Exp Appl Acarol (2007) 43:265–278 DOI 10.1007/s10493-007-9120-z

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

Natalie A. Hummel · Andrew Y. Li · Colleen M. Witt

Received: 14 June 2007 / Accepted: 8 November 2007 / Published online: 27 November 2007 © Springer Science+Business Media B.V. 2007

Abstract 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 identiWed in the synganglion of both tick species. D. albipictus had a signiWcantly higher number of 5HT-IR neurons than A. americanum. The labeling pattern and number of 5HT-IR neurons were sig- niWcantly diVerent between sexes in D. albipictus, but were not signiWcantly diVerent 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,

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N. A. Hummel (&) · A. Y. Li (&) ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory, USDA, 2700 Fredericksburg Road, Kerrville, TX 78028, USA e-mail: [email protected]

A. Y. Li e-mail: [email protected]

Present Address: N. A. Hummel Department of Entomology, Louisiana State University, 404 Life Sciences Building, Baton Rouge, LA 70803, USA

C. M. Witt Department of Biology, RCMI Advanced Imaging Core, University of Texas at San Antonio, 6900 North Loop 1604, San Antonio, TX 78249, USA 1 C 266 Exp Appl Acarol (2007) 43:265–278 pedal, and opisthosomal neuromeres. Double-labeling with neurobiotin backWlled from the Wrst leg damaged at the Haller’s organ revealed serotoninergic neuronal processes surround- ing 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 signiWcant role in tick physiology.

Keywords 5-Hydroxytryptamine · 5-HT · Acari · Amblyomma americanum · Dermacentor albipictus · Synganglion

Introduction

Ticks are among the most signiWcant vectors of arthropod-borne diseases that aVect man and animals (Drummond 2004). The most common method used to control ticks on vertebrate hosts is chemical control (George et al. 2002). Acaricides used to control ticks are mostly nerve poisons that target either the ion channels of neurons, neurotransmitter receptors, or enzymes required for the normal function of ticks (Dekeyser 2005). Resistance to major classes of these acaricides has developed in some tick species, and the resistance problem makes tick control more diYcult and also endangers the continued success of the USDA’s Cattle Fever Tick Eradication Program (CFTEP) (George et al. 2002; Li et al. 2003, 2004). One of the major reasons to study the physiology and molecular biology of ticks is to iden- tify new targets or metabolic pathways for developing novel tick control technology. Serotonin (5-hydroxytryptamine, 5-HT) is an important regulatory molecule in the neuro- nal circuit that controls feeding and salivation in many invertebrates. Serotonin-like immuno- reactivity has been identiWed in the central nervous system (CNS) of many species of invertebrates (see review by Nässel 1988) including spiders (Seyfarth et al. 1990a, b), and is a well-documented neurohormone in insects (Lange et al. 1989; Schachtner and Bräunig 1993; Coast et al. 2002; Lange 2004). The serotoninergic neuronal circuit plays a critical role in regulating the feeding of aphids and whiteXies, and a novel insecticide, pymetrozine, has been developed to speciWcally interrupt such a system, therefore achieving the control of these pest species (Harrewijn and Kayser 1997). Dacks et al. (2003) found that feeding by the Xesh Xy Neobellieria bullata (Parker) (Diptera: Sarcophagidae) decreased following seroto- nin microinjection. Fain and Berridge (1979) found that application of serotonin to isolated salivary glands of the blow Xy Calliphora erythrocephala (Diptera: Calliphoridae) stimulates Xuid secretion. Serotonin does not stimulate salivary secretions by tick salivary glands (see review by Bowman and Sauer 2004). The addition of both serotonin and histamine to a blood-meal, however, resulted in a decrease in the resistance amplitude associated with sali- vation and feeding of the Rocky Mountain wood tick, Dermacentor andersoni Stiles (Acari: Ixodidae), indicating that serotonin and histamine together have an anti-feedant eVect (Paine et al. 1983). Yet, addition of serotonin or histamine in isolation had no signiWcant eVect on the change in the resistance amplitude associated with feeding or salivation. McSwain et al. (1989) identiWed an unknown “brain factor” that induces inositol phosphate production in the salivary glands of the lone star tick Amblyomma americanum L. (Acari: Ixodidae). Inositol triphosphate (IP3) is a second messenger which may be produced following ligand binding to a G-protein coupled receptor in the cellular membrane. The identiWcation of a G-protein coupled serotonin receptor in the southern cattle tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae) (Chen et al. 2004), along with evidence of neurosecretory activity (Binnington and Tatchell 1973) and immunocytochemical studies (Zhu and Oliver 1991, 2001) suggest that neurotransmitters and/or neuromodulators are produced in the tick synganglion. 1 C Exp Appl Acarol (2007) 43:265–278 267

