0{; Reprinted from the JOURNAL OF PARASITOLOGY Vol. 60, No.4, August 1974 p. 687-698 Made in United States of America Copyright @ 1974 by the American Society of Parasitologists FINE STRUCTUREOF THE CENTRALNERVOUS SYSTEMOF DERMACENTORVARIABILIS (SAY),AMBLYOMMAAMERICANUM (L.), AND ARGAS ARBOREUS KAISER,HOOGSTRAAL, AND KOHLS(IXODOIDEA)* Lewis B. Coons,t Mohamed A. Roshdy,:I: and Richard C. Axtell§ ABSTRACT: The central nervous system of unfed male Dermacentor variabilis (Say) and female Am- blyomma americanum (L.) (Ixodidae), and female Argas (Persicargas) arboreus Kaiser, Hoogstraal, and Kohls (Argasidae) was studied by light microscopy using epon sections stained with Azure II and PAS, and by transmission electron microscopy. The fused central nervous system is enclosed within a periganglionic blood sinus and penetrated by the esophagus. This system consists of an outer neural lamella containing layers of homogeneous finely granular material and periodically cross-banded collage- nous fibrils, a cortex of perineurium, glial cells, at least 3 neuronal types, and an inner neuropile of nerve fibers (axons and dendrites) partially ensheathed by glial processes. Large intracellular spaces occur in perineural glial cells in A. arboreus, but heavy glycogen deposits are observed in perineurium of D. vari- abilis and A. americanum. The fine structure of the nervous tissue and of a peripheral nerve is com- pared to that of insects and mites as a basis for investigating structural changes in ticks infected by vi- ruses and rickettsias. The general anatomy and histology of the lular organization and fiber tract intercon- tick central nervous system have been studied nections in the supraesophageal and subesoph- by Robinson and Davidson (1913), Douglas ageal portions of the nerve mass of several ( 1943), Sonenshine (1970), and Eichenberger ixodid and argasid ticks. Neurosecretory cells (1970). Ioffe (1963), Tsvilleneva (1965), in nervous tissues were demonstrated by Gabe and Eichenberger (loc. cit.) described the cel- (1955), Ioffe (1964, 1965), Dhanda (1967), and Eichenberger (loc. cit.). Received for publication 14 December 1973. However, the fine structure of the central * From Research Projects MR041.09.01-0037 A 6HJ nervous system is less known in acarines than and MF51.524.009-301OB F61, Bureau of Med- in insects. Recently, Eichenberger (loc. cit.) icine and Surgery, Department of Navy, Wash- ington, D. C. The opinions and assertions con- and Chow et aI. (1972) gave brief electron tained herein are the private ones of the authors microscope descriptions of some structures in and are not to be construed as official or as re- the nervous tissue of Ornithodoros moubata flecting the views of the Department of Navy or (Murray) and Rhipicephalus sanguineus Latre- of the naval service at large. This work was also ille, respectively, and Coons and Axtell (1971) supported in part by a special grant from the studied the cellular organization in the syn- Office of Naval Research and by Agreement 03-005-01 between the NIAID ( NIH ) and ganglion of the mite Macrocheles muscae- NAMRU-3. domesticae (Scopoli). t Electron Microscope Center, Department of Ento- In studies to increase knowledge of tick mology, Mississippi State University, Drawer internal organs at the electron microscopic EM, Mississippi State, Mississippi 39762. Pub- level, we described the fine structure of Der- lication No. 2747. Mississippi Agricultural and Forestry Experiment Station. macentor variabilis (Say) salivary glands :I:Department of Zoology, Faculty of Science, Ain (Coons and Roshdy, 1973) and of a neuro- Shams University, and Consultant, Medical hemal organ in Argas (Persicargas) arboreus Zoology Department, U. S. Naval Medical Re- Kaiser, Hoogstraal, and Kohls (Roshdy et aI., search Unit Number Three (NAMRU-3), U. S. 1973). The present paper describes the fine Interests Section, c/o Spanish Embassy, Cairo, Arab Republic of Egypt. structure of the central nervous system of Der- § Department of Entomology, North Carolina State macentor variabilis (Say) and Amblyomma University, Raleigh, North Carolina 27607, americanum L. (Ixodidae) and of A. (P.) where this microscopy was conducted with sup- arboreus (Argasidae) and provides basis for port from the Office of Naval Research, Naval Biology Program Contract NOOO14-70-A-0120- further investigations of this organ in pathogen- 001 (R. C. Axtell, principal investigator). infected ticks. 