Synganglial Morphology and Neurosecretory Centers of Adult Amblyomma americanum (L.) (Acari: Ixodidae) JOSEPH B. PRULLAGE,1 J. MATHEWS POUND, 1 AND SHIRLEE. M. MEOLA2

J. Med. Entomol. 29(6): 1023-1034 (1992)

ABSTRACT Lone star ticks, Amblyomma americanum (L.), were processed by standard histological means for paraffin embedding, sectioning, and staining by the paraldehydefuchsin technique. The synganglion is highly condensed around the esophagus and possesses paired optic, cheliceral, palpal, pedal I—IV nerves, and opisthosomal nerves and a single unpaired esophageal nerve. Although optic nerves were observed leading from the eyes to the protocerebrum, distinct optic ganglia were not seen in any of the preparations examined. The paraldehyde-fuchsin technique revealed 14 neurosecretory centers (11 paired, 3 unpaired) within the synganglion, which are described in relation to the underlying neuropilar structure. A previously undescribed internal subesophageal center that consists of a single cell was observed within a cluster of perikarya lying posteriorly adjacent to the esophagus. A three-dimensional reconstruction of the internal neuropilar structure of the synganglion was made, and the included neurosecretory centers were mapped. Comparisons are made to previous work on other ticks, and physiological relationships are considered. KEY WORDS Ixodidae, lone star tick, synganglion

SEVERAL MORPHOLOGICAL HOMOLOGIES have been established on the gross level among the central neural ganglial structures of acarines, crustaceans, and insects. Within the insects, and to a lesser extent the crustaceans, much has been published on the structure and function of the neuroendocrine system, and rapid progress is being made at the cellular and biochemical levels relative to neurohormonal synthesis and regulation (Holman et al. 1990). In contrast, relatively little is known about the neuroendocrine systems of acarines. The neurosecretory centers of several species of argasid (Gabe 1955, Pound & Oliver 1982, Shanbaky et al. 1990) and ixodid ticks (Ioffe 1964, Binnington & Tatchell 1973, Chow & Wang 1974, Obenchain & Oliver 1975, Marzouk et al. 1987b) have been described, and attempts have been made to relate their function to physiological events (Ioffe 1965; Eichenberger 1970; Marzouk et al. 1987a,b). Throughout these studies, certain common features of the synganglia as well as differences at the genus level of the Ixodida have emerged. With extensive research being conducted on isolation and characterization of neurosecretory This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA. 1 Knipling-Bushland U.S. Livestock Insects Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Southern Plains Area, 2700 Fredericksburg Road, Kerrville, TX 78028-9184. 2 Food Animal Protection Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Southern Plains Area, Route 5, P.O. Box 810, College Station, TX 778459594.

products of many insects and elucidation of their physiological roles with a view toward novel control strategies (Menn & Borkovec 1989, Penzlin 1989, Holman et al. 1990), it is becoming more important to have a detailed understanding of the tick neurosecretory system. If functional homologies and biochemical similarities can be established, they may be used as indicators of how broadly the discoveries made on similar insect systems may be generalized among the Ixodida and subsequently assist in their evaluation or modification for use in tick control. The present work was done to develop a map of the neurosecretory system as a first step toward studies to elucidate the functions of the neurosecretory centers of the lone star tick, Amblyomma americanum (L.). Materials and Methods Ticks used in this study were selected from a laboratory strain maintained at the KniplingBushland U.S. Livestock Insects Research Laboratory at Kerrville, TX. The ticks were fourthgeneration offspring of a strain that originated from adult ticks collected near Fredericksburg, TX. These ticks were maintained in environmental chambers with a photoperiod of 12:12 (L:D) h, 85% RH, and 21°C (Winston & Bates 1960, Pound et al. 1989). Adult male and female ticks were allowed to feed and mate in an orthopedic stockinette sleeve secured with contact cement to a stanchioned Hereford steer. Ticks in the process of feeding were removed from the steer during the

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period of rapid engorgement (11—13 d after infestation), but before repletion, and dissected. Feeding ticks were chosen because of the number and increased activity of paraldehydefuchsin positive neurosecretory centers at this stage as seen in preliminary work and in other Ixodid species (Dhanda 1967, Panfilova 1980, Marzouk et al. 1987a). Ticks were mounted dorsum up in paraffin and covered with physiological saline (1.00 g KC1, 0.135 g CaCl2, 0.120 g NaHCO 3 , 7.500 g NaCL in 1.0 liter of distilled water) (Morgan & LaBrecque 1964). The dorsal cuticle was removed, then the majority of the gut was removed with watchmaker's forceps, being careful not to damage the synganglion. The ticks were then fixed in aqueous Bouin's fixative at room temperature and under vacuum for a minimum of 48 h, but not longer than 4 d. The ticks were prepared and stained by the paraldehydefuchsin staining method of Meola (1970) or the azan staining method of Hubschman (1962). Paraldehyde-fuchsin is a broad-spectrum, nonspecific neurosecretory stain that has been used previously to locate and describe neurosecretory regions in many arthropod species. The paraldehyde-fuchsin was prepared with basic fuchsin dyes from Allied Chemical (Morristown, NJ) (C.I. 42500) and from Fisher Scientific (Pittsburgh, PA) (C.I. 42500). The sections were analyzed using Java Video Analysis Software (Jandel Scientific, Corte Madera, CA). Density measurements of individual cells were taken in absolute gray scale increments (black, 0; white, 255) from an image recorded by a Microimage Video Systems video camera, model I HR, (World Video Sales, Boyertown, PA) mounted on an AO Spencer, Series 10 Microstar, compound microscope at a magnification of 600x and captured by a PCVISIONplus Frame Grabber video digitizer board (Imaging Technology, Woburn, MA) in a Dell 310 computer (Dell Computer, Austin, TX). Cell dimensions were measured on the captured image with the software calibrated to convert the dimensions to micrometers. Nuclear diameter was measured as the longest straight-line distance across the nucleus. Cell length was measured as the longest straight-line distance across the cell. Cell width was measured as the longest straight-line distance at a right angle to the line measuring cell length. Synganglial measurements were made using the same system but with the software calibrated in micrometers at a magnification of 343x. Synganglial length was measured as the longest distance along the long axis of the synganglion; width was measured as the longest distance across the synganglion in the transverse plane. Synganglial height was measured in sagittal sections as the longest straight-line distance at a right angle to the long axis of the synganglion.