Furthermore, these data suggest that serotonin may be produced in neurosecretory cells in the tick CNS. To our knowledge, the distribution of serotonin-like immunoreactivity has not been described in the tick CNS. The synganglion is the highly condensed CNS of the tick and contains neurosecretory centers that produce neurohormones (Sonenshine 1991). In ticks, all of the ganglia have been condensed to the point that they are associated directly with the “true brain” which is located in the dorsal aspect of the synganglion. The entire brain is divided into two primary tissue types: the cortex, which contains the somata (neurons), and the neuropils, which con- tain the neuronal projections of these somata. A neurilemma surrounds the CNS. The tick synganglion is divided into two regions by the esophagus which enters ventrally and exits dorsally. The region dorsal and anterior to the esophagus is the supraesophageal ganglion and the region ventral and posterior to the esophagus is the subesophageal ganglion. The objective of this study was to detect the presence of neurons and neuronal processes containing serotonin in the tick CNS, the synganglion, and compare the distribution and density of serotonin-like immunoreactive (5HT-IR) neurons and neuronal processes in both sexes of unfed winter ticks Dermacentor albipictus (Packard) (Acari: Ixodidae) and Ambly- omma americanum. D. albipictus is a one-host tick which completes its entire life-cycle on a single vertebrate host while A. americanum is a three-host tick that parasitizes many small and large vertebrates including deer and humans (Drummond 2004). In order to determine if serotoninergic neuronal processes were associated with the glomeruli in the olfactory lobe, we used a double-labeling technique involving neurobiotin backWlls from the Haller’s organ in the Wrst leg to identify the association between the olfactory glomeruli and 5HT-IR neuronal processes in the tick synganglion. This study provides a description of 5HT-IR structures in the brain of two important tick species and may provide a founda- tion from which we can develop hypotheses and test the possible function of serotonin as a regulatory molecule in tick physiology.

Materials and methods

Experimental animals

The unfed adult A. americanum ticks were from a colony maintained at United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Knipling-Bush- land U.S. Livestock Insects Research Laboratory (KBUSLIRL) in Kerrville, Texas. One- to three-month old, unfed adults were used in this study. All unfed adult ticks were main- tained at 27 § 2°C, 14:10 L:D cycle and a RH of 85% in an incubator with a saturated salt solution. The unfed adult D. albipictus ticks were obtained by infesting larvae under a mus- lin sleeve on the back of a stanchioned Hereford heifer calf at the KBUSLIRL. Larvae were allowed to develop to the metanymphal stage, after which they were forcibly removed from the host (Li et al. 2005) and maintained under the same conditions as for A. americanum until they molted to the adult stage. All unfed D. albipictus adult ticks used in this study were dissected within 24 h after molting.