687 688 THE JOURNAL OF PARASITOLOGY,VOL. 60, NO.4, AUGUST 1974 COONS, ROSHDY,AND AXTELL-FINE STRUCTUREOF THE CNS OF VARIOUS TICKS 689 MATERIALS AND METHODS the cortical region of each tick species cor- Ticks investigated were unfed male D. variabilis respond to the motor or association-motor and virgin female A. americanum collected from neurons, neurosecretory neurons, and globuli nature near Raleigh, North Carolina, and unfed cells, respectively, described by previous au- virgin female A. (P.) arboreus from a laboratory thors. The globuli cells with characteristic, colony originating from the type locality of this species near Cairo and maintained in the NAMRU-3 intensely staining nuclei, are packed in a paired Zoology Laboratories. mass lying in the first pedal ganglia (Fig. 2). For electron microscopy, materials were fixed, Glial cell bodies and processes are often ob- dehydrated, embedded, and sectioned as previously served between the neurons and in the neuro- described (Coons and Roshdy, 1973). Epon-em- pile. bedded sections, 0.5 I-' thick, were stained with Azure II as a general survey stain and with Transmissioneledron microscopy(Figs. 5-22) periodic acid-Schiff ( PAS) reagent to demonstrate carbohydrates. Neural lamella: The ultrastructure of the neural lamella is the same in each species RESULTS examined (Figs. 5-8). This external con- Lightmicroscopy(Figs. 1-4) nective tissue sheath (2 to 5 f-tthick) consists The central nervous system of each species of repeated layers of homogeneous, finely consists of a fused ganglionic mass with periph- granular, disorganized material. The outer- eral nerves extending to various body organs. most layer is thicker in the two ixodid than in This mass is traversed by the esophagus and the argasid species. The areas between these thus divided into supraesophageal and sub- layers are occupied by a feltwork of col- esophageal portions which fuse laterally. lagenous fibrils showing periodic cross-banding As seen in Azure II-stained epon sections (Fig. 6) and embedded in an amorphous (Figs. 2-4), the nervous tissue is surrounded matrix. No organelles were observed in the by an extracellular neural lamella and consists neural lamella. of two zones. The outer zone, or cortex, is Axons containing neurosecretory vesicles comprised of perineurium, glial cells, and neu- were frequently observed in the neural lamella ronal cell bodies. The inner zone, or neuropile, (Figs. 5, 7, 8). Examination of serial sections consists of nerve fibers (axons and dendrites) showed these axons ending near the neural and glial cells and contains the esophageal lamella surface with no evidence of axonal canal. The nervous mass is located in a thin- extensions into the periganglionic sinus. walled periganglionic blood sinus connected Perineurium: Beneath the neural lamella to the heart by the aorta (Roshdy et aI., 1973). lies a complex series of glial cell layers forming In D. variabilis and A. americanum, the the perineurium (Figs. 8-10). Cells bordering perineurium is adjacent to the neural lamella the neural lamella have more irregular mem- and some cells contain heavy deposits of PAS- branes enclosing narrow intercellular (or extra- positive material, possibly glycogen (Fig. I). cellular) spaces, axons containing neurosecre- However, ill A. (P.) arboreus, the perineural tory vesicles, and tracheal elements. Junctional cells form large intracellular spaces or "vacu- specializations were not observed between ad- oles" demarcated by a network of cell mem- jacent cell membranes. In both D. variabilis branes (Fig. 2). Few glycogen deposits were and A. americanum, perineaural cells con- observed in the cell cytoplasm. taining heavy glycogen deposits (identified by Three major types of neurons (N 1-3) in characteristic rosette appearance) form an ap- ~ FIGURES1-4. Photomicrographs of cross sections of the tick central nervous system. 1. Dermacentor variabilis showing heavy deposits of glycogen (Gl) in the perineural glial cells (Pn) and processes ( PAS reaction). 2. Argas (Persicargas) arboreus at the level of first pedal nerve (PN,) showing types 1,2, and 3 neurons (N 1- N3) and intracellular spaces (IS) in the perineurium (Pn) (Azure II stain). 3. Am- blyomma americanum showing compact neurons (N) in the cortex and absence of intracellular spaces in the perineurium (Pn) (Azure II stain). 4. Argas (Persicargas) arboreus showing type 1 (N,) and type 2 (N2) neurons and glial cell bodies (GC) in a ganglion. Es, esophagus; Np, neuropile; NL, neu- rallamella; PgS, periganglionic blood sinus; W, wall of blood sinus (Azure II stain). 690 THE JOURNAL OF PARASITOLOGY,VOL. 60, NO.4, AUGUST 1974 .-- COONS, ROSHDY, AND AXTELL-FINE STRUCTUREOF THE CNS OF VARIOUS TICKS 691 parently continuous layer surrounding neuronal similar to those described for the mite Macro- cell bodies in the cortex. However, in A. (P.) cheles muscaedomesticae (Coons and Axtell, arboreus, glial cells in the perineurium form 1971), were observed in the nervous tissues an extensive system of tortuous extensions of both ixodids and the argasid tick. containing large