Neurosecretory system of the American dog tick,Dermacentor variabilis (Acari: Ixodidae). II. Distribution of secretory cell types,axonal pathways and putative neurohemal-neuroendocrine associations; comparative histological and anatomical implications

Histological observations using specialized techniques reveal neurosecretory cells in 18 centers throughout the rind (cortex) of the central nerve mass or synganglion of Dermacentor variabilis. Many cells contribute to complicated networks of neurosecretory pathways and tracts in pre- and post-esophageal portions of the synganglion. The four types of neurohemal-neuroendocrine associations found in Dermacentor resemble structures found in soft ticks (Argasidae) and in other Arachnida, but are more diverse than those described from any other single species. Neurosecretory terminals are distributed diffusely and in two concentrated associations within the perineurium of the synganglion and major peripheral nerves. Terminals are also distributed in the perineurial layers of lateral segmental organs which lie in the general hemocoel at the level of the pedal nerves. A retrocerebral organ complex surrounds the esophagus at its junction with the midgut. The complex includes dorsal and ventro-lateral lobes (containing neurosecretory terminals and intrinsic secretory cells) and the proventricular (neurohemal) plexus. This plexus seems to be a modified (concentrated) cardioglial association. Cardioglial associations are also formed by the neurosecretory innervation of vascular walls of the dorsal aorta and circulatory sinuses which envelope the synganglion and major peripheral nerves. Inferential considerations of neurosecretory and endocrine interactions in the Acari are based on these anatomical and histological data which also provide the basis for evolutionary considerations of anatomical relationships and specializations in the neurosecretory systems of other Arachnida.     

Multiple paternity in the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

The reproductive strategies and variation in reproductive success of ticks are poorly understood. We determined variation in multiple paternity in the American dog tick Dermancentor variabilis . In total, 48 blood-engorged female ticks and 22 male companion ticks were collected from 13 raccoon ( Procyon lotor ) hosts. In the laboratory, 56.3% of blood-engorged females laid eggs, of which 37.0% hatched or showed signs of development. We examined the presence of multiple paternity in the ensuing clutches by genotyping groups of eggs and larvae at 5 microsatellite loci and subtracting the known maternal alleles, thereby identifying male-contributed alleles. Seventy-five percent of the clutches presented multiple paternity, with a mode of 2 fathers siring the clutch. Males associated with the females on the host always sired some offspring. In 1 case, a male was the sire of clutches derived from 2 females, indicating both polygyny and polyandry may occur for this species. These results, combined with those of several other recent studies, suggest that multiple paternity might be frequent for ixodid ticks.     

Allomonal defence secretions of the American dog tick Dermacentor variabilis (Acari: Ixodidae) promote clustering

The release of the defence secretion from the large wax glands (sensilla sagittiformia) of Dermacentor variabilis ticks modifies the behaviour of other ticks by inducing clustering. A coating of natural tick secretion onto test objects (delipidized ticks, glass beads or filter paper discs) also elicits a clustering response, but a topically applied squalene, its major component, does not have this effect. The clustering response appears to be species specific: D. variabilis cluster on conspecific ticks that secreted but they fail to cluster on Amblyomma americanum or Ixodes scapularis ticks unless coated with secretions from D. variabilis. Volatile components in the defence secretion are involved in recruiting conspecific ticks to those that secreted. When attacked by predatory fire ants, Solenopsis invicta, D. variabilis clustered on individuals that had released the defence secretion. This suggests that the secretion protects ticks from predation by functioning as an alarm pheromone. If confirmed, this is the first report of an alarm pheromone and its glandular source in ticks. The terminology applicable to the integumental glands of ticks is discussed.     

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.     

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.     

Internal and external mycoflora of the American dog tick,Dermacentor variabilis (Acari: Ixodidae), and its ecological implications

Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.     