Neurobiotin and general anatomy

An unfed adult female A. americanum was placed dorsal side up on a strip of utility wax (Heraeus Kulzer, Armonk, NY, USA) on a glass slide. Ticks were fed 1% Neurobiotin (Vector Laboratories, Inc., Burlingame, CA, USA) in 0.1 M KCl through a #2 capillary tube 1 C 268 Exp Appl Acarol (2007) 43:265–278

(Drummond ScientiWc Co., Broomall, PA, USA) placed over the hypostome. Neurobiotin is an intracellular neuronal tracer molecule that has been used widely for labeling neurons through either intracellular injection or backWll from the end of a nerve. The capillary tubes were removed after 1.5 h, and the slides placed on ice to anesthetize ticks before dissection in cold 4% paraformaldehyde followed by Wxation in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C overnight. They were then washed multiple times in Millonig’s Phosphate V Bu er (MPB; 0.164 M NaH2PO4·H2O:0.63 M NaOH (4:1); pH 7.0) followed by dehydra- tion in a graded ethanol series, acetone, and rehydration in a graded ethanol series. The specimens were then washed in MPB and incubated in Avidin-conjugated AlexaFluor 488 (1:25, Molecular Probe, Carlsbad, CA, USA) for Neurobiotin detection overnight at 4°C. They were then washed in MPB and dehydrated in a graded ethanol series, acetone and Wnally embedded in Spurr’s Mod. C (Electron Microscopy Science, Fort Washington, PA, USA) in a bottleneck Beem capsule (Electron Microscopy Science, Fort Washington, PA, USA). They were sectioned at 14
m, 18° blade angle and the sections imaged on an Olym- pus BX60 epiXuorescent microscope (Olympus America Inc., Center Valley, PA, USA).

5-HT immunocytochemistry

Methods to detect serotonin-like immunoreactivity on whole mounts were modiWed from Davis et al. (1996) as follows. Adult D. albipictus and A. americanum ticks of both sexes were chilled 30 min at 4°C before dissection in cold physiological saline (PBS 20X: 160.0 g NaCl, 4.0 g KCl, 23.0 g Na2HPO4–7H2O, and 4.0 g KH2PO4 in 1.0 l dd H2O, pH 7.2–7.4), followed by Wxation in 4% paraformaldehyde in 0.1 M PBS overnight at 4°C. Synganglia were washed multiple times in PBST (PBST: 4.0 g NaCl, 0.1 g KCl, 0.6 g

Na2HPO4–7H2O, and 0.1 g KH2PO4 in 500 ml ddH2O with 0.5% Triton X-100, pH 7.2–7.4), followed by pre-incubation in a blocking solution containing 5% normal donkey serum in PBSAT (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Primary antibody generated in a rabbit against serotonin was then added to the blocking solution (rabbit anti-serotonin, Immunostar, Inc., Hudson, WI, USA) and incubated over- night at a ratio of 1:2,000. Tissues were then washed multiple times in PBST followed by preincubation in blocking solution. Secondary antibody (donkey anti-rabbit Cy2, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was added to the blocking solution at a ratio of 1:800 and incubated. The tissue was washed in PBST, PBS, and cleared with 40%, 60%, and 80% glycerol. Finally, the synganglia were mounted on glass slides in 80% glycerol. In total, nine female and twelve male D. albipictus synganglia and 12 female and 24 male A. americanum synganglia were examined for serotonin-like immu- noreactivity. In order to count the number of 5HT-IR neurons in the synganglion, Wve female and Wve male of each of these species were analyzed in detail. Neurons were counted by studying stacks of laser scanning confocal slice images taken at 0.5
m inter- vals. Controls without the primary antibody were prepared to verify the speciWcity of label- ing. Controls without the primary or secondary antibody were also prepared to determine the amount of background labeling present. The controls did not show any speciWc labeling in the synganglion when the primary or secondary antibody was excluded.