The means of the density measurements were analyzed by analysis of covariance (ANCOVA) to adjust for variation in staining intensity between ticks. Tukey's honestly significant difference (Tukey's HSD) test was then conducted on the adjusted means according to the methods of Kramer (1956). Differences between adjusted means of the density measurements of centers 7 and 9 for males versus females were tested by the Student's t test (Sokal & Rohlf 1969). Cellular dimensions were analyzed by analysis of variance (ANOVA), and Tukey's HSD test was conducted on the means. Statistical analyses, including analysis of covariance of density measurements for unequal sample sizes, ANOVA for unequal sample sizes, and Tukey's HSD for the unadjusted means were conducted using PC-SAS (SAS Institute 1987). Three-dimensional reconstructions were made from the captured section images using PC3D three-dimensional reconstruction software (Jandel Scientific, Corte Madera, CA). Outlines were traced on the captured image using a Numonics 2210 digitizing tablet and 16 button puck, and were imported into the PC3D software for reconstruction. Drawings of the three-dimensional structure of the synganglion were made from the reconstructions. Results Synganglial Morphology. The synganglion of A. americanum is located medially, just above the genital aperture, at the level of the coxae of the second pair of legs. In adult females it has an approximate length of 424 ± 4 1 /urn, width of 467 ± 41/Ltm, and height of 255 ± 34 /urn. In adult males the approximate length is 338 ± 26 /im, width is 343 ± 26 /urn, and height is 232 ± 19 /im. Externally, there is no evidence of segmentation in the synganglion because of the high degree of condensation of the ganglia that compose it. The synganglion surrounds the ascending esophagus just ventral to the proventriculus (Fig. 1A). The long axis of the synganglion is oriented at an acute angle with respect to the longitudinal axis of the tick, with the anterior of the synganglion being somewhat elevated in relation to the posterior. The two major subdivisions of the synganglion are the supraesophageal and subesophageal ganglia. These are further subdivided into various ganglia, related to gnathosomal, pedal and idiosomal structures of the tick, include various glomeruli, and lobes. In many ways, the external morphology of the tick is reflected in the morphology of the synganglion. The entire synganglion is enclosed within a periganglionic sinus membrane, which is continuous with the dorsal aorta, and extends anteriorly as the periesophageal sinus membrane that encloses the esophagus, and adjacent esophageal nerve, and palpal, optic, and cheliceral nerves.

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Fig 1. Synganglial morphology of A. americanum. (A) Midsagittal section through synganglion of a female (anterior left), ae, ascending esophagus; de, descending esophagus; ol, optic lobe; pv, proventriculus; roc, retrocerebral organ complex; vv, vestibular vagina; 6, neurosecretory center 6; 7, neurosecretory center 7; 10 neurosecretory center 10. (B) Horizontal section of anterior left portion of synganglion (anterior up). Also, anterior lateral segmental organ; nl, neurilemma; p, perineurium; pgl, pedal ganglion I. (Scale lines = 50 fim).

The periganglionic sinus membrane also encloses the bases of the pedal and opisthosomal nerves. The neurilemma is a uniform acellular layer that forms the outer layer of the synganglion and the peripheral nerves and measures 1.5-3.5 fim in thickness (Fig. 1A). Immediately beneath this layer is the perineurium, a layer consisting of glial cells that measures 2—6 fim in thickness and stains best with the azan procedure (Fig. IB). Immediately beneath the perineurium is the cellular cortex or rind consisting primarily of the perikarya of the neurons making up the bulk of the synganglion. The cortex varies in thickness and includes trachea, tracheoles, and glial processes. Separating the cortex from the core of nerve fibers (neuropile) is the subperineurium (Fig. 2D), which contains some glial cells but also more frequently contains trachea and tracheoles. The subperineurium forms processes that extend into the cortex much more frequently

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i Fig. 2. Individual structures of synganglion of A. americanum. (A) Horizontal section of opisthosomal nerve (on) bifurcation inside neurilemma (nl) (anterior up). (B) Horizontal section of posterior lateral segmental organ (plso) showing relation to synganglion and pedal nerve III (pn3) (anterior up). (C) Sagittal section of neurosecretory center 7 (7) (detail of Fig. 1A) (anterior left), ae, ascending esophagus. (D) Sagittal section of neurosecretory center 9 (9) showing relationship to neurilemma (nl) (anterior left), sp, subperineurium. (scale lines = 25 fim).

than do those of the perineurium. The subperineurium varies considerably in thickness ranging from < 1 fim to 7 fim in thickness. The neuropile consists almost entirely of neural processes, the axons, and dendrites. The neuropile consists of the bilaterally symmetrical lobes, ganglia, glomeruli, and the system of commissures and connectives (Fig. 3). It is the arrangement of the internal structure of the neuropile that facilitates morphological division of the synganglion into its various ganglia. Supraesophageal Ganglion. The supraesophageal ganglion is composed of those elements of the synganglion lying anterodorsal to the esophagus. Therefore, the supraesophageal ganglion consists of the protocerebrum, the stomodeal bridge, and paired cheliceral ganglia. Paired optic nerves entering the region are evidence that the synganglion contains optic regions, but no optic lobes could be discerned in any of the ticks

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esophegeel nerve cheKcerai nerve palpal nen pedal I nerve chelioeral ganglion palpal ganglion anterodorsai gtomerufi cortex

pedal I ganglion posterior gtomeruK

pedal H nerve—£f— pedal II ganglion i0trooerebral organ complex pedal III nerve proventriculus

pedal III ganglion oommieaurea & oonnectrves

pedal IV nerve

padal IV ganglion optathoaomat ganglion

opisthosomal nerves

A. ophagua cheficeral nerve

palpal nerve

pedal I nerve chellceral ganglion palpal ganglion stomodeal bridge padal I ganglion globuh oeHs olfactory lobe pedal II nerve padal II ganglion oortex pedal III nerve

padal III ganglion commissure* & connectives padal IV ganglion

pedal IV nerve opbtnoeomal ganglion

opisthoeomal nerves

B. Fig. 3. Views of synganglion of A. americanum with neurosecretory regions indicated. Neurosecretory center 7 is internal and, therefore, not shown. Redrawn from 3-dimensional reconstruction (anterior up). (A) Dorsal view. (B) Ventral view. Neurosecretory center 7 is internal and therefore not shown.

