Physiology of Ticks: Current Themes in Tropical Science

Science

PREFACE

THE earliest reference to ticks in Hoogstraal’s Bibliography of Ticks and Tick-borne Diseases is that of Homer from the year 850 B.C. In the reference, Homer mentioned the occurrence of ticks on Ulysses’ dog. An even earlier reference to tick fever is from an Egyptian papyrus scroll dated 1550 B.C. Early scholars were familiar with tick ectoparasites of a variety of domestic animals and described the damage caused by them. Indeed, Cato, Aristotle, and Pliny referred to ticks as disgusting parasitic animals which were very troublesome. Despite this early recognition of the damage to animals caused by ticks, an understanding of the magnitude of the problem was gained only at the end of the nineteenth century with the historic discovery of Smith and Kilbourne (1893) that Boophilus ticks transmit the Texas fever pathogen, Babesia bigemina. This was followed by a series of discoveries that ticks also transmit various other pathogenic filaria, protozoa, bacteria, rickettsiae, and viruses.

Ticks surpass all other arthropods in the number and variety of diseases which they transmit to domestic animals, and rank second to mosquitoes as vectors of human diseases. The unique number of disease agents which ticks transmit both to their hosts and, transovarially, to their offspring increases the potential health hazard manyfold since they are both the vectors and the reservoirs of these diseases. The annual global cost of controlling ticks and tick-borne diseases, together with the loss to mankind of significant amounts of animal protein due to cattle death or diminished productivity, is in the range of thousands of millions of dollars.

A variety of tick-control programmes have been incorporated into modern livestock management practice in order to counteract the adverse effects of ticks. These have mainly involved the use of chemical acaricides. Arsenic salts were the main control agents from the early 1900s until the thirties when Boophilus ticks developed resistance to arsenicals almost simultaneously in Africa, Australia, and South America. Organo-chlorine compounds replaced arsenicals during the years of 1945–55 and were, in turn, replaced by organo-phosphorous compounds and carbamates in the 1955–70 period; these have since been replaced by formamidines and related chemicals in some areas after 1970. The rapid turnover in control agents was due to the development of resistance in ticks, to increasingly unacceptable organo-chlorine residues in meat, milk, and their by-products, as well as to the intense competition for sales among acaricide producers. The environmental impact of acaricide residues, along with the genetic plasticity of ticks which leads to their rapid acquisition of pesticide resistance, has created an acute need for the development of alternative methods of control.

To date, alternative approaches to the control of livestock ticks, based on successful models for insect control utilizing sterile males, genetic manipulations, or pheromone attractants, offer little promise. Attractants may, however, have some potential for use on domestic pets or against ticks with specialized attachment sites. Furthermore, the most logical new approaches to the alleviation of tick depredations in the near future will probably be based on the exploitation or manipulation of natural host-tick vector-pathogen relationships, or of those special physiological and biochemical adaptations which allow ticks to digest and metabolize enormous volumes of host blood.

In 1977 an International Study Workshop on the Physiological Significance of Tick Behaviour was convened by the International Centre of Insect Physiology and Ecology (ICIPE) in Nairobi, under the chairmanship of Professor Andre Aeschlimann, of the University of Neuchatel. The participants spent a most fruitful week discussing some of the aspects of tick physiology which not only contribute to their evolutionary success but may make them vulnerable to control. One of the recommendations of that group was that a book, based on the papers presented at the workshop but which summarized the discoveries in the field of tick physiology over the past 20 years, bringing together work which has been published in many different journals and in a diversity of languages, would be valuable and timely. In order to identify appropriate topics and respective contributors for inclusion in a volume on the physiology of ticks, a number of workshop participants, Professor A. Aeschlimann (of the University of Neuchatel, Switzerland) Professor J. H. Oliver, Jr. (Georgia Southern College, USA), and Dr. D. H. Kemp (CSIRO, Australia), together with the eventual co-editors, Dr. F. D. Obenchain (of the ICIPE) and Professor R. Galun (of the Hebrew University, Jerusalem, Israel), were invited by the chief editor of the series on Current Themes in Tropical Science (Professor Thomas R. Odhiambo) to undertake this task. Rather than presenting physiological information in a more or less classical form, it was jointly concluded that the unique (and probably the most vulnerable) features of tick physiology and the physiological aspects of tick interactions with their hosts should be given a more intensive coverage. It is believed that this form of presentation will appeal to readers–both those with specialized as well as those with more general interest.

A more thorough appreciation of the entire field of tick biology may be obtained by using this volume in conjunction with the English translation of Balashov’s (1972) monograph on Blood-sucking Ticks (Ixodoidea)– Vectors of Disease to Man and Animals.

This book represents an international effort by twenty-five experts. Each contribution has been written as an up-to-date review of its subject, including literature up to 1980, and much unpublished data. Among other relevant topics, this volume includes detailed accounts of the mechanisms used by non-feeding ticks to maintain their water balance as well as the salivary mechanisms used by feeding ixodid ticks for excretion of the enormous excess volumes of water and salts taken in during blood sucking. Two chapters relate electrophysiological data to the sensory biology of host finding and to the control of feeding behaviour and its mechanics, while a third chapter documents the effects of the immune responses of the host on tick feeding, amechanism underlying the development of host resistance to tick infestation. We envisage that these discussions, together with the contributions of other chapters, will draw attention to future areas of productive research, and that the application of recently developed technologies will lead to new methods for the rational manipulation of tick populations.