Neurobiotin backWll/5-HT double-labeling technique

A calf-reared unfed adult female A. americanum was placed dorsal-side up on a strip of utility wax (Heraeus Kulzer, Armonk, NY, USA). The right, front leg was then cut at the Haller’s organ and incubated with a drop of ddH2O for 30 s, after which the drop of ddH2O 1 C Exp Appl Acarol (2007) 43:265–278 269 was removed with a KimWipe (Kimberely-Clark Professional, Mississuga, Ontario, Canada) and replaced with a drop of 1% Neurobiotin (Vector Laboratories, Inc., Burlin- game, CA, USA) in 0.1 M KCl. The posterior end of the scutum was then pierced with a Wne tungsten needle and a silver wire electrode (7.62 mm diameter; A-M Systems, Inc., Carlsborg, WA, USA) was inserted. A second silver wire electrode was inserted in contact with the damaged leg in the pool of neurobiotin. The pool of neurobiotin was sealed with Vaseline (Unilever, Greenwich, CT, USA), and the silver wires were connected to a Grass S88 Stimulator (Grass Medical Instruments, Quincy, MA, USA). Electric pulses (1 V and 700 ms duration) were passed through the circuit at a rate of 2 pps for 2 h to facilitate neu- robiotin backWll of the neurons. After the backWll was concluded, ticks were processed by the same methods as used for 5-HT immunocytochemistry. The only exception was the addition of Avidin-conjugated AlexaFluor 488 (1:25, Molecular Probe, Carlsbad, CA, USA) for the detection of neurobiotin. After being incubated overnight at room temp, tis- sues were washed in PBST, PBS, and cleared with 40%, 60%, and 80% glycerol. Finally, the synganglia were mounted on glass slides in 80% glycerol.

Image processing and evaluation

Digital images were made using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc., Oberkochen, Germany). Image contrast was adjusted using Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) and line drawings were produced in CorelDraw (Corel Corporation, Ontario, Canada). The image stacks captured on the Zeiss LSM were used to count the number of 5HT-IR neurons in the synganglia of these two tick species.

Statistics

Data were analyzed by ANOVA and paired t-tests (means class/lsd) using SAS statistical software (SAS 1998) proc GLM. The classes were species and sex and the response was number of neurons. Five specimens of each sex for each species were analyzed by counting the number of 5HT-IR neurons in the synganglion. The number of 5HT-IR neurons was determined by analyzing stacks of images a slice at a time, with the step intervals of 0.2–0.5
m. We tested to see if there was a signiWcant diVerence in the number of 5HT-IR neurons between species, between sexes, and between sexes within each species. The null hypothesis was that there was no signiWcant diVerence between the mean number of 5HT-IR neurons in each of these comparisons. The null hypothesis was rejected if P <
(
=0.05).

Results

5HT-IR neurons

The synganglion of unfed male and female D. albipictus and A. americanum adults con- tained many 5HT-IR neurons and neuronal processes (Fig. 1a–c). There was a signiWcant V di erence in the number of 5HT-IR neurons when comparing species (tcrit =2.12, P = 0.0016). There was no signiWcant diVerence in the number of 5HT-IR neurons between sexes when species was not taken into consideration (tcrit = 2.998, P =0.09). There was a signiWcant diVerence in the number of 5HT-IR neurons when comparing female D. albipic- tus (mean § SD: 51.3 § 1.3, n = 5; Fig. 2a) to male D. albipictus (56.6 § 4.7, n = 5; W V Fig. 2b) (F1,8 =5.73, P = 0.044). There was no signi cant di erence in the number of 1 C 270 Exp Appl Acarol (2007) 43:265–278

