Histology of digestion in nymphs of Rhipicephalus appendiculatus fed on rabbits and cattle naive and resistant to the ticks

InfemafionalfournalforParasitology Printed in Great Britain.

Vol.

1987. 17,No.8,pp.1393-1411,

0020-75 19/87 $3.00 + 0.00 Pergomon Journnls Ltd. 0 1987 Aurrrolion Societyfor Parasitology

HISTOLOGY OF DIGESTION IN NYMFHS OF RH.P1Cl3WALUS APPENDICULATUS FED ON RABBITS AND CATTLE NAIVE AND RESISTANT TO THE TICKS and J. D. FLETCHER

A. R. WALKER

Centre for Tropical Veterinary Medicine, University of Edinburgh, Roslin, EH25 9RG, U.K. (Received 7 July 1986) AbSfraCt-WALKER A. R. and FLETCHERJ. D. 1987. Histology of digestion in nymphs of Rhipicephalus appendiculatus fed on rabbits and cattle naive and resistant to the ticks. International Journal for Parasitology17: 1393-1411. Nymphs feeding on ears of four rabbits and four calves (Bos tuuncr) were examined during first and third or fourth infestations and also during the moulting period. The gut caecae were removed and examined by histochemistry and light and electron microscopy. Attachment sites of the nymphs were biopsied from all hosts and cell counts made by light microscopy. Resistance was expressed by reduction in numbers of ticks completing engorgement and reduced engorgement weights. The gut was comprised of a proliferative stem cell; a digestive cell that differentiated into a sessile type ingesting by pinocytosis and a motile type ingesting by phagocytosis; and a cell secreting a glycoprotein with acid phosphatase activity into the lumen. The gut grew during the early stages of feeding to accommodate the expansion during engorgement. On rabbits and cattle resistant to ticks the stem cells were damaged, with moribund nuclei and poorly differentiated cytoplasm. Thus there were fewer digestive and secretory cells and the gut did not expand to accommodate a full blood meal. The attachment sites were dominated by mononuclear cells and neutrophils in both host species at the first infestations but at the third or fourth infestations there was a considerable increase in proportions of eosinophils and basophils. Host granulocytes were traced to the lumen of the tick gut and to motile digestive cells which destroyed them by phagocytosis. INDEX KEY WORDS: Rhicephalus uppendicukrtus; nymphal ticks; digestion; feeding; moulting; cattle; rabbits; host resistance; attachment sites; histochemistry; ultrastructure.

INTRODUCTION

THE STUDYof the structure and function of the gut in hard ticks has usually been conducted in the context of anatomical studies on the whole tick. However the gut of ticks is an important potential target for the defences of the host in expressing acquired resistance to ticks. There is also an increasing interest in the use of vaccination against ticks based on the use of antigens derived from the gut (Johnston, Kemp & Pearson, 1986; Kemp, Agbede, Johnston & Cough, 1986; Agbede & Kemp, 1986). It would not only be interesting to find out how naturally acquired resistance was related to the effects of vaccination but by studying digestion useful sources of antigens could also be revealed. The mechanisms of naturally acquired resistance are varied and may act in sequence. But one important component for R. appendiculutus is the reduction in 1974; Chiera, engorgement weight (Branagan, Newson & Cunningham, 1985). There is much information on the reactions at the attachment sites of hard ticks that may affect engorgement but little specifically on what happens in the gut of ticks feeding on resistant hosts. The main studies of the structure and function of tick gut cover Ixodes ricks, by

Hughes (1954); Haemaphysalis spinigera, by Chinery (1964); Hyalomma asiaticum, by Balashov (1983), Balashov & Raikhel (1976), Raikhel (1974, 1975, 1978); and Boophilus microplus, by Agbede & Kemp (1985). To study the effects of host resistance on the gut of the tick it was found necessary to have a detailed description of the ultrastructure of the species and instar used. Till (196 1) included the gut of nymphal R. appendiculatus in her study of the morphology of the tick’s viscera but did not extend this to ultrastructural detail. The purpose of the present study was to describe the ultrastructure of the gut of R. uppendiculutus during feeding and moulting. Comparisons were then made of the gut from ticks fed on naive (no previous exposure to ticks) and resistant hosts. In the context of tick feeding it was useful to know the contents of the feeding lessions from a histological study of the attachment sites, and to have the weight and haematological content of the excreta produced during feeding. Nymphs were studied because they were the easiest instar to handle, considered likely to be representative of digestion in larvae and adults and are an easy stage