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Fig. 4. Morphological structures of synganglion of A. americanum. (A) Anterodorsal glomeruli (agl) and posterior glomeruli (pgl) shown in horizontal section (anterior up), ae, ascending esophagus. (B) Esophageal nerve (en) shown in sagittal section displaced laterally from esophagus (anterior left), sb, stomodeal bridge. (C) Horizontal section of synganglion of female (anterior up), ae, ascending esophagus; cc, commissures and connectives; on, opisthosomal nerves; pgl—pg4, pedal ganglia I-IV; vgl, ventral glomeruli; 6, remnant of single cell of neurosecretory center 6. (scale lines = 50 /im).

examined. The nerves enter the protocerebrum but cannot be traced to a specific region within, or associated with, this ganglion. The route of the optic nerves from the protocerebral regions to the eyes follows the path of the cheliceral nerves as far as the pharynx and the pharyngeal dilator muscles, then proceeds laterally in a slight anterior and dorsal arc to the eyes. The protocerebrum is a single ganglion located dorsomedially at the anterior third of the neuropile. Within this ganglion are commissures and connectives; these are not part of the system of commissures and connectives of the subesophageal ganglion, but they perform the same task in the supraesophageal ganglion and other nerve tracts and glomeruli. The glomeruli of the protocerebrum are bilaterally symmetrical regions of dense neuropile and consist of the anterodorsal glomeruli, the posterior glomeruli, and the ventral glomeruli.

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The anterodorsal glomeruli are three pairs of nearly cylindrical neuropilar structures that protrude slightly from the anterodorsal surface of the protocerebrum (Figs. 3A and 4A). The posterior glomeruli are a single pair of globular neuropilar structures that protrude less from the dorsal surface of the protocerebrum than the anterodorsal glomeruli and are located anteriorly adjacent to the esophageal entry into the neuropile and at the lateral boundaries of the esophagus (Fig. 4A). The ventral glomeruli are also a single pair of pyriform neuropilar structures with their longitudinal axes oriented in a dorsoventral plane. Each ventral glomerulus consists of three dense cylindrical substructures. The anterior pair of substructures within each glomerulus is oriented dorsoventrally, and the single smaller posterior structures are oriented in a longitudinal plane with respect to the synganglion. The ventral glomeruli are located near the ventral surface of the protocerebrum immediately anterior and laterad of the esophagus (Fig. 4C). The cheliceral ganglia lie medial to the palpal ganglia, are slightly more dorsal, and are in the same plane as the ventral surface of the protocerebrum and adjacent to its lateral surface (Fig. 3). Posteriorly, the cheliceral ganglia are joined by commissures that are predominantly supraesophageal, but with a few fibers passing subesophageally. The cheliceral nerves proceed medially upon exiting the neurilemma and converge medially before proceeding anteriorly to the chelicerae. The stomodeal bridge lies posterior to the bases of the cheliceral ganglia, ventral to the protocerebrum, and is anteriorly adjacent to the esophagus. Laterally, the margins of the stomodeal bridge join the palpal ganglia. The single esophageal nerve arises from the stomodeal bridge at its posterior margin, proceeding ventrally, and exits the neurilemma anterior and adjacent to the esophagus. It lies adjacent to the dorsal surface of the esophagus proceeding to the pharynx where it branches into numerous small nerves (Fig. 4B). The recurrent nerves as described by Obenchain (1974) and Binnington & Tatchell (1973) were not apparent in any of the preparations observed. Subesophageal Ganglion. The subesophageal ganglion is composed of all ganglial elements that lie posteroventral to the esophagus. Although the bulk of the paired palpal ganglia lies anterior to the esophagus, the palpal ganglia are considered to be a component of the subesophageal ganglion because the commissures connecting their bases lie posteroventral to the esophagus. Therefore, the subesophageal ganglion is composed of the palpal ganglia, pedal ganglia I—IV, opisthosomal ganglion, and the system of commissures and connectives. The palpal ganglia lie lateral and somewhat ventral to the cheliceral ganglia. The commis-

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sures connecting the bases of these ganglia lie posteriorly adjacent to the esophagus. The palpal nerves exit the neurilemma at the level of the ventral surface of the cheliceral ganglia mediodorsal to the pedal I nerves. The nerves proceed anteriorly in an arc toward the midline and branch posteriorly to the descending esophagus. One branch proceeds to the palps within the periesophageal sinus; the other immediately exits the sinus and proceeds laterally to the area of the salivary glands. The pedal ganglia are arranged in bilaterally symmetric pairs along the ventrolateral margins of the subesophageal ganglion (Figs. 3 and 4C). Pedal I ganglia lie along the lateral surface of the supraesophageal ganglion and the palpal ganglia, and are oriented at an angle of —45° with the origin of the pedal I nerve oriented anteriorly. The pedal I ganglia are larger than the other pedal ganglia, probably because of the presence of nerve fibers from the Haller's organ (a sensory structure located on tarsi I). The pedal II and III ganglia are oriented somewhat anterior and posterior, respectively, to a line perpendicular to the longitudinal axis of the synganglion. Pedal IV ganglia are oriented at a somewhat more oblique posterior angle. The pedal II nerves exit the synganglion and proceed laterally to the bases of leg II. Pedal III and IV nerves proceed to their respective leg bases at progressively more oblique angles to the pedal II nerve. Paired olfactory lobes protrude from the ventral surface of the neuropile ventral to the pedal I ganglia and the commissures connecting pedal I and II ganglia. Medially, the olfactory lobes are adjacent at their largest diameters. The lobes are subspherical and contain numerous dense glomeruli (Figs. 1A and 3B). Lateral to the olfactory lobes and ventral to the pedal I ganglia are the globuli cells, paired subovate clusters of perikarya within the cortex of the subesophageal ganglion (Fig. 3B). Each cluster is composed of numerous small perikarya of ~8 fim in diameter, with darkly staining nuclei of approximately =3.5-4.5 jum in diameter. Nerve tracts proceed medially from the globuli cells and join the lateral borders of the olfactory lobes. The paired opisthosomal ganglia appear as a single band of nerve fibers at the posterior margin of the neuropile without a distinct commissure connecting the two ganglia. The opisthosomal nerves arise from the posterolateral margins of the ganglia at a single root, and divide immediately before reaching the perineurium (Figs. 2A and 4C). The anterior opisthosomal nerves exit the neurilemma dorsomedially to the pedal IV ganglia and the posterior opisthosomal nerves exit the neurilemma at the posterior margin of the synganglion. The opisthosomal nerves branch upon leaving the periganglionic sinus membrane.