Fig. 1 A plastic embedded horizontal section of the synganglion of a female A. americanum labeled with neu- robiotin (a). This specimen shows the cheliceral (C), opisthosomal (Op), and pedal (I–IV) neuromeres (dashed lines). The commissures and connectives (CC) and esophagus (Es). General illustrations, which are a compos- ite of male and female, of the serotonin-like immunoreactive (5HT-IR) cells and processes in the synganglion of (b) D. albipictus, (c) A. americanum drawn from the dorsal perspective. These drawings were prepared after viewing many stacks of slices of whole mounts of synganglion which were 5HT-IR labeled and imaged on a Zeiss LSM 510 laser scanning confocal microscope. The esophagus appears twice in the drawings and is la- beled as a marker for where the other regions are with respect to the esophagus. The esophagus (Es) enters the synganglion postero-dorsally and exits the synganglion antero-ventrally. I–IV are Wrst to fourth pedal neuro- mere; C, cheliceral neuromeres; Es, esophagus; Op, opisthosomal neuromere; OL, olfactory lobe; P, protoce- rebrum; SB, stomadeal bridge; *, posterior dorsal neuromeres; empty arrow, ventro-medial neurosecretory tract; Wlled arrow, lateral neurosecretory tract; empty arrowhead, 5HT-IR neurons at base of pedal nerves; Wlled arrowhead, posterior 5HT-IR neurons. Solid lines, regions of the synganglion which contain an abun- dance of 5HT-IR neuronal processes; dashed lines, 5HT-IR neuronal processes projecting from neurons to neuromeres or between neuromeres spheres, 5HT-IR neurons; dashed Wlled spheres, 5HT-IR neurons in the ventral aspect, which would lie below the regions illustrated above these 5HT-IR neurons. Scale bar = 100
m

5HT-IR neurons when comparing female A. americanum (43.2 § 6.3, n = 5; Fig. 2c) to male A. americanum (46.6 § 8.4, n = 5; Fig. 2d) (F1,8 =0.64, P = 0.449). We observed a spatial diVerence in the distribution of peripheral 5HT-IR neurons when comparing species. In both male and female D. albipictus, 5HT-IR neurons were present near the base of the second, third and fourth pedal nerves (Fig. 1b, open arrowheads) as opposed to A. america- num synganglia, in which these cell bodies were not present. A pair of 5HT-IR neurons was 1 C Exp Appl Acarol (2007) 43:265–278 271

Fig. 2 Laser scanning confocal images of tick synganglia showing 5HT-IR in ixodid tick synganglia. Images are z-projections from the ventral perspective, which are a composite of a series of Z-slices through the syn- ganglion. Ventral view of the synganglion of D. albipictus (a) female, (b) male; A. americanum (c) female, (d) male. Arrow, 5HT-IR neuronal processes in the stomadeal bridge; empty arrow, cheliceral neuromeres; Wlled arrowheads, 5HT-IR pedal neurosecretory cells; *, olfactory lobes. Scale bar = 100
m present in the posterior region of the synganglion of D. albipictus and A. americanum (Fig. 1b, c; Wlled arrowheads).

Supraesophageal ganglion

The supraesophageal ganglion was divided into many neuromeres, which were diYcult to distinguish using the immunocytochemical technique alone. Based on previous research studying paraYn sections (Pound and Oliver 1982), the supraesophageal ganglion was divided into the antero-dorsal, postero-dorsal, and ventral glomeruli; cheliceral, palpal, pro- tocerebral, and ventral neuromeres; and stomadeal bridge. The supraesophageal ganglion had the greatest concentration of 5HT-IR neurons in both species examined (Fig. 3a–d). These 5HT-IR neurons projected neuronal processes into the multiple neuromeres of the