Genetic variation in the mitochondrial 16S rRNA gene of the American dog tick,Dermacentor variabilis (Acari: Ixodidae)

Genetic variation in the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene was examined for the American dog tick, Dermacentor variabilis (Say, 1821). Nine different haplotypes were detected among 369 adult D. variabilis collected from four localities in Canada. There were eight variable nucleotide positions in the 404 bp sequence alignment. Individuals of haplotype 1 occurred at frequency of >75% at all localities. Five haplotypes were detected at only one of the four localities. High haplotype diversity and low nucleotide diversity, combined with significantly negative F_s values for ticks at three localities, suggest a recent population expansion. Genetic differences were found between populations at different localities, but a Mantel regression analysis revealed no association between genetic differences and geographical distances. There was also no association between tick haplotype and the prevalence of the bacterium, Rickettsia montanensis Weiss and Moulder, 1984, in D. variabilis among localities or on opposite sides of Blackstrap Lake (Saskatchewan). The 16S rDNA haplotypes from Canadian populations of D. variabilis formed a clade with those from the eastern and central U.S.A., to the exclusion of D. variabilis from geographically isolated populations in the western U.S.A. Although sample sizes for D. variabilis in the eastern U.S.A. are small, there may be genetic divergence between populations in Canada and those in the eastern U.S.A., which may have implications for studies on the pathogenic agents transmitted by D. variabilis to its hosts.     

Feeding and respiratory gas exchange in the American dog tick,Dermacentor variabilis

This study investigated the effect of blood feeding on respiratory gas exchange in the dog tick Dermacentor variabilis. Adult male and female ticks were fed on bovine hosts from 1 to 11days. Females fed slowly for the first 6days and then rapidly engorged on blood 2-3days prior to dropping from the host. Ticks were removed at daily intervals during feeding, weighed and CO(2) emission measured at 25 degrees C using flow-through respirometry. During feeding, females (N=39) showed a 100-fold gain in mass from 5.78+/-1.05mg to 541.15+/-18.60mg while standard metabolic rate (Vdot;co(2)) increased from 0.179+/-0.030&mgr;lh(-1) in unfed ticks to 87.32+/-5.72&mgr;lh(-1) in fully engorged ticks. CO(2) release prior to feeding was highly discontinuous with discrete spiracular bursts of CO(2) emission approximately every 30min. For CO(2) emission measured in detached partially or completely fed ticks, burst frequency became more and more rapid as feeding progressed and changed to continuous sustained CO(2) output during rapid engorgement. In contrast to females, male ticks (N=20) showed little change in mass and maintained discontinuous CO(2) throughout the 11day attachment period on the host. The switch from discontinuous to continuous CO(2) release and presumed increase in respiratory water loss in female ticks is correlated to an increase in metabolic expenditure associated with blood meal digestion rather than any factor relating directly to maintenance of water balance.     

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.     

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.     

Absence of insect juvenile hormones in the American dog tick,Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae)

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with L-[methyl-(3)H]methionine and farnesoic acid or with [1-(14)C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12-168 h after attachment to host) and mated replete females (2 d after detachment from the host). Those from O. parkeri were third and fourth stadium nymphs and female O. parkeri, 1-2 d after detachment. Corpora allata from Diploptera punctata, Periplaneta americana and Gromphadorina portentosa were used as positive controls in these experiments. No farnesol, methyl farnesoate, JH I, JH II, JH III, or JHIII bisepoxide was detected by radio HPLC from any tick analysis while JH III, methyl farnesoate, and farnesol were detected in the positive controls. To examine further for the presence of a tick, insect-juvenilizing agent, Galleria pupal-cuticle bioassays were conducted on lipid extracts from 10 and 15 d old eggs, unfed larvae (1-5 d after ecdysis), unfed nymphs (1-7 d after ecdysis), and partially fed, unmated female adults (completed slow feeding phase) of D. variabilis. Whole body extracts of fourth stadium D. punctata and JH III standard were used as positive controls. No juvenilizing activity in any of the tick extracts could be detected. Electron impact, gas chromatography-mass spectrometry of hemolymph extracts from fed, virgin (forcibly detached 7 d after attachment) and mated, replete (allowed to drop naturally) D. variabilis and fully engorged (1-2 d after detachment) O. parkeri females also failed to identify the common insect juvenile hormones. The same procedures were successful in the identification of JH III in hemolymph of fourth stadium D. punctata. Last stadium nymphal (female) O. parkeri implanted with synganglia from second nymphal instars underwent normal eclosion to the adult. The above studies in toto suggest that D. variabilis and O. parkeri do not have the ability to make the common insect juvenile hormones, and these juvenile hormones do not regulate tick metamorphosis or reproduction as hypothesized in the literature.     

RdlDv,a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, RdlDv. The closest homologue of the predicted gene product of RdlDv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, RdlDv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that RdlDv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.     

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