1393

A. R. WALKER and J. D. FLETCHER

1394

for the experimental monitoring of acquired resistance in this species in the laboratory. Rabbits were used as hosts in addition to cattle because they are so widely used as an experimental host for this tick that comparative data was considered useful. to use

MATERIALS

AND METHODS

Ticks and hosts. Rhipicephalus appendiculatus in the colony at this laboratory were derived from a colony at Muguga in Kenya. Larvae were fed as one batch on a naive rabbit and the resulting nymphs were stored at 18”C, 85% RH for between 2 and 4 months after completion of post moult development before being used. The experimental animals were four female New Zealand white rabbits of between 2.5 and 3 kg, and four Bos taurus calves comprising a Friesian heifer and a bull, a Hereford heifer, and a Jersey bull, of between 82 and 113 kg. Both rabbits and calves were kept in similar controlled environments, fed on hay and concentrates, and used according to the sanctions of the Cruelty to Animals Act (1876) and the guidelines of the Universities Federation for Animal Welfare, of the United Kingdom. Nymphs, in batches of 100, were confined to ears of rabbits and calves using linen bags or the neck of calves using linen patches. The patches were attached with neoprene rubber contact adhesive (‘Evostik’, Evode Ltd., Stafford, U.K.). The infestations were at 2-week intervals and with feeding lasting up to 7 days the interval between completion of one feed and the start of the next was about 7 days. Three infestations were applied to the rabbits and four to the calves. The aim was to produce a strong expression of resistance at a similar level in the two groups of host species, but also to provide enough ticks for study. Nymphs of R. appendiculatus have been shown to have an approximate 50% reduction in both numbers engorging and engorged weights when fed in earbags of cattle made resistant by feeding 400 adults of the same species per week for 24 weeks (de Castro, Cunningham, Dolan, Dransfield, Newson & Young, 1985). The effect on the tick gut of individual differences in resistance between the hosts was not studied because of the difficulties of processing many individual specimens. The infestation sequence on the rabbits was as follows: the first two on the left ear then the final on the right ear. On the calves the sequence was: the first two on the left ear and neck, then right neck only, then right ear only. The last infestation on the calves was only of 100 nymphs whereas the earlier infestations were of 100 on the ears plus 100 on the neck or 200 in a larger neck patch. Live nymphs for examination were removed from the ears at days 1,3 and 5 of the first and third or fourth infestations; two nymphs from each host at each sampling day. These times roughly corresuonded with the phases 1.2 and 3 of feeding for hard ticks described by Balashov (1972) and mea&red by Purnell, Boarer & Peirce (19711 for R. auuendiculatus nymphs. For the purposes of this study we were unable to select effectively nymphs in different feeding stages on criteria of weight or size, this problem was complicated by the differences due to host species and resistance status. Instead they were selected visually: nymphs at stage 1 of feeding showed slight expansion of the idiosoma in proportion to the scutum; at stage 2 much expansion of the idosoma and of gut caecae separately visible through the translucent integument; stage 3 taut distention of the idiosoma with black to white homogenous colour of replete and contiguous caecae. On naive cattle these stages were on average 1.2,2.1 and 3.8 mmlongfromcornua toposteriorof idiosoma respectively, and on resistant cattle they were 1.1,