1 C 272 Exp Appl Acarol (2007) 43:265–278

Fig. 3 Laser scanning confocal images which provide detail of the 5HT-IR neurons and neuronal processes (regions which are white) in the supraesophageal ganglion of the tick synganglion. Images are z-projections from the dorsal perspective, which are a composite of a series of Z-slices through the synganglion. Dorsal view of the supraesophageal ganglion of a D. albipictus (a) female, (b) male; A. americanum (c) female, (d) male. Arrow, post-oral commissure containing 5HT-IR neuronal processes; arrowhead, posterior-dorsal glomeruli; empty arrowhead, cheliceral neuromeres; *, anterior-dorsal glomeruli. Scale bar = 50
m supraesophageal ganglion including the cheliceral and protocerebral neuromeres (Fig. 3a– d) and also ventrally to the olfactory lobes (Figs. 4a and 5b; *). Dorsally, the antero-dorsal and postero-dorsal glomeruli exhibited extensive serotonin-like immunoreactivity (Fig. 3a– d). Serotonin-like immunoreactivity was present in many of the commissures and connec- tives present between the bilaterally paired neuromeres in the dorsal aspect of the supra- esophageal ganglion including the pre-oral commissure which communicated between the 5HT-IR cheliceral neuromeres postero-dorsally (Fig. 3a–d; arrow). Ventrally, the 5HT-IR cheliceral neuromeres were joined by the 5HT-IR stomadeal bridge (Fig. 4a, b; arrow).

Subesophageal ganglion

The pattern of 5HT-IR neurons and neuronal processes in the subesophageal ganglion region was similar in both species studied. The olfactory lobes, opisthosomal, and pedal neuromeres were located in the subesophageal ganglion. All of these regions contained 1 C Exp Appl Acarol (2007) 43:265–278 273

Fig. 4 Laser scanning confocal images of tick synganglia whole mounts showing 5HT-IR highlighting sub- esophageal regions. Images are Z-projections from the ventral perspective. Subesophageal regions of the syn- ganglia which include the stomadeal bridge and pedal neuromeres. (a) Stomadeal bridge in a male A. americanum; arrow, 5HT-IR neuronal processes in the stomadeal bridge; Es, esophagus; *, olfactory lobes. (b) Stomadeal bridge in a female D. albipictus; arrow, 5HT-IR neuronal processes in the stomadeal bridge; Es, esophagus; arrowheads, 5HT-IR neurons which send 5HT-IR neuronal projections to the Wrst pedal neu- romeres. (c) A female D. albipictus synganglion containing 5HT-IR neurons (arrowheads) which send 5HT- IR neuronal projections (empty arrowhead) to the pedal neuromeres (I–III); *, olfactory lobes. Scale bars = 50
m

5HT-IR neuronal processes. Ventrally, a cluster of three 5HT-IR neurons was present pos- terior to the esophageal entrance in the ventro-medial region of the synganglion and there were two pairs of 5HT-IR neurons present lateral to the esophagus (Figs. 1b, c and 5a). The pedal neuromeres (Figs. 1b, c and 4c) and associated commissures and connectives were also 5HT-IR (Fig. 1b, c). Posteriorly, the opisthosomal neuromeres contained many 5HT-IR neuronal processes (Fig. 1b, c).

Pedal 5HT-IR neuron

In both species studied, we found four sets of 5HT-IR neurons which projected neuronal processes to the pedal neuromeres (Figs. 1b, c, 2a–d and 4c). In D. albipictus, the pedal 5HT-IR neurons that innervated the Wrst, third and fourth pedal neuromeres were paired. Thus we observed a minimum of fourteen 5HT-IR neurons innervating the pedal 1 C 274 Exp Appl Acarol (2007) 43:265–278

Fig. 5 Laser scanning confocal images of tick synganglia showing 5HT-IR (magenta) and a Neurobiotin backWll (green) from the Haller’s organ in the Wrst leg. These images are z-projections which are a composite of a series of z-slices through the olfactory lobe region of the synganglion. (a) The olfactory lobes of an A. americanum containing glomeruli (green) that were backWlled from the Haller’s organ with neurobiotin tracer surrounded by 5HT-IR neuronal processes (magenta). (b) The olfactory lobe of female A. americanum back- Wlled with neurobiotin from the Haller’s organ. The glomeruli (green) are closely associated with 5HT-IR (magenta) neuronal processes. The matching unWlled olfactory lobe (which would receive inputs from the Haller’s organ in the paired Wrst leg) is outlined with a dashed white line neuromeres (Fig. 1b, c). In contrast, all four sets of 5HT-IR neurons associated with the pedal neuromeres were paired in A. americanum, resulting in a total of sixteen 5HT-IR neu- rons. The pedal 5HT-IR neurons directly projected neuronal processes to the lateral neuro- secretory tract which in turn connected with the pedal neuromeres (Figs. 1b, c and 2a).