ICIPE, Nairobi, June 1980

F.D. OBENCHAIN and R. GALUN

Editors

CHAPTER 1

The Tick Cuticle

R.H. HACKMAN and B.K. FILSHIE,     Division of Entomology, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia

Publisher Summary

This chapter describes the structure, syntheses, and composition of the tick cuticle. The success of ticks as terrestrial arthropods is related directly to the properties of their cuticles. Like other arthropods, ticks have an external skeleton or integument, which is skin, skeleton, and, if necessary, a food reserve. The cuticle is that part of the integument external that is secreted by the epidermis, which is itself supported on an internal basement membrane. The cuticle is not uniform over the entire animal but varies from hard sclerotized plates to soft extensible membranes. To allow movement, rigid plates are connected together by flexible membranes that act as hinges. There are some differences in structure between the cuticle of the alloscutum of the nymph and the female of B. microplus. In the female, maximum cuticle thickness is reached when the tick is about 4.5 mm long. Ticks, like insects, possess dermal glands throughout the epidermal layer. In insects, the dermal glands are supposed to produce the cement layer over the surface of the wax layer and this is correlated with their wide dispersal over the body surface.

CONTENTS

1.1 Introduction

1.2 Structure

1.2.1 Morphology of the Cuticle

1.2.1.1 Epicuticle

1.2.1.2 Procuticle, Exocuticle, Mesocuticle, Endocuticle, and Subcuticle

1.2.1.3 Pore Canals and Wax Canals

1.2.1.4 Muscle Attachments, Glands, and other Integumental Structures

1.2.2 Deposition of the Cuticle

1.2.2.1 Formation of the Epicuticle

1.2.2.2 Formation of the Procuticle

1.2.2.3 Modifications to Cuticle Structure during Feeding

1.3 Cuticular Components

1.3.1 Chitin

1.3.2 Protein

1.3.3 Lipids

1.3.4 Other Components

1.3.5 Sclerotization

1.3.6 Penetration of Acaracides

1.3.7 Water Exchange

1.4 Conclusions

References

1.1 INTRODUCTION

Initial descriptions of the tick cuticle, which date from around the beginning of this century, were based on histochemical studies using the light microscope, but in the last decade further progress has been made in our understanding of the structure, synthesis, and composition of the cuticle by use of newer techniques such as electron microscopy, chromatography, and electrophoresis. Like other arthropods, ticks have an external skeleton or integument which is skin, skeleton, and, if necessary, a food reserve. The cuticle is that part of the integument external to and secreted by the epidermis (the epidermal cell layer), which is itself supported on an internal basement membrane. In insects there is evidence which suggests that some cuticular proteins may not be synthesized by the epidermal cells but are transported across them from the haemolymph, having been synthesized elsewhere. However, tissue culture experiments have established that epidermal cells secrete cuticle. Cuticle is a heterogeneous, non-cellular membrane, which is not only the external covering but extends into the fore- and hind-guts and lines the ducts of dermal glands and the tracheal system.

The cuticle is not uniform over the entire animal but varies from hard sclerotized plates to soft extensible membranes. To allow movement, rigid plates are connected together by flexible (intersegmental) membranes, which act as hinges. Ixodid ticks are referred to as hard ticks because of their sclerotized (hardened) capitulum, scutum, appendages, and other small areas. On the other hand, argasid ticks are the leathery or soft ticks because sclerotized areas are relatively small. The cuticle, being an exteneral skeleton, determines the maximum size and the shape that a particular developmental stage can attain, and further growth is possible only by moulting, i.e. shedding the cuticle and replacing it by a larger one. Ixodid ticks undergo two moults in their progress from larvae to nymphs to adults. Argasid ticks undergo three or more moults. Some species have two nymphal instars, others up to six or more. Respiration in nymphal and adult ticks is by way of tracheae which terminate in the spiracular plates on the sides of the body. Larval ticks, except for those of some argasid species, do not appear to have spiracles, and it is assumed that gaseous exchange occurs through the intact cuticle.

The cuticle is divided into two layers, a thin outer epicuticle and a thicker inner procuticle. The epicuticle is complex and contains several layers, viz. lipid, polyphenol, and cuticulin; in argasid ticks a cement layer overlies the lipid layer. The procuticle is formed of protein and chitin. Its outer region may be sclerotized to form an exocuticle; in which case the remaining inner region is known as the endocuticle. In some cuticles the outer part of the endocuticle may show different staining properties from the inner part. The outer part is then referred to as mesocuticle. Mesocuticle is described as a stabilized but non-sclerotized region. Ticks, in common with other cheli-cerates, possess an endosternite or internal skeletal support for some prosomal muscles. Contrary to earlier claims, Cutler and Richards (1974) report that the endosternite does not contain chitin; its chemical composition is unknown. Water loss in ticks is almost exclusively through the cuticle (Lees, 1946), and the lipid layer of the epicuticle plays a major role in regulating the movement of water.

Setae or bristles are present on the cuticle and each is set in a socket on top of a canal leading from an epidermal cell. Pore canals extend through the procuticle from the epidermis up to the epicuticle. A number of sense organs and dermal glands open onto the surface of the cuticle. Some setae are thought to be temperature sensors and others tactile sensors. Figure 1.1 is a diagrammatic representation of the arthropod integument, Fig. 1.2 an electron micrograph of a transverse section of the integument from the dorsal surface of the alloscutum of the nymph of Boophilus microplus. For recent reviews on the arthropod cuticle, reference can be made to Hackman (1971, 1974a) and Neville (1975). References to the early work on tick cuticle are to be found in Richards (1951).

FlG. 1.1 A diagrammatic representation of the arthropod integument. (From Hackman, 1971.)

FIG. 1.2 Transverse section of the cuticle of the alloscutum of an engorged nymph. Inner endocuticle (inner) is distinctly lamellate. Outer endocuticle (outer) is not lamellate but bundles of microfibrils (arrows) tend to occur in the same orientation at regular spacings through the thickness of the outer endocuticle, with a period approximately the same as the lamellar spacing of the inner endocuticle. Epicuticle, epi; pore canals, pc; wax layer, w. Scale = 2.5μm.

