Cockroach allatostatin-like immunoreactivity in the synganglion of the American dog tick Dermacentor variabilis (Acari: Ixodidae)

Using immunocytochemistry based on a monoclonal antibodyagainst Diploptera punctata allatostatin I and horseradishperoxidase-diaminobenzidine reaction, the presence of allatostatin-likeimmunoreactivity is demonstrated in the synganglion of Dermacentorvariabilis females. The immunoreactive cells are located in theprotocerebral, cheliceral, palpal, stomodeal, postesophageal, and opisthosomalregions of the synganglion. Strongly immunoreactive granules accumulate in theboundary area of the subganglia in the preesophageal part of the synganglion.This suggests that the immunoreactive materials may be released directly fromthere. In addition, a putative neurohemal area is found in the anterior area ofthe opisthosomal ganglion, where abundant immunoreactive materials are stored.Weak immunoreactivity and fewer immunoreactive cells are seen in newly moltedfemales compared with one month old, unfed females. Thus, the immunoreactiveproducts may be depleted during molting and synthesized in females beforefeeding.     

Evidence of a maternal effect that protects against water stress in larvae of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

We report that the ability to absorb water vapor from the air in larvae of the American dog tick, Dermacentor variabilis, changes depending upon moisture conditions where the eggs develop. When development occurs at lower relative humidities, resultant larvae can replenish water stores, maintain water balance, and survive at relative humidities as low as 75-85% RH, a range that agrees with previously published values for the critical equilibrium humidity or CEH. In contrast, exposure to high relative humidity conditions during development elevates the CEH to 93-97% RH. These larvae can survive only at relative humidities that are close to saturation, as 93% RH is a dehydrating atmosphere. For these larvae, absorption at 97% RH can be prevented by blocking the mouthparts with wax, indicating that an upward shift has occurred in the moisture threshold where the active mechanism for water vapor absorption operates. Based on transfer experiments between low and high relative humidities, the CEH of larvae is determined by the relative humidity experienced by the mother rather than the moisture conditions encountered by eggs after they are laid. The fact that no changes in body water content, dehydration tolerance limit and water loss rate were observed implies that adjustments to the CEH conferred by the mother have the adaptive significance of enabling larvae to maintain water balance by limiting the range of hydrating atmospheres.     

Attraction of immature stages of the American dog tick (Dermacentor variabilis) to 2,6-dichlorophenol

To determine whether 2,6-dichlorophenol is solely a sex pheromone, the response to it by the various stages of the American dog tick, Dermacentor variabilis, were compared. In contrast to adults, 2,6-dichlorophenol was attractive to unfed nymphs and to unfed larvae. Use of this chemical also prompted the expression of a novel type of feeding posture behavior in adults. The overlap in attraction to other substituted phenols plus the lack of functional value of this response for larvae and nymphs rules out the possibility that 2,6-dichlorophenol is a general attractant. However, 2,6-dichlorophenol likely plays a dual role as an attachment stimulant in the adult tick.Undergraduate Research Program in Biology     

Immunocytochemical mapping of FMRFamide-like peptides in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis

FMRFamide-like immunoreactivity was studied in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis using immunocytochemistry based on the peroxidase-antigeroxidase method. FMRFamide-like immunoreactive cells are widely distributed in various regions of the tick synganglion including protocerebral, cheliceral, stomodeal, palpal, pedal I–IV, and opisthosomal regions in both species. However, there is one layer of immunoreactive cells located on the dorsal surface of the postoesophageal part of the synganglion that is found only in D. variabilis. Besides the immunoreactivity within the cell body and its axons, the neuropile and the neural lamella (the extracellular sheath of the synganglion) are rich in immunoreactive materials. Some coxal muscles are innervated by the FMRFamide-like immunoreactive processes of the nerve from the pedal ganglion.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

Development and characterization of 12 polymorphic microsatellite loci in the American dog tick (Dermacentor variabilis)

Twelve polymorphic microsatellite loci were developed for the American dog tick (Dermacentor variabilis), an important vector of infectious diseases in humans and animals. Four multiplexed panels comprising the loci were developed and 45 ticks collected from two raccoons (Procyon lotor) were genotyped. The number of alleles per locus ranged from nine to 30, and single locus heterozygosities ranged from 0.18 to 0.93. Data generated using these markers will further our understanding of factors affecting gene flow in D. variabilis, thus helping to elucidate the transmission dynamics of diseases associated with this vector.     

