout the slow feeding period followed by a rapid and substantial rise during the rapid engorgement feeding period and a continuation of this rise in titer through the most productive egg laying part of oviposition. The vitellogenin titer rapidly falls toward the end of oviposition as the tick nears death. There was a 170-fold increase in vitellogenin concentration as measured from the onset of rapid engorgement to the maximum vitellogenin titer. The rapid engorgement feeding period signaled a sharp increase in vitellogenin production.

Hemocytic encapsulation of implants in the tick Dermacentor variabilis.

Implants of Epon, inserted in Dermacentor variabilis (Say) through incisions in the cuticle, were encapsulated by hemocytes. We followed this process at intervals of 1,3,6, 12 and 24 h, and every 24 h thereafter up to 120 h. Degranulation of Type 1 granulocytes and coagulation of hemolymph were first seen at 1 h after implantation and were the earliest evidence of encapsulation. By 3 h after implantation, the degranulation and disintegration of granulocytes had formed a matrix at the Epon surface. From 6 h until encapsulation was completed, plasmatocytes and granulocytes continued to respond to degranulation and formed multiple cell layers around the Epon implant. The capsule was complete at 72 h after implantation. Completion was marked by decreasing degranulation, migration of hemocytes from the outermost layers of the capsule, and by the appearance of loosely attached hemocytes on the outer surface of the capsule. The most common junctional complex observed was gap junctions.

Fine structure of the fat body and nephrocytes in the life-stages of Dermacentor variabilis.

The fine structure of the fat body and associated nephrocytes of the American dog tick, Dermacentor variabilis (Say), was described in unfed larvae, unfed nymphs, and in unfed and fed adults of both sexes. The fat body consisted of one type of cell, the trophocyte. Morphological changes that occurred in the trophocytes of both sexes were dependent on feeding. The ultrastructure of feeding male trophocytes was distinct from trophocytes of feeding females. In the feeding female, the trophocyte developed an ultrastructure characteristic of cells that produce secretory proteins. A type of scalariform cell junction was found associated with rough endoplasmic reticulum of the trophocytes. Nephrocytes were closely associated with trophocytes but were not part of the fat body. Nephrocyte ultrastructure was unaltered throughout the life-stages we examined, except at the end of oviposition. Organelles in the nephrocytes were not randomly distributed, but were found in distinct regions of the cytoplasm. Slit diaphragms at the surface of the nephrocytes were extracellular specializations that had a periodic ultrastructure.

Relationship between feeding, mating, vitellogenin production and vitellogenesis in the tick Dermacentor variabilis.

Anti-vitellin IgG directed against Dermacentor variabilis egg vitellin was used in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gradient gel immunoblots to detect the presence of vitellin and its precursor, vitellogenin, in the organs of feeding adults and in the immature stages of this tick. Vitellin polypeptides were found in the egg, larvae, nymph, and in the unfed adult stages of both sexes. Vitellin polypeptides were first detected in the ovary of mated females during the rapid-engorgement feeding period. These polypeptides were also present in the ovaries of ovipositing females, unmated females fed for extended periods, and fed unmated females that were detached from the host and held for 12 h before dissection. The same anti-vitellin antibody was used in immunoblots to monitor the appearance of vitellogenin in the organs and hemolymph of female ticks. Immunoreactive peptides of vitellogenin were found in the fat body, midgut, and hemolymph of pre-rapid-engorging mated and unmated females. These polypeptides were not found in fed males nor in Malpighian tubes of feeding or ovipositing females. Our data supported the following conclusions: 1) presence of immunoreactive vitellogenin in the adult female fat body, hemolymph, and midgut was dependent upon feeding; 2) in mated feeding females, we could not detect the uptake of vitellogenin by the ovary until rapid engorgement; 3) in unmated females, vitellogenesis did not begin unless prolonged feeding occurred; and 4) during the early developmental stages of this tick, vitellin served as an embryonic nutrient reserve and as a reserve against starvation between feedings.

Onset of vitellogenin production and vitellogenesis, and their relationship to changes in the midgut epithelium and oocytes in the tick Dermacentor variabilis.

In Dermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (uptake of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.

Ultrastructure of the midgut and blood meal digestion in the adult tick Dermacentor variabilis.

Digestive cells in the midgut of male and female Dermacentor variabilis (Say) took up the blood meal in coated vesicles and smooth flask-shaped vesicles, and deposited it in endosomes which were digested via heterophagy. Iron was concentrated in residual bodies. Digestion occurred in three distinct phases in mated females: (1) continuous digestion (initiated by feeding) occurred during slow engorgement; (2) reduced digestion (initiated by mating) occurred in mated females during the period of rapid engorgement; (3) a second continuous digestion phase (initiated by detachment from the host) occurred throughout the post-feeding periods of preoviposition and oviposition. It proposed that the stem cells in the midguts of unfed females were progenitor of digestive, replacement, and presumed vitellogenic cells in midguts of mated feeding females. Digestive cells were present in all three digestion phases. Only during the first continuous digestion phase did digestive cells fill up with residual bodies, rupture and slough into the lumen, or did whole cells slough into the lumen. During the other two digestion phases no sloughing of digestive cells was observed. At the end of oviposition the digestive cells were filled with residual bodies. Replacement cells were present only during the first continuous-digestion phase. Presumed vitellogenic cells were present only during the reduced-digestion phase and during the second continuous-digestion phase. Stem cells in unfed males developed only into digestive cells in feeding males. Fed males and fed unmated females had only the first continuous-digestion phase. After being hand-detached from the host, unmated 13-day-fed females went through cellular changes associated with the reduced-digestion phase and second continuous-digestion phase of fed mated females, then began ovipositing. Maximum development of the basal labyrinth system and lateral spaces matched the known time of maximum water and ion movement across the midgut epithelia. Spectrophotometric analyses of lumen contents and midgut cells, sampled after detachment from the host, showed that concentrations of protein and hemoglobin at day 1 post-detachment decreased by one-half at the beginning of oviposition, while hematin increased about twofold by the end of oviposition. This supported the idea of the presence of a second continuous-digestion phase.

The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 23. Fine structure of the salivary glands of unfed A. (P.) Arboreus Kaiser, Hoogstraal, and Kohls.

Salivary glands of the unfed adult Argas (Persicargas) arboreus (family Argasidae) contain 2 types of alveoli, one nongranular and one granule-secreting. The fine structure of the nongranular alveolus is similar to that of the family Ixodidae. In the granule-secreting alveolus, the presence of 3 types of secretory cells, each with morphologically distinct granular inclusions, confirms histological and histochemical observations on argasid salivary glands. Epithelial cells with numerous membranous infoldings, mitochondria, microtubules, and a complex canalicular system probably concerned with fluid regulation and secretion are located between granule-secreting cells and form caps over their basal regions. The luminal border of both secretory and epithelial cells is microvillate. The alveolar lumen leads into the chitinous alveolar duct which lacks the complex valvular structure of ixodid alveoli. Axons containing neurosecretory material occur in both nongranular and granule-secreting alveoli and probably control salivary secretion.