1.2 STRUCTURE

Our present knowledge of tick cuticular structure comes from earlier studies with the light microscrope combined with a few recent descriptions of the fine structure from transmission and scanning electron microscope studies. By comparison with insect cuticle, published information on the fine structure of the tick integument is sparse indeed. These few studies indicate that many parallels may be drawn between insect and tick cuticles. However, several differences have been discovered, particularly in the structure and development of the cuticle of the alloscutum of female ixodid ticks. This cuticle has an enormous capacity for growth, expansion, and stretching during engorgement of blood in the adult instar. The following description of the structure of the cuticle combines a review of published information augmented by some new descriptions and micrographs of the cuticle of the adult female cattle tick, B. microplus.

1.2.1 Morphology of the Cuticle

In common with similar descriptions of insect cuticle, difficulties exist in correlating the interpretation of structures seen in the light microscope with those seen at higher resolution in the electron microscope. These difficulties result from the different methods of preparation and examination which are used with the two techniques and the different ways in which images are formed. For instance, different staining methods applied to paraffin sections have allowed the procuticle to be subdivided into a number of layers– exocuticle, mesocuticle, endocuticle, and subcuticle–but with electron stains these layers are difficult or even impossible to distinguish from one another. The wax and cement layers, both largely soluble in lipid solvents, are not preserved by the normal methods used to prepare cuticle for transmission electron microscopy. Because thin (50–100 nm) sections must be used for electron microscopy, difficulties exist in the interpretation of three-dimensional structures such as the shape of pore canals or the disposition of fibrous elements within the procuticle.

1.2.1.1 Epicuticle

In arthropods generally, light-microscope studies have demonstrated the presence of four distinct layers, viz. cement, wax, cuticulin, and polyphenol. The presence of these layers has been inferred from histo-chemical reactions. Individual layers are too thin to be resolved separately by light microscopy (Richards & Anderson, 1942; Wigglesworth, 1947; Richards, 1951). Polyphenols have been demonstrated by their ability to reduce ammoniacal silver nitrate. The cuticulin layer, which is supposed to be a tanned lipoprotein complex, is defined by its resistance to lipid solvents such as boiling chloroform, the lipid only being released after warming with potassium chlorate and nitric acid (Wigglesworth, 1947). Wax layers are seen only in transverse sections of frozen cuticle, using lipid stains such as osmium tetroxide, or those of the Sudan series (Richards, 1951). Cement, when present, forms the outermost layer, and is produced by dermal gland secretions poured out onto the cuticle surface through fine ducts shortly after moulting.

Light-microscope studies of ixodid and argasid ticks have demonstrated all four of these layers. The cement layer is present only in argasids (Lees, 1947; Balashov, 1968). Nothing is known of its structure or composition, but it is assumed that it has a similar function to the homologous layer of insects, namely the protection of the underlying wax layer. The cement layer is more resistant to lipid solvents than is wax but can be removed with boiling chloroform (Arthur, 1962).

The waterproofing properties of the cuticle lie in the surface wax layer, situated outside the cuticulin (Lees, 1947). Although some work has been carried out to characterize the lipids extracted from the cuticle (see §1.3.3), the location of these lipids is not known absolutely. It is usually assumed that they originate entirely from the surface wax layer. It is not known whether the layer deposited on the surface of the cuticulin is composed entirely of lipid or whether additional components are present. One would not normally expect to find, in sections prepared for electron microscopy, a layer of wax in this position, but a well-preserved layer is seen despite the application of lipid solvents during the embedding procedure (Nathanson, 1967). In B. microplus the layer is clearly seen in post-ecdysial cuticle of nymph and adult (Figs. 1.2 and 1.6). It is irregular in thickness, varying from 0.1 μ m up to 1 μ m or more, generally poorly stained but sometimes containing laminated electron-dense plates, arranged in small crystallites which are not oriented in any preferred direction (Fig. 1.6). Similar layers are absent in insects.

FIGS. 1.5 and 1.6 Transverse sections of the outer part of the cuticle of the alloscutum of a partially fed adult. The epicuticular layers consist of an outermost wax layer (w) which sometimes has a lamellated substructure (Fig. 1.6), the cuticulin (c) and outer epicuticle (oe) (detail seen in higher magnification insert to Fig. 1.6), the dense layer (d), and tubular network (tn) at interface of dense layer and outer endocuticle (outer). Arrows indicate junction of pore canals (pc) with wax canals (wc) which pass through the epicuticle as far as the outer epicuticle. Figure 1.5, scale = 10 μm; Fig. 1.6, scale = 25 μm (insert X5).

The cuticulin and polyphenol layers, as defined for insects by Wigglesworth (1947), have beeen demonstrated by Lees (1947) in several ixodid and argasid species. Balashov (1968) reported the presence of amber-coloured cuticulin layers in ixodids and argasids varying between 1 and 3 μm thick. However, this thickness is greater than that of the whole epicuticle, as determined by other authors. Following the early applications of electron microscopy to arthropod cuticle structure, Locke (1957, 1966) re-defined the term cuticulin to refer to the thin electron dense layer of the epicuticle of insects that is first laid down on the surface of the epidermis at moulting. Locke referred to the much thicker internal layer comprising the remainder of the epicuticle, as the protein epicuticle or the dense homogeneous layer. The latter term is the one currently in use.