Olfactory lobes

The glomeruli of the olfactory lobes were located in the ventral aspect of the synganglion, posterior to the esophageal entry (Fig. 1b, c). The neurobiotin backWll from the Haller’s organ of A. americanum revealed that these glomeruli were the terminal arborizations of sensory neurons arising from the sensillae located within the Haller’s organ. A net-like arrangement of 5HT-IR neuronal processes enmeshes the glomeruli in the olfactory lobes (Figs. 4a and 5a, b). There was also a commissure between the olfactory lobes which labeled in the neurobiotin backWlls (Fig. 5a; arrow). The pattern of serotonin-like immuno- reactivity in the olfactory lobes was similar in both sexes of the two species studied (Figs. 1b, c and 2a, c, d). The olfactory lobes were occupied by the 5HT-IR neurons associ- ated with the Wrst pedal ganglion as well as 5HT-IR neurons in the supraesophageal gan- glion. The 5HT-IR ventro-lateral neurosecretory tracts arise from the posterior aspect of the olfactory lobes (Fig. 4a; arrowhead).

Discussion

In this study, we found that 5HT-IR neurons and neuronal processes were present in the large part of the synganglion of two ixodid tick species: D. albipictus and A. americanum. The distribution of 5HT-IR neurons and neuronal processes was similar to that observed in 1 C Exp Appl Acarol (2007) 43:265–278 275 spiders (Seyfarth et al. 1990a, b). The primary diVerence between ticks and spiders was in the number of 5HT-IR neurons associated with the speciWc neuromeres. In the subesopha- geal ganglion of Cupiennius salei Keys. (Chelicerata: Araneae), Seyfarth et al. (1990b) found Wve 5HT-IR neurons associated with each pedipalpal neuromere. This contrasts with up to two 5HT-IR neurons associated with each pedal neuromere in the ixodid ticks we examined. Seyfarth et al. (1990b) also found 5HT-IR neuronal processes near the central, longitudinal tracts in the subesophageal ganglion. This is similar to the abundance of sero- tonin-like immunoreactivity in the ventromedial and lateral neurosecretory tracts of the ixodid ticks we examined. Seyfarth et al. (1990b) also identiWed three 5HT-IR neurons associated with each opisthosomal neuromere. Seyfarth et al. (1990b) also noted that there were no 5HT-IR nerves exiting the subesophageal ganglion, which was similar to our observation that there were no 5HT-IR nerves exiting the subesophgeal ganglion in ixodid ticks. In the supraesophageal ganglion, the “brain”, of C. salei, 5HT-IR neurons were iden- tiWed in the cheliceral neuromeres and the “central body” (Seyfarth et al. 1990b). Similarly, in ixodid ticks, we identiWed dense ramiWcations of 5HT-IR neuronal processes in the supraesophageal ganglion. In particular, we identiWed abundant 5HT-IR in the cheliceral neuromeres. Serotonin-like immunoreactivity was also identiWed in the cheliceral nerves of C. salei (Seyfarth et al. 1990b). Overall, the pattern of serotonin-like immunoreactivity in the CNS of ticks and spiders was very similar. Very little is known about the distribution of speciWc neurotransmitters in the tick CNS. The distribution of neurosecretory centers in the synganglion of both ixodid (IoVe 1964; Dhanda 1967; Binnington and Tatchell 1973; Chow and Wang 1974; Obenchain 1974a, b; Obenchain and Oliver 1975; Binnington 1983; Prullage et al. 1992; Szlendak and Oliver 1992) and argasid (Gabe 1955; Eichenberger 1970; Eisen et al. 1973; Roshdy et al. 1973; Gabbay and Warburg 1977; Pound and Oliver 1982) ticks has been described using light microscopy and transmission electron microscopy. The number of neurosecretory cell centers ranged from 11 in Ornithodoros moubata (Murray) (Eichenberger 1970) to 24 in Ixodes scapularis Say (Szlendak and Oliver 1992). There were 14 neurosecretory cell cen- ters in the synganglion of A. americanum (Prullage et al. 1992). The paraldehyde-fuschin labeling technique (Meola 1970) has been used to identify neurosecretory cell centers. This method identiWes general neurosecretory activity in the synganglion but does not provide information on the type of neurotransmitter produced in the neurosecretory cell centers. Overall, the one-host tick (D. albipictus) had a signiWcantly greater density of 5HT-IR neurons. In both species examined, the supraesophageal ganglion had the highest concen- tration of 5HT-IR neurons, a result which was in agreement with previous studies of tick neuroanatomy which reported concentrations of NSC in the supraesophageal ganglion (Gabe 1955; Eisen et al. 1973). Serotonin-like immunoreactivity was absent in the nerves exiting the synganglion suggesting that serotonin may function locally within the syngan- glion rather than as a neurotransmitter at the neuromuscular junction. The distribution of 5HT-IR was signiWcantly diVerent between species of ticks, particu- larly with respect to the number of 5HT-IR neurons which project their processes into the pedal neuromeres. The location of the 5HT-IR neurons associated with the pedal neuro- meres is conserved across species. Furthermore, 5HT-IR neuronal processes were abundant in the pedal neuromeres of both species examined. The one-host tick (D. albipictus) con- tained fewer pedal 5HT-IR neurons than the three-host tick (A. americanum). The life his- tory of three-host ticks requires that they drop oV the host at the conclusion of engorgement of each life stage (larval, nymphal and adult) and require additional mobility to locate the next vertebrate host. Thus, this observation suggests that serotonin may have a role in regu- lating mobility, much as it does in insects (Harrewijn and Kayser 1997; Dacks et al. 2003). 1 C 276 Exp Appl Acarol (2007) 43:265–278