Structure and anatomical relationships of the synganglion in the American dog tick,Dermacentor variabilis (Acari: Ixodiadae)

The synganglion of Dermacentor variabilis Say is a single nerve mass, condensed around the esophagus and within the periganglionic sinus of the ciculatory system. Protocerebral, cheliceral (including stomodeal bridge), and pedipalpal ganglia lie in the pre-esophageal portion of the nerve mass and bear optic, cheliceral, and pedipalpal nerves respectively. The unpaired stomodeal and the recurrent nerve which forms the hyper-esophageal ganglion arise from the stomodeal bridge. Paired primary and accessory nerves to the retrocerebral organ complex have mixed protocerebral-cheliceral origins. Pedal ganglia (including ventral olfactory lobes of pedal ganglia I) and composite opisthosomal ganglion lie in the post-esophageal nerve mass and bear pedal nerve trunks and two pairs of opisthosomal nerves respectively. Internally, the synganglion consists of cellular rind and fibrous core. A welldefined neurilemma with a laminar matrix covers nerve mass and peripheral nerves. The rind contains the somata of ganglionic neurons and ensheathing glial cells and is restricted to the synganglion mass. It is limited by two specialized glial layers, the external perineurium and internal subperineurium. Discrete glomerular formations are present within the protocerebrum and olfactory lobes. Olfactory glomeruli located in pedal ganglia I are associated with a pair of globuli cell groups. Possible physiological relationships between anatomical specializations of the synganglion, extraneural sinuses and circulating hemocytes are considered. The evolutionary significances of condensation in the stomatogastric neuropile regions and throughout the synganglion, together with the simplification and loss of glomerular formations, are discussed.     

The geographic distribution and ecological preferences of the American dog tick,Dermacentor variabilis (Say), in the U.S.A.

Equine piroplasmosis (EP), caused by two parasitic organisms, Theileria equi and Babesia caballi, is a tick‐borne disease of recent concern in horses in the U.S.A. Outbreaks of EP have been detected in Florida, Missouri, Kansas and Texas. In 2009, EP transmission in Texas occurred through the adults of two tick species, Amblyomma mixtum [formerly known as Amblyomma cajennense (Fabricius, 1787)] Koch (Ixodida: Ixodidae) and Dermacentor variabilis (Say) (Ixodida: Ixodidae), the American dog tick (ADT). In this study, we developed a continent‐scale map for the distribution of the EP vector species D. variabilis, using a presence‐only modelling approach to assess the habitat preferences of this tick. We used identification records from our tick geodatabase of locations in which the presence of the ADT had been noted. The potential distribution of the ADT in the U.S.A. was estimated from environmental factors using the maximum entropy approach based on localities in which there is a high probability of occurrence according to habitat suitability. Elevation and temperature were found to be biologically significant environmental variables influencing the presence of this tick species. Properly designed and constructed probability surfaces using maximum entropy offer one useful approach to the mapping of distribution ranges of tick species based on suitable habitat in the U.S.A.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Underwater survival in the dog tick Dermacentor variabilis (Acari:Ixodidae)

Ticks are blood-feeding arthropods known for their long survivability off the host. Although ticks are terrestrial, they can survive extended periods of time submerged underwater. A plastron is an alternative respiration system that can absorb oxygen from water via a thin layer of air trapped by hydrophobic hairs or other cuticular projections. The complex spiracular plate of ticks has been postulated to serve as a plastron but that function has not been verified. This study provides evidence of plastron respiration in the American dog tick, Dermacentor variabilis, and for the first time confirmed the existence of plastron respiration in Ixodidae. Longer survival rates in oxygenated water indicate that underwater respiration requires oxygen. Wetting the spiracular plate with alcohol debilitates any potential plastron function and lowers the survival rate. Survival underwater may also be enhanced by metabolic depression and possibly anaerobic respiration. This study describes the first example of plastron respiration in the Ixodidae.     