1.5 and 2.6 mm long respectively. Ticks were sampled as unfed nymphs; as nymphs in the three stages of feeding; as mobile engorged nymphs (2 days post detachment, p.d.); as pharate adults (2 weeks p.d.); as newly moulted females (4 weeks p.d.), and as females at the completion of post moult development (8 weeks p.d.). Developing ticks were kept at 23-C, 85% RH until completion of post moult development. Female ticks fed as nymphs on a naive rabbit were fed on another naive rabbit and their guts compared to those of nymphs by light microscopy at stages l-3 of feeding and at 2 days post engorgement. During feeding excreta from the nymphs was collected from the confinement bags. Biopsies of attachment sites of nymphs was taken as described for adults by Walker & Fletcher (1986), from two sites, at days 1, 3 and 5 from each host at first and third or fourth infestations. Control samples were taken at 3 days before the first infestation. Processing. The biopsies were processed for light microscopy and cell counts made on a total of 104 samples as described by Walker & Fletcher (1986). The tick viscera were exposed by removal of the dorsum and immobilized by flushing with dilute Karnovsky’s fixative (1: 1 with water). The entire viscera were removed from the tick and fixed for 1 h in Kamovsky’s fixative in 0.1 M phosphate buffer at pH 7.4. One sample from each pair was embedded in hydroxyethyl methacrylate (‘Historesin’, L.K.B., Bromma, Sweden) with infiltration in the monomer over 4 days. The remaining tick of each pair was processed for electron microscopy by post fixation in osmium tetroxide, staining en bloc in uranyl acetate, dehydrating in acetone, infiltrated over 4 days and embedding in Araldite. Ultrathin sections were stained with uranyl acetate and Reynold’s lead citrate. By light microscopy samples of the guts of 64 nymphs were examined and by electron microscopy samples of the guts of a separate total of 64 nymphs were examined. Gut from nymphs at stage 2 of feeding through to the mobile engorged nymph stage at 2 days post detachment from naive rabbits was prepared for scanning electron microscopy by tearing open caecae, flushing out the ingesta with phosphate buffered saline at pH 7.4, fixing in Karnovsky’s fixative, critical point drying and coating with gold and palladium. The lumen contents from similar specimens were suspended in phosphate buffered saline pH 7.4, any cells were isolated by centrifugation then fixed and processed for light and transmission electron microscopy as described above. Histology and histochemistry. Samples of gut at all stages and from all infestations were stained for light microscopy with Giemsa’s stain in alkaline buffer; for proteins with mercuric bromophenol blue (MBPB); for polysaccharides with periodic acid Schiff’s reagent (PAS); for lipid with oil red 0 and Sudan black; for proteoglycans (=acid mucopolysaccharides) with a combination of Azure A, PAS, and MBPB on serial sections (alcian blue for control proteoglycans was ineffective with specimens in methacrylate); for acid phosphatases with naphthol phosphate and Schiffs reagent; peroxidase with diaminobenzidine, and esterases with indoxyl acetate. All methods were from Bancroft (1975) or Bancroft & Stevens (1982). Cryostat sections and whole gut specimens could not be prepared satisfactorily. Specimens for electron microscopy from stages 2 and 3 of feeding at first infestations were stained for acid phosphatase by Gomori’s lead method and specimens from completion of the post-moult development stage for peroxidase with diaminobenzidine. Blood and excreta. Blood samples were taken by venipuncture from all hosts after final infestation and analysed by haemoglobinometer (Coulter Electronics Ltd.,

Digestion in ticks

1395

Bedford, U.K.). The excreta from first and third or fourth infestations was accumulated and pooled by host species

difference was also noted in the histological sections of guts.

group. It was diluted with distilled water to give a dilution of dry wt in water approximate to that of the host blood. The haemoglobin content of these solutions were measured on the haemoglobinometer by the cyanomethaemoglobin conversion method.

Structure of gut of nymphs fed on naive rabbits and cattle The gut went through considerable morphological

RESULTS

Expression of resistance

Table 1 summarizes the feeding performance of the numphs. Resistance was expressed in all hosts by a reduction in numbers attaching, premature detachment in a partially engorged state too small to enter into the moult, a tendency for detachment to be later on rabbits and earlier on cattle, and a reduction in engorgement weight by about 40-50%. Pruritus was evident from the increased grooming by the hosts but the ticks were partly protected by the cloth bags. The ranges given in Table 1 for percentage detaching engorged and engorgement weights indicate that individual differences in resistance between the hosts were not great. Moulting was slightly affected; if a nymph detached at a weight sufficient to enter into moult (3 mg smallest recorded) then it was likely to complete moult to a viable adult. The percentages for moulting in ticks fed on resistant hosts would of course have been lower if all the small detached ticks had been included. Approximately 10% of engorged nymphs were abnormally pale to white. These nymphs also moulted successfully. The nymphs of rabbits at first infestation passed through stage 1 of feeding earlier than those on calves at first infestation. This