The olfactory lobe structure we observed in tick synganglia through neurobiotin backWll and 5-HT immuncytochemistry was similar to that described in the olfactory lobes of the predatory mite, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) (van Wijk et al. 2006) and the antennal lobe of insects (Dacks et al. 2006). The olfactory lobe contains the terminals of sensory neurons in the Haller’s organ which is in the Wrst leg (Steullet and Guerin 1994). In insects, serotonin has been found to modulate the response to chemical signals received by the antennal lobes. Furthermore, diurnal releases of serotonin from 5HT-IR neurons present in the antennal lobes of Manduca sexta L. (Lepidoptera: Sphingi- dae) increase excitability of both local interneurons and projection (output) neurons (Kloppenburg and Hildebrand 1995; Mercer et al. 1995, 1996; Kloppenburg et al. 1999). The extensive amount of 5HT-IR neuronal processes enmeshing the olfactory lobes of ixo- did ticks we examined, which contained the terminals of the sensory neurons from the Hal- ler’s organ (the olfactory organ of ticks), suggested that serotonin may also modulate the olfactory response in ticks. Future research will examine the roles of serotonin in modulat- ing tick behaviors, including the olfactory response of ticks to pheromones and other chemosensory cues.

Acknowledgements We thank K. M. Collins, J. B. Welch, Z. Syed and two anonymous reviewers for critically reviewing this manuscript. We thank N. Davis for generously training N. A. H. in immunocyto- chemical methods; the RCMI Advanced Imaging Core Facility at University of Texas, San Antonio for use of the Zeiss LSM 510; and J. M. Pound for thoughtful discussion. We also thank D. Burchers for rearing ticks and technical support. N. A. H. is supported by a USDA, ARS, Postdoctoral Research Associate Program award to A. Y. L.

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