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The system of commissures and connectives is located ventromedially within the subesophageal ganglion medial to the pedal ganglia and is bounded posteriorly by the opisthosomal ganglia (Figs. 3 and 4C). It is composed of numerous nerve tracts arranged in five layers. The commissures connect the bilaterally paired ganglia and the connectives provide the interganglial communication, not only between nonsymmetric ganglial pairs, but also between the supraesophageal and the subesophageal ganglia. Peripheral Nervous Structures. The retrocerebral organ complex lies ventral and adjacent to the proventriculus at the point where the ascending esophagus exits the dorsal periganglionic sinus membrane (Figs. 1A and 3A). The complex is formed by an outpocketing of the outer layers of the esophagus which contains three or four large cells. The cells are spherical and have a chromophobic cytoplasm with a centrally located nucleus. Surrounding these large cells are numerous small cells with a chromophilic cytoplasm. The lateral segmental organs are paired structures that lie near the outer surface of the periganglionic sinus membrane, between pedal nerves I and II (Fig. IB) and between pedal nerves II and III (Fig. 2B). The lateral segmental organs are connected to pedal nerves I and II and pedal nerves II and III byfinehemal nerves. The posterior pair are the largest and are spherical in shape containing 4^5 large subovate cells. The anterior pair are much smaller and range from subspherical to cylindrical in shape and contain 3-4 subovate cells. The cells are bounded by a thin basal lamina. The axons of the hemal nerves do not appear to penetrate the basal lamina of the lateral segmental organs. The cytoplasm of the cells stains green by the pareldyhyde-fuchsin procedure, and the nucleus stains a dark red-brown. No paraldehyde-fuchsin positive staining was noted in any of the lateral segmental organs examined for this study. Neurosecretory Centers. Fourteen paraldehyde-fuchsin positive neurosecretory centers were identified in the synganglia examined (Fig. 3; Tables 1 and 2). The neurosecretory granules within the cells usually stained bluish-purple to a dark purple, but occasionally the neurosecretory granules in neurosecretory center 6 stained reddish-purple. The number of cells, dimensions, and staining intensity within each neurosecretory center varied between individuals. However, a repeatable pattern could be discerned among the neurosecretory centers within and between the sexes, with the exception of centers 7 and 9, which differed in mean density (Tables 1 and 2). The dimensions of the cells of the neurosecretory centers tended to be smaller in the males, as did the overall dimensions of the synganglion. The centers are associated with underlying regions of the neuropile; therefore, lo-

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Table 1. Locations, densities, and dimensions of neurosecretory centers in the synganglion of adult female americanum

Diagram number 1 2 3 4

5 6 7 8 9 10 11 12 13

14

LjOCcition

Anterodorsal protocerebral Anterolateral protocerebral Midlateral protocerebral Posterolateral protocerebral Anterolateral cheliceral Anteromedial stomodeal Internal subesophageal Dorsal Posterodorsal commissure and connective Ventrolateral esophageal Pedal I Pedal II Pedal III Pedal IV

Mean density" Mean cell (urn) (gray scalec) (±SEMr 109 (3.3)abc 108 (3.4)abc 117(3.5)ab 104 (4.2)abc 100 (3.3)bc 111 (1.8)abc 115(7.0)ab 121 (5.3)a 122 (4.0)a 110(4.2)abc 93 (3.4)c 117(3.2)ab 118(3.3)ab 115(3.3)ab

15.8 (1.4)b 18.7(1.4)b 20.5(1.4)b 18.5(1.7)b 19.3 (1.4)b 32.1(0.7)a 16.4 (2.9)b 22.0 (2.2)b 34.8 (1.7)a 15.1 (1.7)b 20.7 (1.4)b 15.3(1.3)b 15.0(1.4)b 15.5(1.4)b

Mean cell (urn) (±SEM)fe

Mean cell ( p ) (±SEM)fc

10.7 (0.8)de 13.1 (0.8)cde 14.7 (0.9)bcd 10.8(l.l)de 11.7(0.8)de 18.2 (0.5)b 9.7 (1.7)e 17.0(1.3)bc 23.8 (1.0)a 9.5 (1.0)e 9.9 (0.9)e 9.1 (0.8)e 9.1(0.8)e 8.9 (0.8)e

6.7 (0.3)bc 7.0 (0.3)b 7.9 (0.3)ab 6.7 (0.4)bc 6.4 (0.3)bc 7.9 (0.2)ab 5.2 (0.7)c 7.9 (0.5)ab 9.6 (0.4)a 6.7 (0.4)bc 6.5 (0.3)bc 6.7 (0.3)bc 6.5 (0.3)bc 6.5 (0.3)bc

" Mean density, least squares mean density adjusted by covariate analysis to standardize, between tick, staining intensity. b Within each column, means having the same letter are not significantly different (P < 0.05; Tukey's honestly significant difference). c Gray scale: black, 0; white, 255.

cations of individual centers will be described in relation to the nearest neuropilar structure. The dorsal surface of the protocerebrum has a single pair of neurosecretory centers associated with it. The anterodorsal protocerebral centers (1) (Fig. 3A; Tables 1 and 2) are paired centers consisting of single cells located anterior and dorsal to the anterodorsal glomeruli. The cells stain a medium purple and contain a fine granular neurosecretion that is evenly distributed throughout the cytoplasm and extends into the axon hillocks of the cells. The cells are subspherical and have centrally located nuclei. The lateral surfaces of the protocerebrum have three paired neurosecretory centers associated with them. The anterolateral protocerebral centers (2) (Fig. 3A; Tables 1 and 2) are paired centers consisting of single cells located dorsally on the lateral surface of the protocerebrum and dorA.

sal to the cheliceral ganglia. The cells stain a medium purple and contain finely granular neurosecretion that is evenly distributed throughout the cytoplasm of the cells. The cells are ovate to pyriform in shape with acentric nuclei. The midlateral protocerebral centers (3) (Fig. 3A; Tables 1 and 2) are paired centers consisting of single cells located approximately at the midlateral surface of the protocerebrum. The cells stain light purple and contain a fine-to-medium granular neurosecretion that occurs in diffuse clumps throughout the cytoplasm of the cells. The cells are subspherical tending toward ovate in shape and have acentric nuclei. The posterolateral protocerebral centers (4) (Fig. 3A; Tables 1 and 2) are paired centers consisting of single cells located toward the posterolateral surface of the protocerebrum. The cells stain dark purple and contain a coarsely granular neurosecretion that

Table 2. Locations, densities, and dimensions of neurosecretory centers in the synganglion of adult male americanum

Diagram number 1 2 3 4 5 6 7 8 9 10 11

12 13 14

Location

Mean density" (gray scalec)

Mean cell ( p ) (±SEM)'J

Mean cell (urn)

Mean cell ( p )

Anterodorsal protocerebral Anterolateral protocerebral Midlateral protocerebral Posterolateral protocerebral Anterolateral cheliceral Anteromedial stomodeal Internal subesophageal Dorsal subesophageal Posterodorsal commissure and connective Ventrolateral esophageal Pedal I Pedal II Pedal III Pedal IV

96 (2.7)de 102(3.1)cde 99 (2.7)cde 110(3.7)bcd 91 (2.8)e 113(1.9)bc 141 (4.9)a 119(4.1)b 99 (3.9)cde 113(2.9)bc 94 (2.7)e 100 (2.7)cde 114 (2.7)bc 114 (3.5)bc