TABLE

~-EXPRESSION OFRESISTANCE CATTLE

AT FIRST,

THIRD

Attachment % at 24 h Rabbits 1st infestation

100

To

changes during feeding and moulting and these are illustrated in Fig. 1. In Figs. 2-5 are illustrated the normal ultrastructural changes during feeding and moulting. No obvious differences were noted between nymphs fed on rabbits or calves except that the nymphs fed on calves spent longer in stage 1. Stem cells (=reserve cells of some authors). These

were proliferative and since only one could be distinguished structurally it was considered pluripotent. These cells were not evident in unfed or moulting ticks but became numerous by stage 2 of feeding (Table 2). They showed no signs of digestive or secretory function, and. had only small areas of microvilli exposed to the lumen and contained no heterolysosomes (Figs. 6, 7 and 27). Lipid droplets accumulated however. Mitotic figures were seen by light microscopy and spindle microtubules by electron microscopy. It was not determined whether these cells were continuously present during development or whether they originated at feeding from digestive cells of the previous instar. Secretory cells. These were conspicuous by light microscopy because of their intensely basophilic cytoplasm and enzymic activity of their secretory granules (Figs. 2, 12, 13 and 17). By electron microscopy they showed cytoplasm dominated by rough

R. appendiculatu.vNnwns

OR FOURTH

INFESTATIONS

Time of peak detachment (days)

4.8

AS REDUCED

WITH

INFESTATIONS

% Detached engorged by day 7

100

FEEDING AT

PERFORMANCE

l&DAY

ON RABBITS

AND

INTERVALS

Mean engorged weight (mg)

% Mouh of engorged nymphs

7.8

100

(7.z.2) (4.$;.6)

96

(706;8) (57-77)

(4.014.1)

(71Q

(9.E.9)

(576:6)

(8.Z.8)

(4OZ5)

(5.iii.7)

(54?2)

(6.1-6.6)

W$’ 2nd infestation 3rd infestation Cattle 1st infestation 2nd infestation 3rd infestation 4th infestation

95 88

100 72 57 89

5.8 5.8

100

5.3

100

4.6

99

4.8

94

4.0

100

Means from four rabbits and four calves up to day 7 of feeding. Ranges of percentage detached engorged and of weights for engorged ticks are highest and lowest means from within each group of hosts. Detachment recorded as percentage detached engorged at a weight sufficient to enter into moulting stage out of total applied. Percentage moulting recorded as percentage of the total that entered into the moulting stage of development.

A. R. WALKER

1396

a

and J.

D. FLETCHER

Digestion

endoplasmic reticulum with numerous ribosomes and also many Golgi bodies, both features characteristic of cells producing protein for secretion (Figs. 8 and 9). These cells were sparse (Table 2) and transient and only fully active at stages 2 through to 3 of feeding and were not seen in non-feeding stages. No intracellular digestive activity was seen and the lumenal plasma membrane was dominated by exocytosis of the secretory granules. The granules were composed of a glycoprotein showing strong reactions for protein and faint reactions for polysaccharide, and always showed strong reactions for acid phosphatase. Senile digestive cells. These were present at the beginning of feeding, and were the same cells surviving from the previous instar (Figs. l-5). They discharged residual bodies into the lumen by exocytosis and it is presumed that they commenced a new cycle of digestion. Sessile digestive cells at stages 1 and 2 of feeding were seen budding off large membrane bound vesicles containing an amorphous flocculent material derived from the cytoplasm. These vesicles were commonly seen free in the lumen as shown in Figs. 17 and 18. The flocculent material was a nonproteinaceous polysaccharide. It showed no metachromasia characteristic of the other proteoglycans seen in this study such as in cartilage or mast cell granules. The rapid expansion of the gut during stages l-2 was achieved by the proliferation of stem cells and their differentiation into sessile digestive cells. The presence of residual sessile digestive cells suggested that generations of sessile cells were produced. The massive passage of material through the gut indicated by weighing the excreta suggests that the residual cells seen were very transient and thus many generations of sessile digestive cells must have been produced. Sessile digestive cells showed intense digestive activity with very large areas of active microvilli and coated pinocytotic pits (Figs. 2, 3, lo14). Heterolysosomes were very common but never massive during the feeding stages. Primary lysosomes with acid phosphatase activity were seen but the acid