Filshie (1970a, b) described an additional unstained layer on the outside of the cuticulin of insects, called the outer epicuticle. In the rabbit tick Haemaphysalis leporispalustris, Nathanson (1967) described four regions or layers. His regions II, III, and IV correspond in position to the outer epicuticle, cuticulin, and dense homogeneous layers of the insect epicuticle, respectively (Figs. 1.5 and 1.6. for B. microplus). Filshie (1976), in a detailed study of the structure of the epicuticle of the adult female of B. microplus, found the same layers, but the outer epicuticle and the dense layer both contained additional complexities not seen previously in other arthropod cuticles. The outer epicuticle, when first formed, contains regular striations with a period of about 5 nm. Traversing the dense layer are electron opaque filaments about 17 nm in diameter with a spatial density of about 800 per μn² (Figs. 1.7 and 1.9). The above results were obtained by staining sections with salts of uranium and lead. If, instead, sections are treated with 1–2% potassium permanganate followed by lead citrate, different elements of the dense layer of the epicuticle are revealed. In transverse sections of the dense layer, faint striations can be seen perpendicular to the cuticular surface (Fig. 1.8). When the dense layer is sectioned parallel with the cuticle surface (Fig. 1.10), it can be seen that the striations are caused by a close-packed array of electron lucent microfibrils sectioned transversely. Each microfibril is approximately 3 nm in diameter and neighbouring microfibrils are spaced about 6 nm apart. This is the first report of microfibrils in the epicuticle of any arthropod, but ones similar in size and oriented more or less parallel to the cuticular surface (Neville, 1975), are seen in the procuticle of insects (Fig. 1.4). It is usually assumed that the unstained microfibrils of arthropod procuticles are composed of chitin and that the dense matrix material is protein (Rudall, 1967). Apart from the above structural evidence there is none to suggest that chitin is present in the epicuticle of ticks. Although there have been isolated reports of chitin in the epicuticle of other arthropods (Krishnan et at., 1955) these have been shown to be incorrect (Kennaugh, 1959), the chitin-containing layer having been wrongly identified as part of the epicuticle. Nevertheless, it is interesting that the microfibrils of the epicuticle and the procuticle of B. microplus are of the same diameter.

FlG. 1.4 Higher magnification of portion of Fig. 1.3 showing individual, unstained microfibrils cut in cross-section (arrows) surrounded by electron-dense granular material. Scale = 100 μm.

FIGS. 1.7–1.10 The epicuticle of the alloscutum of an early post-moult adult, sectioned and stained in different ways to reveal different substructures. Figures 1.7 and 1.8 are transverse sections; Figs. 1.9 and 1.10 are sections parallel with the cuticle surface. Figure 1.7 and 1.9 are stained with uranyl acetate and lead citrate; Figs. 1.8 and 1.10 are stained with potassium permanganate and lead citrate. Faint vertical striations in the dense layer (d) of Fig. 1.7 are the dense filaments (df) seen in Fig. 1.8 (outlined area shown at high magnification in insert) are microfibrils seen in cross-ection in Fig. 1.10 (higher magnification in insert). Cuticulin, c; pore canals, pc; tubular network, tn; wax canals, wc. All figures to same magnification; scale = 25 μm (inserts X2.5).

A further structural feature, apparently peculiar to the tick epicuticle, is a tubular network at the interface between the dense layer and the procuticle (Filshie, 1976). Individual tubules are approximately 15 nm in diameter. They interconnect with one another and follow closely the irregular profile of the inner face of the dense layer (Figs. 1.5, 1.6, 1.7, and 1.10). In general, the features peculiar to the epicuticle of B. microplus described above–dense filaments, microfibrils, and the tubular network–are most easily distinguished in the cuticle of either the pharate adult or the early post-ecdysial adult female (compare Figs. 1.5, 1.6, 1.7, and 1.10). Apparently, chemical changes that take place after moulting render the epicuticle less reactive to electron stains, so that the separate components, particularly the microfibrillar structure, cannot be clearly distinguished. The epicuticle of nymphs of Hyalomma asiaticum has been studied by Amosova (1975). Its fine structure is essentially identical to that of B. microplus.

1.2.1.2 Procuticle, Exocuticle, Mesocuticle, Endocuticle, and Subcuticle

The terms procuticle, exocuticle, mesocuticle, and endocuticle all apply to the chitin-protein layers beneath the epicuticle of arthropods. Richards (1951) first defined the procuticle as the original, undifferentiated, non-sclerotized chitin-protein material secreted by the epidermis. As mentioned in the introduction to this chapter, the outer parts of the procuticle over certain regions of the exoskeleton may become sclerotized following ecdysis. This outer part is then known as the exocuticle and the inner soft cuticle secreted after ecdysis is known as the endocuticle. Frequently, the region between exocuticle and endocuticle stains differently and has been called the mesocuticle. The thin layer immediately outside the epidermis, presumably containing the unpolymerized precursors of the chitin-protein complex, also stains differently from the rest and was recognized by Schmidt (1956) as a separate layer, the subcuticle.

In previous literature on the tick cuticle, the terminology applied to these layers is very confused. Lees (1947) refers to exocuticle as sclerotized endocuticle and to endocuticle as non-sclerotized endocuticle, but in a subsequent paper (Lees, 1952) he refers to the same layers as outer endocuticle and inner endocuticle, respectively. Nathanson (1967, 1970), Beadles et al. (1973), and Beadle (1974) correctly use the terminology of Richards (1951) for the sclerotized regions, but they use the term exocuticle incorrectly to refer to the outer endocuticle of soft cuticle. Balashov (1968) refers to a layer of partly sclerotized mesocuticle in the alloscutum cuticle of ixodid ticks. This cuticle must undergo considerable stretching during feeding (see §1.2.2.3), a process that could not occur in even a partly sclerotized cuticle. This confusion indicates the desirability of adopting a standard terminology. We suggest that the one proposed by Richards (1951) be used.

In the argasid or soft ticks, Balashov (1968) reports that the extensible body cuticles of different species have different surface structures, although internal structures are similar between species. The surface may be folded or tuberculate in unfed ticks, the folds tending to flatten in some regions upon engorgement. The endocuticle appears to be non-lamellate throughout its entire thickness (15–30 μm in adults) except for a narrow region 1–3 μm thick on the inner border. The capitulum and legs of argasid species are formed of hard cuticle (Balashov, 1968). In these regions, the cuticle is completely sclerotized, consisting entirely of exocuticle and epicuticle. A thin endocuticle is present in some other sclerotized areas such as the dorsal and spiracular plates. The articulated cuticle in argasids is composed entirely of endocuticle (and epicuticle), 20–30 μm thick, with a very distinct lamellate structure (Balashov, 1968). There have been no fine structural studies of argasid tick cuticle. In the ixodid ticks, sclerotized cuticle occurs over the capitulum, scutum, appendages, anal valves, male ventral plates, and several other small sclerites. According to Balashov (1968), the procuticle becomes entirely sclerotized and, although lamellae are not seen in the light microscope, the resulting exocuticle is highly birefringent. Beadle (1974) examined the sclerotized cuticles of B. microplus and Boophilus decoloratus with the electron microscope. In the adult, the cuticle thickness ranged between 3–6 μm over most areas but reached up to 10 μm in some regions of the mouthparts and appendages. Lamellae or fibrous laminae were not resolved.