13.2 (l.O)cd 12.7(1.2)cd 14.2 (l.O)bcd 13.0 (1.4)cd 15.3(l.l)bcd 19.7 (0.7)b 14.7(1.8)bcd 18.0(1.5)bc 31.5(1.5)a 13.8(l.l)cd 13.2(1.0)cd 12.0(1.0)d 11.5(1.0)d 12.7(1.3)cd

9.4 (0.6)bcd 7.3 (0.7)d 9.0 (0.6)cd 8.4 (0.8)cd 9.5 (0.7)bcd 13.0 (0.4)b 10.0(l.l)bcd 11.8 (l.O)bc 19.5 (0.9)a 8.9 (0.7)cd 8.2 (0.6)d 8.7 (0.6)cd 8.3 (0.6)cd 8.7 (0.8)cd

6.6 (0.2)b 5.7 (0.3)b 6.6 (0.2)b 6.1 (0.3)b 6.4 (0.2)b 6.7 (0.2)b 5.7 (0.4)b 6.3 (0.4)b 8.3 (0.3)a 6.7 (0.2)b 6.6 (0.2)b 6.1 (0.2)b 6.3 (0.2)b 5.8 (0.3)b

° Mean density, least squares mean density adjusted by covariate analysis to standardize, between tick, staining intensity. Within each column, means having the same letter are not significantly different (P < 0.05; Tukey's honestly significant difference). c Gray scale: black, 0; white, 255. b

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occurs in discrete clumps in single bands around the nuclei. The cells are subspherical and have centrally located nuclei. The anterior surface of the protocerebrum has two paired neurosecretory centers associated with it. The anterolateral cheliceral centers (5) (Fig. 3A; Tables 1 and 2) are paired centers consisting of single cells located anterior and adjacent to the ventral glomeruli and medial to the cheliceral ganglia. The cells stain dark purple and contain medium-sized granular neurosecretion that is evenly distributed throughout the cytoplasm of the cells. The cells are roughly pyriform tending toward subspherical in shape with acentric nuclei. The anteromedial stomodeal centers (6) (Figs. 1A, 3A, and 4C; Tables 1 and 2) consist of paired multicellular regions. The regions consist of 3—4 cells each, with the medial pair of cells laterally adjacent to each other. This center is located at the level of the stomodeal bridge and at the anterior margin of the cortex dorsal to the entrance of the esophagus. The cells stain medium purple to reddish-purple and contain medium-sized granular neurosecretion that is evenly distributed throughout the cytoplasm of the cells and into the axon hillocks. The cells are the second largest (32.1 by 18.2 /urn in females and 19.7 by 13.0 /im in males) of the neurosecretory centers and are pyriform with the narrow part of the cells oriented posteriorly and containing the acentric nuclei with the axon hillocks at the apices. The axons of these cells are oriented toward the stomodeal bridge. There are two unpaired and one paired centers closely associated with the esophagus. The internal subesophageal center (7) (Figs. 1A and 2C; Tables 1 and 2) consists of a single medial cell. It is situated within a small cluster of perikarya that are located internally and are not contiguous with the cortex of the synganglion. This center is located posterior and adjacent to the esophagus and anterior to the system of commissures and connectives at a level even with the ventral surface of the ventral glomeruli and dorsad of the olfactory lobes. The cell stains a significantly darker purple in the female (115 gray scale units) than in the male (141 gray scale units) (Student's t test: t = 3.217, P < 0.05) and contains coarse granular neurosecretion that is evenly distributed throughout the cytoplasm and extends into the axon hillock. The cell is subspherical with an acentric nucleus. The axon appears to be oriented toward the esophagus but could not be traced to its destination. The dorsal subesophageal center (8) (Fig. 3A; Tables 1 and 2) consists of a single medium-to-large cell (22.0 by 17.0 /Mm in females and 18.0 by 11.8 /xm in males). It is located posterior to the esophagus and ventrally adjacent to the perineurium at the thin layer of cortex found in this area. The cell stains moderate to light and contains a fine granular neurosecretion that is evenly distributed

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throughout the cytoplasm. The cell is subspherical in shape with a centrally located nucleus. The ventrolateral esophageal centers (10) (Figs. 1A and 3B; Tables 1 and 2) are paired centers consisting of single cells located just laterally adjacent to the esophagus at its entrance into the synganglion. The cells stain deeply-tomoderately purple and contain a fine granular neurosecretion that is evenly distributed throughout the cytoplasm. The cells are weakly pyriform to subspherical in shape, with acentric nuclei located toward the anterior portion of the cells. There is a single center associated with the system of commissures and connectives. The posterodorsal commissure and connective center (9) (Figs. 2D and 3; Tables 1 and 2) consists of a single cell, which is the largest cell of all the neurosecretory centers (34.8 by 23.8 /un in females and 31.5 by 19.5 /un in males). It is located dorsad and at the posterior margin of the system of commissures and connectives internally adjacent to the perineurium. The cell stains mediumto-light purple and varies significantly in staining between males and females (122 gray scale units in females and 99 gray scale units in males) (Student's t test: t = 3.962, P < 0.05). The cell contains a flocculent neurosecretory material that appears to be randomly distributed throughout the cytoplasm of the cell. There are four centers associated with the pedal ganglia, one of which is also closely assor ciated with the globuli cells. The pedal I centers (11) (Fig. 3B; Tables 1 and 2) are paired centers consisting of single cells located ventral and adjacent to the pedal I ganglia and posteriorly adjacent to the globuli cells. The cells stain deeply (a dark purple) and are always among the most densely staining in an individual tick (93 gray scale units for females, 94 gray scale units for males). The cells contain a fine granular neurosecretion that is densely packed and evenly distributed throughout the cytoplasm and into the axons. The axons can be traced, because of the presence of dark-staining paraldehyde fuchsin positive granules, to where they join the system of commissures and connectives near the dorsal aspects of the olfactory lobes. The cells are subcylindrical with centrally located nuclei. The pedal II (12), III (13), and IV (14) centers (Fig. 3B) are similar to each other with no significant differences in dimensions, shape, or staining properties (Tables 1 and 2). Because of this similarity, they will be described together. Each center is paired and consists of single cells located ventral to their respective ganglia and lateral to the ventral layer of the system of commissures and connectives. The cells stain medium-to-light purple, and contain fine granular neurosecretion that is evenly distributed throughout the cytoplasm and into the axon hillocks. The axons are oriented medially toward