in ticks

1397

phosphatase activity of these cells appeared slight and diffuse in the same light microscopy preparations which showed intense activity in the secretory cells (Fig. 12). Pinocytotic and lysosomal activity was very similar to that described in detail for H. asiaticum by Raikhel (1974, 1975). Lipid droplets and numerous haematin and other residual bodies from the heterolysosomes accumulated in older cells and these cells detached from the basal lamina (Figs. 2, 1.5 and 16). These residual cells had reduced pinocytotic activity and became free in the lumen and apparently destined for excretion. Sessile digestive cells at engorgement became very stretched but retained pinocytotic activity and by the middle of pre-moult development they became enormously distended with large heterolysosomes. Although there was little evidence of lysosomal activity during moulting, by the completion of post-moult development there were very few proteinaceous heterolysosomes left in these cells, the main reserve material being lipid droplets (Figs. 5 and 23). Membrane bound residual bodies were mainly numerous small dense bodies of a natural blue/black colour containing what appeared to be lipofuschin and thus presumably were autolysosomes. Motile digestive cells. These resembled residual sessile digestive cells but were only produced at and just after engorgement (Figs. 1,3,4,5,19-22). At this stage the stem cells were scarcely active and the motile digestive cells appeared to originate from sessile digestive cells that detached from the basal lamina. On detachment the plasma membrane changed activity from exclusively pinocytotic to mainly phagocytic activity, but after moulting the motile digestive cells were no longer phagocytic and regained pinocytotic activity. The motile cells were readily distinguished in sections by their inclusions of host leucocyte nuclei in their heterolysosomes and under the scanning electron microscope by larger and more irregular microvilli and folds of the plasma membrane (Figs. 20 and 22). During the preparation of the specimens for scarming electron microscopy the lumen could be

FIG. 1. Representative cross-sections of gut caecae at stages of feeding and digestion to show size changes and arrangement of cell types in chronological order oh unfed nymph (UFN), feeding states l-3 (PSI to 3), pharate adults (PA), newly moulted females (NMF), and at completion of post moult development (CPMD). mdc = motile digestive cell, rmdc = residual motile digestive cell, rsdc = residual sessile digestive cell, sect = secretory cell, sdc = sessile digestive cell, stc = stem cell. FIG. 2. Representative segment of gut caecum of nymph at the second stage of feeding on a susceptible host. ax = axon, bl = basal lamina, cpm = convoluted basal plasma membrane at periphery (adlumenal) of all epithelial cells, daut = dense autophagosome, exe = exocytosis of secretory granules from secretory cell into the lumen, exv = excretory or secretory vesicles budding off from sessile digestive cells as membrane bound bodies, Go1 = Golgi bodies common in perinuclear area of sessile digestive cells and throughout cytoplasm of secretory cells, gi = gap junctions rare and distribution not obvious, het = heterolysosomes formed by accretion of pinocytotic vesicles and primary lysosomes, laut = lucent autophagosome, lip = lipid droplets in all cells, mit = mitochondria, mt = microtubules seen at recently divided nuclei of stem cells, must = muscle cells form an anastomosing net around gut caecae, npm = non-pinocytotic plasma membrane of a residual cell, nucl = nucleoli large in most cells particularly sessile digestive and secretory cells, pin = pinocytotic plasma membrane of sessile digestive cells, rer = rough endoplasmic reticulum well developed throughout secretory cells and in perinuclear area of sessile digestive cells, res = residual bodies from digestion of heterolysosomes, rsdc = residual sessile digestive cells form when sessile digestive cells become overloaded with residual bodies and lipid and then detach from the epithelium, sect = secretory cells rare and producing acid phosphatase into lumen, sepd = septate desmosomes connect epithelial cells, sev = secretory vesicles synthesised in secretory cells, sdc = sessile digestive cell, stc = stem cells commonly seen paired.