Cold-hardiness of Dermacentor marginatus (Acari: Ixodidae)

The cold-hardiness of Dermacentor marginatus using laboratory-reared offspring of ticks collected in Germany was characterized. Investigations of unfed stages revealed that adult ticks suffered 50% mortality at –10°C after 4–5 months, but larvae and nymphs suffered mortality within few days, whereas –15°C was lethal for all stages within a very short period. Larval hatch and moulting of engorged larvae and nymphs did not occur at 10°C. Embryonic development of eggs with larval hatch was considerably reduced by exposure of eggs to 10°C. Engorged females did not lay eggs at 10°C, the oviposition capability, however, persisted over 6 months at 10°C, 5 months at 5°C, 3 months at 0°C and 2 months at –10°C without substantial decrease of the oviposition capacity or reduction of viable eggs. These results present evidence that unfed adult ticks are the ecoepidemiologically most effective stages, which are capable to tolerate long and extremely cold winters without substantial impairment of the population density. It is also considered that engorged females interrupt their oviposition at low and subzero temperatures delaying it for months and so contribute in bypassing winter conditions. None of the stages survived supercooling indicating that D. marginatus is freeze intolerant. Mean supercooling point (SCP) ranged between –26°C in eggs and –12, 6°C in engorged females. Compared with eggs, the SCP of the other stages was significantly higher. In conclusion, the SCP is considered to have no predictive value in the context with cold-hardiness.     

Serotonin-like immunoreactivity in the central nervous system of two ixodid tick species

Immunocytochemistry was used to describe the distribution of serotonin-like immunoreactive (5HT-IR) neurons and neuronal processes in the central nervous system (CNS), the synganglion, of two ixodid tick species; the winter tick, Dermacentor albipictus and the lone star tick, Amblyomma americanum. 5HT-IR neurons were identified in the synganglion of both tick species. D. albipictus had a significantly higher number of 5HT-IR neurons than A. americanum. The labeling pattern and number of 5HT-IR neurons were significantly different between sexes in D. albipictus, but were not significantly different between sexes in A. americanum. 5HT-IR neurons that were located in the cortex of the synganglion projected processes into the neuropils, invading neuromeres in the supraesophageal ganglion including the protocerebrum, postero-dorsal, antero-dorsal and cheliceral neuromeres. In the subesophageal ganglion, dense 5HT-IR neuronal processes were found in the olfactory lobes, pedal, and opisthosomal neuromeres. Double-labeling with neurobiotin backfilled from the first leg damaged at the Haller’s organ revealed serotoninergic neuronal processes surrounding the glomeruli in the olfactory lobes. The high number of the 5HT-IR neurons and the extensive neuronal processes present in various regions of the synganglion suggest that serotonin plays a significant role in tick physiology. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. The U.S. Government’s right to retain a non-exclusive, royalty free license in and to any copyright is acknowledged.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.     

Kinetics of ingested host immunoglobulin G in hemolymph and whole body homogenates during nymphal development of Dermacentor variabilis and Ixodes scapularis ticks (Acari: Ixodidae)

Patterns in the utilization of host immunoglobulin G (IgG) during nymphal development differed between Dermacentor varibilis (Say) and Ixodes scapularis Say ticks. In unfed nymphs of D. variabilis, host IgG was readily detectable in both hemolymph and whole body homogenates. In unfed nymphs of I. scapularis, host IgG was absent in hemolymph and at very low concentrations in whole body homogenates. Host IgG in unfed nymphs was undoubtedly the remnants of IgG acquired during the larval bloodmeal that persisted through metamorphosis to the nymphal stage. In both tick species, host IgG crossed the midgut into the hemocoel during the latter phases of engorgement. Concentrations of host IgG in I. scapularis declined considerably after replete nymphs molted to the adult stage. In contrast, concentrations of host IgG in D. variabilis remained elevated throughout metamorphosis to the adult stage. When larval D. variabilis were fed on a rat, then 2 months later as nymphs on a rabbit, the rat IgG (“old IgG”) present in unfed nymphs was totally replaced by rabbit IgG (“new IgG”) within 2 d of nymphs attaching to the rabbit. Presumably, the old IgG acquired from a previous bloodmeal was secreted via saliva into the new host. This revised version was published online in July 2006 with corrections to the Cover Date.