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the system of commissures and connectives, but the paths of axons from these centers could not be traced. The cells are subspherical to subcylindrical in shape with acentric nuclei. Discussion In many respects the morphology of the synganglion of A. americanum is essentially similar to previously published reports of other ixodid tick species (Ioffe 1963, Binnington & Tatchell 1973, Chow & Wang 1974, Obenchain 1974). Previous authors, with the exception of Chow & Wang (1974), have reported the presence of distinct optic lobes or paired optic ganglia in all other ixodid ticks examined. Binnington (1972) specifically addressed the question of the presence of eyes and optic nerves in several tick species with eyes and several species thought to lack eyes. In all of the species examined, he was able to find paired optic ganglia. Although optic nerves could be seen in several preparations examined for this study, they could not be traced to distinct lobes or glomerular structures within the protocerebrum. It was theorized by Pound & Oliver (1982) that the lack of development of optic lobes in several argasid species was related to the lesser development of the photoreceptor organs. In A. americanum the eyes are more highly developed than those of the argasids examined by Pound & Oliver (1982) but are still primitive (Phillis & Cromroy 1977). Further, Carroll & Pickens (1987) have found that Dermacentor variabilis (Say) and Hyalomma dromedarii Koch have unimodal wavelength sensitivity responses, indicating that ticks have only one visual photochemical receptor system. This state of primitive development of the eyes may indicate that distinct optic lobes may not have developed or were lost in A. americanum. Therefore, the interpretation of impulses from these photoreceptor cells may be accomplished over a more diffuse area than in optic lobes alone, or the interpretation of visual information that is performed does not require specialized areas of the synganglion. The evolutionary trend of the chelicerate central nervous system is the anterior placement and condensation of ganglia that in the more primitive condition existed in the abdominal nerve cord (Ioffe 1963, Eichenberger 1970). Accompanying this trend is the progressive movement of subesophageal elements anteriorly into the supraesophageal ganglion (Ioffe 1963). The tick synganglion epitomizes this trend and is a highly condensed organ composed of fused ganglia and an intricate system of commissures and connectives. It is the location of the commissures relative to the esophagus that is used to establish the association of the ganglia with the supra- versus subesophageal nerve mass. Previous reports are divided as to whether the palpal ganglia are as-

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sociated with the supra- or subesophageal nerve mass (Ioffe 1963, Tsvileneva 1964, Obenchain 1974). In A. americanum only subesophageal palpal commissures were detected. In horizontal sections, nerve fibers could be seen branching from the palpal ganglia and proceeding toward the anterior of the esophagus, but no evidence of a supraesophageal commissure was observed. Tsvileneva (1964) has confirmed supraesophageal commissures in Boophilus calcaratus (=B. annulatus [Say]), Hyalomma anatolicum Koch, and Hyalomma detritum Schulze, indicating that gradation exists in the anterior movement of the palpal ganglia among the ixodids. The opisthosomal ganglion of acarines is one of the most condensed of all chelicerates. It is the result of fusion of all the previously free opisthosomal ganglia of the primitive chelicerates (Ioffe 1963). Even within the Ixodoidea, there are apparent gradations in the degree of condensation of this ganglion. The least condensed ganglia reported to date occur in Ornithodoros kelleyi Cooley & Kohls (Sonenshine 1970) and Argas persicus (Oken) (Eisen et al. 1973). Both of these argasids have two bilaterally symmetrical pairs of opisthosomal ganglia with a single opisthosomal nerve arising from each ganglia. Within the Ixodidae, Dermacentor (Ioffe 1963, Obenchain 1974) and Hyalomma species (Tsvileneva 1964, Marzouk et al. 1987c) have bilaterally symmetrical opisthosomal ganglia that are very similar to A. americanum, except that the two opisthosomal nerves arise from separate roots. However, in Boophilus microplus (Canestrini) (Binnington & Tatchell 1973) Argas radiatus Railliet (Obenchain & Oliver 1976) and Ornithodoros parkeri Cooley (Pound & Oliver 1982), only a single pair of nerves arises from the opisthosomal ganglion. The opisthosomal nerves of these latter species branch a short distance outside the perineurium. The opisthosomal ganglion of A. americanum appears to be intermediate in its degree of condensation because the nerves arise from a single root but branch before reaching the perineurium (Fig 2A). The structure of the retrocerebral organ complex of A. americanum is similar to that described in D. variabilis (Obenchain 1974, Obenchain & Oliver 1975), H. dromedarii (Marzouk et al. 1985), several Ornithodoros species (Gabe 1955, Eichenberger 1970, Pound & Oliver 1982, 1984), and Argas arboreus Kaiser (Roshdy et al. 1973). In several of the preparations examined for this study, faint paraldehyde-fuchsin positive staining was observed in the smaller cells of this complex, but neurosecretory granules resembling those observed in neurosecretory centers in the synganglion were not seen. Positive paraldehyde-fuchsin staining is also observed outside the synganglion, and, in these cases, it does not always indicate the presence of neurosecretion. The absence of accumulation of neurosecre-

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tion in A. americanum indicates that the retrocerebral organ complex is not a homologue of the insect corpus cardiacum as previously suggested (Roshdy et al. 1973, Obenchain 1974, Obenchain & Oliver 1975). Other studies have shown that the structure and dynamics of the retrocerebral organ complex of O. parkeri and B. microplus suggest a hormonal-neurohormonal regulatory function, rather than a storage function (Binnington 1983, Pound & Oliver 1984). However, studies by Marzouk et al. (1985) have indicated that neurosecretion as indicated by paraldehydefuchsin and chrome-hematoxylin-phloxine staining does accumulate in the retrocerebral organ complex of partially fed virgin female H. dromedarii, the same stage examined in these studies. More specific work is necessary to ascribe a function to the retrocerebral organ complex. The structure of the lateral segmental organs of A. americanum is essentially that described for B. microplus (Binnington & Tatchell 1973, Binnington 1981) and the external morphology and location agrees with that reported for Amblyomma tuberculatum Marx (Obenchain & Oliver 1976). Other authors have described the lateral segmental organs of several species of ticks and much variation in morphology, chromophilia, and number of organs appears to exist. In D. variabilis the basic structure of the lateral segmental organs agrees with other descriptions except that four pairs of organs are reported (Obenchain 1974, Obenchain & Oliver 1975). In D. variabilis (Obenchain & Oliver 1975), H. dromedarii (Marzouk et al. 1985), and Ixodes persulcatus Schulze (Panfilova 1980) varying degrees of positive staining for neurosecretion were observed. These accumulations were seen to increase during feeding by Marzouk et al. (1985) and Panfilova (1980). However, in ultrastructural studies of B. microplus (Binnington 1981) and in the present study, no evidence of neurosecretion was found in the lateral segmental organs of feeding ticks. The results of this study indicate that the lateral segmental organs of A. americanum is not a homologue of the corpus cardiacum or other neurohemal organs of insects. Neurosecretory Centers. The neurosecretory centers appear quite similar to those previously reported for other ixodid and argasid ticks. Variations in the number of regions reported and their placement are common, but the general plan appears to be conservative. The largest number of neurosecretory centers (22) has been reported in H. dromedarii (Marzouk et al. 1987b) while 18 centers have been reported in Dermacentor pictus (=D. reticulatus [F.]) (Ioffe 1964), D. variabilis (Obenchain & Oliver 1974,1975), /. persulcatus (Panfilova 1980), and 7. parkeri (Pound & Oliver 1982); 16 centers have been reported in Ornithodoros tholozani (Laboulbene & Megnin) (Gabbay & Warburg 1977) and 15 in B. microplus (Binnington & Tatchell 1973) and