1398

A.R.WALKERandJ.D.FLETCHER

FIG. 3. Representative segment of gut caecum of a nymph at about the engorgement stage of feeding on a naive or a resistant host. Abbreviations as in previous figures but also: er = erythrocytes sometimes pack lumen tightly and often form rouleaux, het = heterlysosomes massive in the sessile digestive cells and in these cells they are formed only from the pinocytotic vesicles but in the motile digestive cells earlier heterolysosomes are of pinocytotic origin and later ones of phagocytic origin, mdc = motile digestive cells form from sessile digestive cells which change activity of the plasma membrane when they lose contact with the basal lamina and some detach into the lumen, monl = mononuclear cell from attachment site on host, npm = non-pinocytotic plasma membrane of motile digestive cell when detaching from the basal lamina, pher = phagocytosis of erythrocytes, phl = phagocytosis of leucocytes, sect = secretory cells not active, stc = stem cells not active after engorgement.

seen to contain large free cells within the blood meal. Histological preparations of these isolated cells showed them to be of the same type as motile digestive

cells seen in sections of intact gut. However many of the motile digestive cells remained adherent to the lumenal surface of sessile digestive cells. Residual sessile digestive cells were never seen during or after the engorgement phase of feeding. The number of both types of digestive cells appeared to be fixed at engorgement. The motile cells ingested whole erythrocytes and leucocytes from the lumen and expanded to pack the centre of the lumen (Figs. 5 and

23). At completion of moulting the gut epithelium became disorganised with gaps where the basal lamina was exposed to the lumen. Some motile digestive cells re-attached to the basal lamina, others remained adherent to sessile digestive cells and the remainder disintegrated in the lumen. This repositioning of the two types of digestive cells during the post-moult development period was seen well in light microscopy specimens stained with pyronin which revealed pyroniphilic residual bodies characteristic of motile digestive cells. Motile digestive cells accumulated massive numbers of haematin-containing

Digestion in ticks

1399

FIG. 4. Representative segment of gut caecum of a nymph at the middle of the moulting period after feeding on a naive or resistant host. Abbreviations as in previous figures but also: bl = basal lamina as an outer and inner layer separated by a continuous sheet of muscle cells, daut = dense autolysosomes, accumulate at periphery of sessile digestive cells, glc = glycogen granules in cytosol of both digestive cell types, het = heterolysosomes predominate in motile digestive cells, lip = lipid droplets predominate in sessile digestive cells, mdc = motile digestive cells tightly pack centre of caecum, must = muscle cells form continuous sheet, res = residual bodies containing haematin accumulate mainly in motile digestive cells, sdc = sessile digestive cells contain variable numbers of heterolysosomes.

residual bodies during the moulting development. These bodies were voided into the lumen by cell disintegration in some cases but it was not seen how the motile digestive cells which remained into the next stage (questing adults) voided their burden of haematin granules (Fig. 23). This excreta was voided from the tick as the black faeces at the completion of post-moult development. Motile digestive cells contained more proteinaceous heterolysosomes and fewer lipid droplets than the sessile digestive cells at the completion of moulting development. At this stage the heterolysosomes were characterised by myelin bodies which may have been derived from the plasma membranes of intact blood cells ingested. They were

also characterised at this stage by small dense membrane bound residual bodies which appeared to be extruded from the larger heterolysosomes (Fig. 24). These stained purple with Giemsa’s stain and were intensely pyroninophilic suggesting that they contained waste nucleotides derived from nuclei of phagocytosed leucocytes. Other types of membrane bound residual bodies were the same small dense bodies as in the sessile cells but not so numerous, and also lucent bodies of various sixes. Both these types were presumed to be autolysosomes. No other enzymic activity was detected in the gut, neither esterases or peroxidases being found in the gut.

A. R. WALKER and J. D. FLETCHER

FIG. 5. Representative segment of gut caecum of an unfed adult female at completion of post-moult development after feeding as a nymph on a naive or resistant host. Abbreviations as in previous figures but also: ax = axon, bl = basal lamina with epithelial cells not closely apposed, daut = dense autolysosomes mainly at periphery of sessile digestive cells, extr = extruded residual bodies from heterolysomes of motile digestive cells only, gj = gap junctions rare, glc = glycogen in sparse deposits, het = hetelysosomes predominate in motile digestive cells at various stages of digestion but lipid droplets predominate in sessile digestive cells, laut = lucent autophagosome with dense outer membranes, lu = lumen with remains of residual motile digestive cells, mit = mitochondria, mdc = motile digestive cells tend to reattach to the gut epithelium, must = muscle cells form an anastomising net around gut caecae, myl = myelin bodies in some heterlysosomes of motile digestive cells, pin = pinocytosis occurs sparsely in both digestive cell types, res = residual bodies, rmdc = residual motile digestive cells disintegrate in gut lumen, sepd = septate desmosomes, sdc = sessile digestive cells.