The intersegmental cuticle of Boophilus is 3–5 u, m thick and is composed of 5–8 lamellae exhibiting a finer, fibrous parabolic pattern (Beadle, 1974).

The region of the cuticle of ixodid ticks that has received most attention is that of the alloscutum of the female. This cuticle has the peculiar capacity of expansion and stretching during blood-sucking. The following species have been examined: Ixodes ricinus (Lees, 1947, 1952), Boophilus annulatus ( ≡ B. calcaratus) and Hyalomma detritum (Tsvileneva & Mashansky, 1965), Hae-maphysalis leporispalustris (Nathanson, 1967, 1970), Amblyomma americanum (Beadles et at., 1973), B. decoloratus and B. microplus (Beadle, 1974), and Hyalomma asiaticum (Amosova, 1975). The alloscutum of the larvae and nymphs of ixodids also has limited capacity for stretching (Balashov, 1968; Beadle, 1974).

Figures 1.2–1.4 show transverse sections of the alloscutum cuticle of the engorged nymph of B. microplus. Two regions can be seen in electron micrographs–a distinctly lamellate inner endocuticle and a non-lamellate outer endocuticle (Fig. 1.2). The inner endocuticle is composed of 6 or 7 lamellae spaced about 0.7 μn apart with a substructure of helicoidally packed sheets of microfibrils (Fig. 1.3). Microfibrils are relatively electron lucent, about 3 nm in diameter and surrounded by granular electron dense material (Fig. 1.4). In insects and other arthropods, where similar microfibril/matrix systems have been observed, it is thought that the microfibril is composed of chitin and the matrix of protein (Neville, 1975). Similar fibrous material, with a microfibrillar substructure, is also present in the outer endocuticle (Fig. 1.2), but instead of forming a continuous, helicoidally arranged system, it is broken up into small bundles or fibrils. Within each fibril, the microfibrils are oriented parallel to one another, but fibrillar orientation within the outer endocuticle is not constant. However, there is evidence that fibrillar orientation varies systematically; longitudinally sectioned fibrils occur at regular spacings through the outer endocuticle (arrows in Fig. 1.2), the spacings being of the same order as the lamellar spacing of the inner endocuticle. The space between fibrils in the outer endocuticle is filled with amorphous, granular, relatively poorly staining material. Towards the surface, the concentrations of fibrils to amorphous material and the size of individual fibrils decreases markedly. In the outermost 2 or 3 μn, no fibrils are visible at low magnifications (Fig. 1.2).

FIG. 1.3 High magnification of the inner endocuticle of the cuticle shown in Fig. 1.2, including one complete lamella. The microfibrils show the helicoidal packing typical of other arthropod cuticles with microfibrils cut in cross-section (xs) between the dense laminae, where microfibrils are oriented parallel to the section plane (Is). Scale = 25 μm.

There are some differences in structure between the cuticle of the alloscutum of the nymph and the female of B. microplus. In the female, maximum cuticle thickness is reached when the tick is about 4.5 mm long (see §1.2.2). Micrographs of transverse sections of this cuticle are shown in Figs. 1.14, 1.16, and 1.17. Subdivision of the endocuticle into inner and outer layers is more marked than in the nymph. The inner endocuticle (total thickness 18 μm) is made up of a number of distinct lamellae, each about 2 μn thick (Fig. 1.14), but the substructure of these lamellae is different from that of the nymph. At high magnification (Fig. 1.17) it can be seen that about 50% of the volume of the inner endocuticle of the adult female is made up of amorphous material of medium electron density. Some fibrils with a microfibrillar substructure are also present, the remainder of the volume being occupied by the anastomosing pore canal network (discussed below). Some, if not all, of the fibrous material appears to lie within the pore canals, a situation that has not been recorded in other arthropod cuticles. The non-lamellate outer endocuticle (total thickness about 20 μn) is composed almost entirely of amorphous material which is continuous from the inner endocuticle. A small amount of fibrous material is present, and this is again seen largely within the pore canal system (Fig. 1.16). Lees (1952) reported that both the inner and outer endocu tides of I, ricinus females were lamellate, the outer lamellae being the more densely packed. In contrast, with the electron microscope, both we and Beadle (1974) detected lamellae only in the inner endocuticle of B. microplus.

FlG. 1.14 Transverse section through the alloscutum cuticle of an adult at the end of the growth phase, when the cuticle is approximately at its maximum thickness (in this case 45 μm). The inner endocuticle (inner) is clearly lamellate and the outer endocuticle (outer), clearly non-lamellate. Pore canals (pc) of the inner endocuticle are extremely numerous, branched and anastomosed, whereas in the outer endocuticle they are less branched and far less numerous. Epicuticle, epi; subcuticle, sub. Scale = 1.0 μm.

FIG. 1.16 Detail of the other endocuticle of the specimen seen in Fig. 1.14. The bulk of the volume is composed of amorphous material (a). Sparse microfibrillar bundles (f) are present and these usually appear to lie within the pore canals (pc). Pore canals are bordered by an electron-dense membrane (m). Scale = 50 μm.

FIG. 1.17 Detail of the inner endocuticle of the specimen seen in Fig. 1.14. Pore canals (pc) are far more numerous, branched and anastomosed than they are in the outer endocuticle (Fig. 1.16). Microfibrillar bundles (f) are more numerous and amorphous material (a) sparser than in the outer endocuticle. Scale = 50 μm.