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Rhipicephalus sanguineus (Latreille) (Chow & Wang 1974). Differences in numbers of neurosecretory centers may be in part caused by interpretation. Neurosecretory center 7 has not been previously reported in any tick species. This neurosecretory center consists of a single unpaired cell posterior to the esophagus and dorsal to a line drawn across the top of the olfactory lobes (Figs. 1A and 2C). This area is contained within a small pocket of cell bodies and is not in direct contact with the cortex of the synganglion. Among the Acari only Dermanyssus gallinae (De Geer) has been shown to have neurosecretory centers located within the neuropile and adjacent to the esophagus (Severino etal. 1984). However, these neurosecretory centers are supraesophageal and are ventral to the protocerebrum in this mite. The synganglion of D. gallinae shows the same level of anterior movement of the ganglia; however, the esophagus passes through at a more horizontal angle. This difference in geometry may indicate that one or both of the esophageal centers of D. gallinae is a homologue of the area in A. americanum, but it is impossible to determine at this time. This center stained much more intensely in female than in male ticks (Tables 1 and 2) indicating that this center is much more active in either production or storage of neurosecretion in females than in males. This could indicate that this area is active in gametogenesis and especially in oogenesis. Neurosecretory material could be discerned in the axon of this cell, which extended to the esophagus, but the destination of the axon could not be determined, thus providing no further indications as to this center's function. Neurosecretory center 9 also showed an interesting reversal of neurosecretory center content between males and females. However, in this instance the staining was more intense in males than in females (Tables 1 and 2) indicating that this center contained relatively more neurosecretion in males. This could be the result of increased production or decreased release of neurosecretion in males or decreased production or increased release of neurosecretion in females. In other ixodid tick species, a general increase in neurosecretory material within all neurosecretory centers of feeding mated females has been noted (Dhanda 1967, Chow & Wang 1974, Panfilova 1980, Marzouk etal. 1987a). Center 9 is similar to area 17 of Chow & Wang (1974) and Panfilova (1980), who report that during engorgement in fed females the amount of neurosecretion in this area increases. These results favor the possibility of decreased release of neurosecretion in males or the increased release of neurosecretion in females. In other previously mentioned tick species, neurosecretory centers associated with the opisthosomal ganglia are common. However, in

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A. americanum these areas were, at best, indistinct. In preliminary observations of other physiological stages, distinctly staining centers associated with the opisthosomal ganglia are still lacking. Neurosecretory material was observed in the axon hillocks or axons of several of the neurosecretory centers (centers 1, 5, 6, 7, and 11-14) (Fig. 3). Additionally, centers 5, 7, and 11-14 are not in contact with the perineurium of the synganglion. This indicates that the neurosecretion within these cells is perhaps transported to another site where it may be stored and released or is released directly at the site of action. Obenchain & Oliver (1975) successfully traced the axonal pathways of several neurosecretory cells in D. variabilis. Determination of probable homologous neurosecretory areas between D. variabilis and A. americanum, based on cell descriptions and location, are possible because of the detail of the report. Of the centers where neurosecretory material was noted in the axons of A. americanum (1, 5, 6, and 11—14), the probable homologues in D. variabilis had pathways that could be traced at least into the neuropile. An area homologous to the corpus cardiacum of insects has not been determined in ticks, although Marzouk et al. (1985) believe the retrocerebral organ complex may serve this function. Even though neurosecretory material was not observed in axons of cells in other neurosecretory centers, this does not preclude the possibility that the material is transported to another site before release. However, the proximity of centers 8, 9, and 10 to the perineurium does provide some evidence that neurosecretory material released from these cells would have a direct path to the hemolymph bathing the synganglion (Fig. 2D). Also, the morphology of centers 8 and 9 implies the ability of these cells to store and secrete large amounts of material. Binnington (1983) demonstrated the presence of neurosecretory terminals in the neural lamella and perineurium of the synganglion and peripheral nerves in B. microplus. These terminals could provide direct access of these neurosecretory centers to the hemolymph in the periganglionic sinus that bathes the synganglion. Acknowledgment The authors thank Craig A. LeMeilleur for assistance with tick rearing, and Patricia A. Agold for critical review and revision of the manuscript.

References Cited Binnington, K. C. 1972. The distribution and morphology of probable photoreceptors in eight species of ticks (Ixodoidea). Z. Parasitenk. 40: 321-332. 1981. Ultrastructural evidence for the endocrine nature of the lateral organs of the cattle tick Boo-