Pathology ofgut in nymphs fed on resistant rabbits and cattle Gross morphological changes in the gut were evident on dissection; the caecae were very fragile at stage 3 and at all stages of feeding and moulting the caecae were thinner in diameter. The gut epithelium

was thinner, particularly at engorgment, but it was always seen intact. The colour of the ingesta were often pale to white and bubbles of gas (not analyzed) were seen in the lumen on a few occasions. Many more intact leucocytes were seen in the lumen, positively identified types were mononuclear cells, neutrophils

and eosinophils. Ticks with pale ingesta had few host cells of any type in the lumen. Proportions of tick cell types at stage 2 of feeding are shown in Table 2. There were significant differences in the proportions of cells counted by light microscopy in nymphs fed on naive calves compared to nymphs fed on resistant calves. This was not evident in the nymphs fed on rabbits. However, by light microscopy pathological changes were not striking and the gut epithelia were evidently functional (Fig. 25). Stem cells were affected in nymphs fed on resistant

Digestion TABLE

~-PERCENTAGE

OFGUTCELLS~N

R.appendiculatus

1401

in ticks NYMPHSATSTAGE

~OFFEEDINGON

NAIVE

AND

RESISTANT

RABBITSANDCATTLE

Rabbits 1st infestation Sessile digestive Stem cells Secretory cells

cells

Total cells counted

Cattle 3rd infestation

1st infestation

4th infestation

59.5 39.8 0.7

59.6 40.0 0.4

67.8 31.4 0.8

go.5 18.6 0.9

1286

730

971

467

The totals of cells counted were arbitrary samples counted by light microscopy and did not reflect numbers of cells available in a constant area of epithelium. Chi-square calculated for data from nymphs fed on rabbits shows no significant difference in cell proportions between 1st and 3rd infestations but for the data from nymphs fed on calves a significant difference ( P > 0.001) in cell proportions between 1st and 4th infestation.

rabbits and calves when observed by electron microscopy (Figs. 26-29). This was the case in every relevant

tick specimen examined and all stem cells seen showed some effect, usually conspicuously. Their nuclei were poorly defined and poorly staining and often without an intact nuclear membrane. The rough endoplasmic reticulum was poorly defined and sparse, and mitochondria were swollen. Both the microvilli and the convolutions of the peripheral (adlumenal) plasma membrane were poorly developed. Secretory cells were difficult to find by electron microscopy and none were found in nymphs fed on resistant hosts by this method. By light microscopy they were seen poorly developed with fewer secretory granules. Sessile digestive cells showed less pathological change. They appeared to ingest less material by pinocytosis but the plasma membrane, microvilli and pinocytotic pits were always seen intact (Figs. 19, 26, 28 and 29). Some cells had fewer microvilli and pinocytotic pits but it was difficult to judge rates of activity from so few samples. The convolutions of the peripheral (adlumenal) plasma membranes were reduced. At engorgement the sessile digestive cells were stretched very thinly but the gut epithelium always remained intact. By the mid-moulting stage no differences were obvious, the sessile digestive cells appeared normal. Motile digestive cells also showed few pathological changes but they all contained large amounts of cell debris in large heterolysosomes derived from the phagocytosis of erythrocytes, neutrophils and eosinophils (Figs. 20 and 30). Basophils may also have been phagocytosed but could not be identified positively in the gut lumen or in heterolysosomes. At stage 2 of feeding through to early moulting stages of digestion the motile digestive cells contained large amorphous and homogenous areas of proteinaceous material not apparently bound by membranes. It appeared as if some heterolysosomes had burst within the cells. By completion of moulting the motile digestive cells showed few differences from those fed on susceptible hosts except a tendency to contain more of the

pyroninophilic heterolysosomes.