1.2.1.3 Pore Canals and Wax Canals

In most arthropod cuticles a system of fine channels passes through the entire cuticle, possibly establishing connections between the epidermis, the several layers of the cuticle, and the surface. Usually the channels in the endocuticle and exocuticle (where such a subdivision exists) are similar in structure. At the interface with the epicuticle, however, the channels sometimes become finer and frequently branch distally (Locke, 1961). The channels in endo- and exocutides are known as pore canals. The finer channels through the epicuticle have been called wax canals because they are supposedly involved in wax secretion in some insects (Locke, 1960, 1965). Pore canals are relatively large structures and are easily resolved in the light microscope, but wax canals can only be seen in the electron microscope.

In the unfed female tick (I. ricinus) the density of pore canals has been estimated to be greater than 1,400,000 mm−2, or in excess of 60 per epidermal cell (Lees, 1952). The pore canal system may be delineated with the rapid Golgi staining method (chrom-silver) and certain regions stain with hae-matoxylin. If frozen sections are allowed to dry, certain regions may become filled with air, indicating that the canals were previously fluid-filled. In thick sections they appear to be arranged in bundles, each of which may correspond to an underlying epidermal cell. During early feeding, the pore canals become more widely spaced and distended within the outer endocuticle and exceedingly branched distally, where they appear to end blindly beneath the epicuticle. Within the inner endocuticle, Lees (1952) has reported that the pore canals become progressively filled with solid material. Arthur (1962), however, argued that the canals must remain open if, as Lees suggested, growth of the outer layers of the cuticle is to proceed up to the time of final engorgement (see §1.2.2.3). Tsvileneva and Mashansky (1965) observed that, in H. detritum, the distinct lamination in the inner endocuticle, although associated with the presence of fibrous cuticle, was mainly caused by lateral branching of pore canals. Balashov (1968) also reported ramification of the canal system in the inner endocuticle of I. ricinus and a lack of pore canals in the articulation cuticle.

More detail on pore canal structure has been provided by electron microscopy, but information on the three-dimensional structure of the system (branching, anastomosing, twisting, etc.) is hindered by the inability to examine thick sections or whole mounts by this technique. However, finer canals and the content of canals can be examined at higher resolution. Nathanson (1967) examined the pore canals of H. leporispalustris with the electron microscope. They are more numerous in sclerotized areas of the cuticle where their morphology is similar to those of Galleria and Tenebrio (Locke, 1961). They are 0.2–0.4 μm in diameter in the endocuticle and contain filaments 20–30 nm in diameter. In the exocuticle the contents are less clearly delineated and canals branch so that they are about three times more numerous at the epicuticular border. In the cuticle of the alloscutum of H. leporispalustris pore canals are less numerous and thinner (0.15–0.3 μn). They may contain uniformly disperse fibrous material or an electron opaque core. Filaments of diameter 15–20 nm have been observed in the lumen of canals of the inner endocuticle, and these filaments appear to pass into the cytoplasm proper of the underlying epidermal cell. In the sclerotized cuticles of larval B. decoloratus and B. microplus pore canals are about 0.15 μn at their widest and contain dense granular material throughout. Nearer their junction with the epidermis, the material is more obviously of cytoplasmic origin (Beadle, 1974). In the larval non-sclerotized cuticle of the same species, there are large numbers of canals in the inner endocuticle which fuse to produce large porous areas in the outer endocuticle.

Figures 1.2 and 1.14–1.17 illustrate the fine structure of the pore canals of B. microplus. In the inner endocuticle of the engorged nymph (Fig. 1.2) there are large canals up to 0.6 μn in diameter traversing the lamellae with some lateral branching. If these are really pore canals, they are somewhat larger than previously reported. However, this cuticle was taken from a tick at apolysis (when the epidermis retracts from the cuticle at the beginning of the adult moult), so it is possible that moulting fluid may have already begun digestion of the inner lamellae of the cuticle. In the outer endocuticle the pore canals are narrower, branched, and oriented at diverse angles to the cuticle surface. The latter is the result of stretching of the cuticle during the final feeding period of the instar. A similar effect has been observed in the outer endocuticle of the adult (Fig. 1.15).

FlG. 1.15 Transverse section of the fully stretched alloscutum cuticle after final engorgement. The stretching causes the thickness to be reduced to about half that at the end of the growth phase (Fig. 1.14). Pore canals of the inner endocuticle appear to have closed up so that the lamellate appearance is no longer seen. The junction between inner and outer endocuticles is also indistinct but is probably in the region of the dotted line. Pore canals (pc) of both the inner and outer endocuticles are extremely deformed by the stretching. The epidermal layer beneath the cuticle is still intact and the cells appear healthy. Scale = 1.0 μm.

Branching and anastomosing of pore canals is much more pronounced in the inner endocuticle of the partially fed adult (Fig. 1.14). At high magnification (Fig. 1.17) profiles of the canal system are limited by a membrane which has greater electron density than either the granular contents or the amorphous interstitial material. This membrane can be traced back to its continuity with the plasma membrane of the epidermal surface, but the typical trilaminar structure of the epidermal membrane cannot be resolved in the more distal regions of the pore canals. As mentioned in the previous section, the pore canals do appear to contain microfibrils of the chitin-protein complex, lending some support to Lees’ (1952) observation that the pore canals of the inner endocuticle of I, ricinus become fully chitinized. Pore canals of inner and outer endocu tides are continuous but are far less numerous or branched in the outer layer (Fig. 1.14). At higher magnifications (Fig. 1.15) they are seen to contain granular material and are bordered by an electron-dense membrane similar to those of the inner endocuticle.