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philus microplus. Tissue & Cell 13: 475-490. 1983. Ultrastructural identification of neurohaemal sites in a tick: Evidence that the dorsal complex may be a true endocrine gland. Tissue & Cell 15: 317-327. Binnington, K. C. & R. J. Tatchell. 1973. The nervous system and neurosecretory cells of Boophilus microplus (Acarina: Ixodidae). Z. Wiss. Zool. 185: 193-206. Carroll, J. F. & L. G. Pickens. 1987. Spectral sensitivity to light of two species of ticks (Acarina: Ixodidae). Ann. Entomol. Soc. Am. 80: 256-262. Chow, Y. S. & C. H. Wang. 1974. Neurosecretory cells and their ultrastructures of Rhipicephalus sanguineus (Latreille) (Acarina: Ixodidae). Acta Arachnol. 25: 53-67. Dhanda, V. 1967. Changes in neurosecretory activity at different stages in the adult Hyalomma dromedarii Koch, 1844. Nature 214: 508-509. Eichenberger, G. 1970. Das zentralnervensystem von Ornithodoros moubata (Murray), Ixodoidea: Argasidae, und seine postembryonal entwicklung. Acta Trop. (Basel) 27: 15-53, (English translation #419 NAMRU-3). Eisen, Y., M. R. Warburg & R. Galun. 1973. Neurosecretory activity as related to feeding and oogenesis in the fowl-tick Argas persicus (Oken). General and Comparative Endocrinology 21: 331-340. Gabbay, S. & M. R. Warburg. 1977. The diversity of neurosecretory cell types in the cave tick Ornithodoros tholozani. J. Morphol. 153: 371-386. Gabe, M. 1955. Histological data on neurosecretion in arachnids. Arch. Anat. Microsc. Morphol. Exp. 44: 351-353. Holman, G. M., R. J. Nachman & M. S. Wright. 1990. Insect neuropeptides. Ann. Rev. Entomol. 35: 201217. Hubschman, J. H. 1962. A simplified azan process well suited for crustacean tissue. Stain Technol. 37: 379^380. Ioffe, I. D. 1963. The structure of the brain of Dermacentor pictus Herm. (Chelicerata, Acarina). Zool. Zh. 42: 1472-1484. 1964. Distribution of neurosecretory cell in the central nervous system of Dermacentor pictus Herm. (Chelicerata: Acarina). Dokl. Akad. Nauk. SSSR, S. Evoluts. Morfol. 154: 229-232 (English translation #326 NAMRU-3). 1965. Seasonal changes in neurosecretion contents of neurosecretory cells of Dermacentor pictus Herm. ticks (Ixodoidea, Acarina). Med. Parazitol. Moskva. 34: 57-63, (English translation #1445 NAMRU-3). Kramer, C. Y. 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12: 307-310. Marzouk, A. S., F.S.A. Mohamed & G.M. Khalil. 1985. Neurohemal-endocrine organs in the camel tick, Hyalomma dromedarii (Acari: Ixodoidea: Ixodidae). J. Med. Entomol. 22: 385-391. Marzouk, A. S., M. K. Abdel Moez & Z.E.A. Darwish. 1987a. The effect of feeding and mating on the neurosecretory activity in female Hyalomma dromedarii synganglion (Acari: Ixodoidea: Ixodidae). J. Egypt. Soc. Parasitol. 17: 547-570. Marzouk, A. S., G. M. Khalil & Z.E.A. Darwish. 1987b. Neurosecretory cell types and distribution in unfed female Hyalomma dromedarii (Acari: Ixo-

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doidea: Ixodidae) synganglion. J. Egypt. Soc. Parasitol. 17: 41-61. Marzouk, A. S., G. M. Khalil, F.S.A. Mohamed & N. Farid. 1987c. Hyalomma dromedarii (Acari: Ixodoidea: Ixodidae): central and peripheral nervous system anatomy. Exp. Appl. Acarol. 3: 145-161. Menn, J. J. & A. B. Borkovec. 1989. Insect neuropeptides: Potential new insect control agents. J. Agric. Food Chem. 37: 271-278. Meola, S. M. 1970. Sensitive paraldehyde-fuchsin technique for neurosecretory system of mosquitoes. Trans. Am. Microsc. Soc. 89: 66-71. Morgan, P. B. & G. C. LaBrecque. 1964. Preparation of house fly chromosomes. Ann. Entomol. Soc. Am. 57: 794-795. Obenchain, F. D. 1974. Structure and anatomical relationships of the synganglion in the American dog tick Dermacentor variabilis (Acari: Ixodidae). J. Morphol. 142: 205-223. Obenchain, F. D. & J. H. Oliver, Jr. 1975. 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. J. Morphol. 145: 269-294. 1976. Peripheral nervous system of the ticks, Amblyomma tuberculatum Marx and Argas radiatus Railliet (Acari: Ixodoidea). J. Parasitol. 62: 811-817. Panfilova, I. M. 1980. Changes in the neuroendocrine system of female Ixodes persulcatus during the period of feeding. Zool. Zh. 59: 851-858 (English translation #1608 NAMRU-3). Penzlin, H. 1989. Neuropeptidesm—occurrence and functions in insects. Naturwissenschaften 76: 243-252. Phillis, W. A. & H. L. Cromroy. 1977. The microanatomy of the eye of Amblyomma americanum (Acari: Ixodidae) and resultant implications of its structure. J. Med. Entomol. 13: 685-698. Pound, J. M. & J. H. Oliver, Jr. 1982. Synganglial and neurosecretory morphology of female Ornithodoros parkeri (Cooley) (Acari: Argasidae). J.

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Morphol. 173: 159-177. 1984. Morphology of the retrocerebral organ complex in penultimate nymphal and adult female Ornithodoros parkeri (Cooley) (Acari: Argasidae), pp. 295-303. In D. A. Griffiths & C. E. Bowman [eds.] Acarology VI, Vol. I. Ellis Horwood, Chichester, England. Pound, J. M., J. E. George & J. A. Miller. 1989. A programmable environmental system for maintenance and observation of individually housed ticks. J. Parasitol. 75: 994-996. Roshdy, M. A., N. M. Shourkey & L. B. Coons. 1973. The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 17. A neurohemal organ in A. (P.) arboreus Kaiser, Hoogstraal, and Kohls. J. Parasitol. 59: 540544. SAS Institute. 1987. SAS/STAT Guide for Personal Computers, Version 6 Edition. SAS Institute, Cary, NC. Severino, G., J. H. Oliver, Jr. & J. M. Pound. 1984. Synganglial and neurosecretory morphology of the chicken mite Dermanyssus gallinae (De Geer) (Mesostigmata: Dermanyssidae). J. Morphol. 181: 49-68. Shanbaky, N. M., A. El-Said & N. Helmy. 1990. Changes in neurosecretory cell activity of female Argas (Argas) hermanni (Acari: Argasidae). J. Med. Entomol. 27: 975-981. Sokal, R. R. & F. J. Rohlf. 1969. Biometry. Freeman, San Francisco. Sonenshine, D. E. 1970. A contribution to the internal anatomy and histology of the bat tick Ornithodoros kelleyi Cooley and Kohls, 1941. II. The reproductive, muscular, respiratory, excretory, and nervous systems. J. Med. Entomol. 7: 289-312. Tsvileneva, V. A. 1964. The nervous structure of the ixodid ganglion. Zool. Jb. Anat. Bd. 81: 579-602. Winston, P. W. & D. H. Bates. 1960. Saturated solutions for the control of humidity in biological research. Ecology 41: 232-237. Received for publication 7 April 1992; accepted 14 July 1992.

Synganglial morphology and neurosecretory centers of adult Amblyomma americanum (L.) (Acari: Ixodidae).

Lone star ticks, Amblyomma americanum (L.), were processed by standard histological means for paraffin embedding, sectioning, and staining by the para...
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