Wax canals are clearly resolved in electron micrographs, the electron density of their contents depending on the developmental stage of the tick and the type of stain applied to sections. In pre-ecdysial alloscutum cuticle of adult females the wax canals contain a central electron-dense filament about 25 nm in diameter which is revealed by staining with uranyl acetate and lead citrate (Fig. 1.9), but in the same cuticle, the contents of the wax canals appear to be removed if sections are oxidized with potassium permanganate prior to staining with lead citrate (Figs. 1.8 and 1.10). The spatial density of the wax canals, as measured from sections parallel to the cuticle surface, is about 10 μm−2. Wax canals and pore canals join at the interface between endocuticle and epicuticle (Figs. 1.5–1.7), but the tubular network also found at this interface does not interconnect with the pore canal/wax canal system (Filshie, 1976). The wax canals penetrate both the dense layer of the epicuticle and the inner layer of the cuticulin. During later stages of feeding the contents of the wax canals become less reactive to electron staining (Fig. 1.6).

It can be seen, then, that the pore canal/wax canal system of the tick does provide an effective link between the epicuticle and the surface wax layer and that branches of pore canals pass through and between endocuticular layers. The density of wax canals (10 mm−2) in B. microplus epicuticle obtained from electron microscopy approximates the density of pore canals in the outer endocuticle of the unfed female of I ricinus (1.4 μm−2) as calculated by Lees (1952) from light microscopy. The branching of pore canals below the epicuticle observed by Lees may account for the actual difference in the two figures. The possible roles of pore canals and wax canals in the formation of the cuticle and the changes that occur during blood sucking will be discussed in the following sections.

1.2.1.4 Muscle Attachments, Glands, and other Integumental Structures

A number of different specialized cells, group of cells, or cuticular structures are present within the tick integument. The most obvious of these are the various sensory structures, which are areas of cuticle modified into hairs, pegs, pits, etc., and supplied with dendritic nerve endings from cells usually located within or just beneath the epidermal cell layer. The structure and function of these sense organs is discussed separately in Chapter 3.

Ticks, like insects, possess dermal glands, which occur throughout the epidermal layer. In insects, the dermal glands are supposed to produce the cement layer over the surface of the wax layer (Wigglesworth, 1947), and this is correlated with their wide dispersal over the body surface. In the bug Rhodnius the type B glands are composed of four cells: (1) the secretory cell; (2) the saccule-forming cell; (3) the duct-forming cell; and (4) a cell which surrounds the other three (Lai Fook, 1970). The gland cell lies within the epidermal layer and its secretions pass into the saccule (when present) and then via a fine duct to an opening on the cuticle surface. Scanning electron micrographs have confirmed the presence of dermal gland openings on the cuticle surface (Scheie et al., 1968). There is evidence that the secretory activity of dermal glands in insects is not restricted to the time of ecdysis or just after ecdysis when the cement layer is being deposited. In Tenebrio, Delachambre (1973) found that the glands still showed some activity in older adults at a time when one would have expected that additional cement was not required for protection of the wax layer.

Argasid ticks possess a cement layer and ixodids do not, but both groups have dermal glands. In the argasid Ornithodoros moubata, the dermal glands pass through two cycles of activity: just before secretion of moulting fluid and a few hours after moulting during secretion of the cement layer (Lees, 1947). The nymphal glands of Dermacentor andersoni are of two types; large (type A) and small (type B) cells. During the nymph-adult moult, a third, multi-nucleate gland (type C) is formed from nuclei of type B cells. Ixodes ricinus possess only small, type B glands (Lees, 1947).

In ixodids, Lees (1947) found that the dermal glands secrete a thick yellow grease which slowly spreads over the cuticle surface or collects around the duct openings. Discharge of this secretion occurs only after full engorgement but may not occur in all developmental stages. Ixodes ricinus larvae and nymphs do not discharge, but the egg-laying female does. In D. andersoni, on the other hand, grease is secreted from dermal glands in the engorged nymph.

The females of B. microplus secrete a clear fluid from the dermal glands when handled (K. C. Binnington, per. comm.). Dermal glands, forming a lyre-shaped pattern in the ventro-lateral hypodermis of larvae and nymphs of the Australian paralysis tick Ixodes holocyclus secrete copious amounts of a creamy exudate during the period between apolysis and ecdysis and the exudate remains on the exuvium in a pattern reflecting that of the glands (B. F. Stone & K. C. Binnington, pers. comm.) Dermal glands of engorged nymphs and adults of I. holocyclus also secrete a pink fluid which contains a paralysis toxin having similar properties to those of the toxin found in its saliva (Koch, 1967; B. M. Doube & B. V. Goodger, unpublished data). Clearly, the dermal gland secretions may have different roles to play in different tick species and at different developmental stages within the same species. Much more work is required in order to determine the composition and function of these various secretions.

The porose areas are located on the basis capitulum of female ixodid ticks. The cuticle in these regions is depressed slightly and perforated with numerous pores. Earlier authors thought that the fine ducts leading from the pores were innervated (see Arthur, 1962). Consequently, the porose areas were thought to be sense organs until recently. Feldman-Muhsam and Havivi (1960) and Felman-Muhsam (1963) demonstrated that the ducts led to secretory glands and suggested that the secretion was a lubricant which combined with the secretion of Gé né’s organ (the egg-waxing organ–see Chapter 9). Atkinson and Binnington (1973) selectively destroyed the porose areas of B. microplus and showed that this treatment had no demonstrable effect on the size and hatchability of subsequent egg-batches. By analysis of egg-waxes from treated and untreated ticks they showed, however, that antioxidant which originates from the secretions of the porose areas (see § 1.3.3) inhibits the autoxidation of steroids in the egg-waxes.

Ixodid ticks possess a coxal gland which is connected via a duct to the exuvial space and shows maximum activity during moulting (Binnington, 1975). Histochemical tests demonstrated a non-enzymic tyrosine-containing protein within the active gland. Binnington suggested a possible role for the gland in hardening of the cuticle. Argasid ticks also possess an accessory coxal gland. The ducts, unlike those of ixodids, are connected first to the duct of the osmoregulatory coxal organ which in turn passes to the exterior (Lees, 1946).

The dorsoventral muscles of the alloscutum of ixodid ticks are arranged roughly in longitudinal rows but there is no modification to the surface pattern except for the presence of long, shallow furrows or grooves (Lees, 1947). In argasids the boundary of the insertion of each muscle bundle is clearly delineated and encloses a number of polygonal units each representing the insertions of individual fibres (Lees, 1947). According to Balashov (1968), the argasid cuticle is sclerotized in these areas. The structure of muscle attachments in ixodid nymphs and adults has been studied by Yalvac (1939) with the light microscope. He showed that the muscle of nymphs is attached to a tendon-like structure which appears to pass between epidermal cells and through the cuticle to join with the epicuticle. In the adult, however, the tendons are not visible in the cuticle. Results of electron microscopy have shown that all arthropods have a characteristic type of attachment which, because of its structure, can be unmade and remade at each moult. The tendons, or tonofibrillae as they are sometimes called, are not continuous from their point of attachment at the muscle cell to their termination within the cuticle, but are made up of two sets of fibrils; proximally of bundles of microtubules running transversely through the epidermal cells, and distally of dense rodlike tonofibrillae within the cuticle (see Neville, 1975, for summary of structure in insects). Muscle filaments and microtubules are anchored to the internal faces of muscle cells and epidermal cells respectively by structures known as hemidesmosomes. The intercellular space contains a cement-like material. The earlier observations of light microscopists that tendons (or tonofibrils) pass between epidermal cells (Yalvac, 1939) are incorrect. All electron microscopic observations made to date show that tonofibrillar structure in arthropods is as described above. Examination of the attachment in B. microplus has shown that its fine structure follows the general pattern (Filshie, unpublished).

The structure of spiracles in ticks has been studied by a number of authors (Nuttall et al., 1908; Mellanby, 1935; Browning, 1954a, b; Arthur, 1956, 1962; Hinton, 1967; Woolley, 1972; Roshdy & Hefnawy, 1973; Roshdy, 1974). In B. microplus (Hinton, 1967) the larva lacks spiracles, but in both the nymph and adult the spiracle consists of a cribriform plate similar in external structure to that of beetle larvae of the family Scarabaeidae. In the adult there are about 35–40 surface pores, each being an oval or circular hole about 2 μm wide, arranged peripherally around a large central structure called the ostium. Earlier authors (Browning, 1954a, b; Arthur, 1956, 1962) suggested that the ostium was the functional spiracle. This was disputed by Hinton (1967), who claimed the ostium was a collapsed ecdysial tube for extraction of the nymphal tracheal system during the nymph-adult ecdysis. Subsequently Roshdy and Hefnawy (1973) showed that in the spiracle of nymphal Haemaphysalis longicornis the ostium is connected to the tracheal system via an atrial chamber with a valve capable of opening and closing the spiracle. Roshdy (1974) also showed that the peripheral pores are connected by narrow ducts to underlying tegumental glands and concluded that they did not function as aeropyles, as proposed by Hinton (1967). The formation of the spiracle in adult H. longicornis has been described by Roshdy (1974). The recent observations of Rudolph and Knülle (1978) on Amblyomma variegatum appear to support the interpretation of Hinton, while stressing the role of the atrial valve in the regulation of gas exchange between the tracheal system and the atmosphere (see §2.2.3).

1.2.2 Deposition of the Cuticle

The life-cycle of all arthropods is divided into a number of discrete stages or instars separated by periods of moulting, when the cuticle of the previous instar is shed and a new one formed by the underlying layer of epidermal cells. Increase in body-size is achieved in two ways. In those areas where the post-moult cuticle is hardened or sclerotized, expansion to accommodate this increase must occur immediately before hardening, i.e. usually in the 1–2 hours that succeed ecdysis (shedding of the previous cuticle). Expansion under these circumstances usually is brought about by a temporary increase in haemolymph pressure which in turn is achieved by a combination of muscle contraction and air swallowing (Cottrell, 1964). Growth in those parts of the body enclosed by sclerotized cuticle occurs, therefore, in a series of discrete increments brought about by post-moult expansion and hardening of the cuticle. On the other hand, many parts of the cuticle remain soft, untanned, and flexible throughout the instar, and growth can be achieved during the intermoult period by gradual expansion in surface area of the cuticle. During the final feeding stages in some ticks, volume increase is so rapid that it cannot be accommodated by concomitant cuticle growth, and considerable stretching of the cuticle occurs in addition to an earlier growth phase. Although the inner layer of untanned cuticle (the procuticle) is capable of stretching, the epicuticle is always found to be inextensible even though it remains flexible. In those instances where increase in body-size occurs during an instar, the epicuticle is initially folded, the surface becoming progressively smoothed with growth. Consequently, initial deposition of the several layers of the cuticle is intimately related to subsequent requirements for growth, locomotion, and adaptation to the environment of the instar.

1.2.2.1 Formation of the Epicuticle

As discussed in §1.2.1.1, the layers that comprise the epicuticle are–from the outside: cement, wax, cuticulin, and dense layer. Chronologically, however, the layers are not secreted in that order. The first layer to be deposited is the cuticulin. It begins to appear shortly after the epidermis retracts from the previous cuticle (the process known as apolysis) at the beginning of the moult. Cuticulin formation has been studied in detail in an insect by Locke (1966) and more recently in the alloscutum of B. microplus by Filshie (1976). In most respects, the secretory mechanisms are identical. Small areas of the cuticulin membrane appear initially as caps over the tips of small projections or microvilli of the apical plasma membrane of the epidermal cells. As secretion proceeds, these caps become progressively larger in surface area until neighbouring areas fuse to form a continuous layer. The plasma membrane at the tips of microvilli contains localized dense areas (plasma membrane plaques) and it is thought that precursors of the cuticulin pass through the membrane in these areas (Locke, 1966). Many small, dense vesicles are also seen in the apical cytoplasm of the cells at this time and some appear to join with the membrane and discharge their contents into the exuvial space. During the cuticulin formation, moulting fluid is presumably being secreted as well. The dense vesicles may, therefore, be associated with either cuticulin or moulting fluid production; it is not possible to distinguish the separate secretory mechanisms on morphological