bodies

possibly

extruded

from

the

Female ticks The gut in female ticks during feeding was not examined in detail but all the cell types described for the nymphs were present. In addition a basophilic cell of the same type as described by Till (1961) and Agbede & Kemp (1985) for B. microplus was seen. This type of cell has been shown in R. sanguineus to be a vitellogenin-producing cell (Coons, Tarnovski & Ourth, 1982). Feeding conditions at attachment sites Table 3 shows the cell count data as means for each experimental group at the sampled stages of the serial infestations. There were roughly three times more cells infiltrating into the attachment sites in rabbits (48,8,08 cells counted) compared to the calves (18,828 cells counted). Thus the attachment sites on rabbits were more congested and showed less liquid exudate and haemorrhage than the calves. The proportions of cells showed moderate similarity between the rabbits and cattle with a preponderance of mononuclear cells, followed by neutrophils in the case of rabbits and eosinophils in the case of calves. However, although the proportions of eosinophils differed by a factor of three the actual numbers were closely similar in rabbits and cattle and the same applies to the basophils. Numerous granular cells of all types were seen degranulating in the attachment sites. The great majority of attachment sites of all sampled stages showed little haemorrhage. This was most notable in the cases of preparations containing a sectioned nymph with its gut tightly packed with intact erythrocytes. Intra-epidermal vesicles, necrotic areas and areas containing fibrin nets formed in infestations on resistant rabbits and cattle. However distinct feeding cavities did not form. The cone of attachment cement rarely penetrated below the level of the basal lamina and the mouthparts never penetrated below this level. Thus at all stages of feeding in all

CPm

FIG. 6. Electron micrograph to show a stem cell in a nymph at stage 1 of feeding on a naive rabbit. FIG. 7. Electronmicrograph to show asessile digestive cell in a nymph at stage 2 of feeding on a naive rabbit. FIG. 8. Electronmicrograph to show a secretory cell in a nymph at stage 2 of feeding on a naive rabbit. FIG. 9. Electronmicrograph to show a secretory cell in a nymph at stage 2 of feeding on a naive rabbit. ax = axon, bl = basal lamina, exe = exocytosis of secretory vesicle, cpm = convoluted plasma membrane at basal lamina, lip = lipid droplet, lu = lumen, must = muscle cell, pin = pinocytotic plasma membrane of sessile digestive cell, sect = secretory cell, sev = secretory vesicle, sdc = sessile digestive cell, stc = stem cell.

FIG. 10. Electronmicrograph

to show asessile

digestivecell

in anymph

at stage 2 of feeding on anaive

rabbit.

FIG. 11. Electronmicrograph of perinuclear area of a sessile digestive cell in a nymph at stage 2 of feeding on naive rabbit. FIG. 12. Photomicrograph of gut caecum of a nymph at stage 2 of feeding on a naive rabbit to show acid phosphatase activity in a secretory cell and in sessile digestive cells; stained for acid phosphatase with naphthol phosphate and Schiff s reagent; counterstained with methyl green. FIG. 13. Photomicrograph of gut caecum as in Fig. 12, serial section stained with Giemsa’s stain to show cell types. FIG. 14. Electronmicrograph to show acid phosphatase activity in primary lysosomes and in residual bodies, in a sessile digestive cell in a nymph at stage 2 of feeding on a naive rabbit. Stained by Gomori’s lead method only. asterisk = unknown structure possibly a residual body, acph = acid phosphatase activity red stain shows as darker spots in cells, bl = basal lamina, Gol = GoI& body, het = heterolysosome, lip = lipid droplets, lu = lumen, ly = primary lysosome, mit = mitochondrion, nu = nucleus, nucl = nucleolus, rer = rough endoplasmmic reticulum, rsdc = residual digestive cell, sect = secretory cell, sdc = sessile digestive cell, stc = stem cell.

FIG. 15. Electronmicrograph

to show a residual sessile digestive cell still attached to sessile digestive cells, in a nymph at stage 2 on a naive rabbit. FK. 16. Eiectronmicrograph to show a residual sessile digestive cell which has detached from the gut epithelium, in a nymph at stage 2 of feeding on anaive rabbit. FIG. 17. Photomicrograph to show excretory or secretory vesicles budding off from se&e digestive cells and residual sessile digestive cells in a nymph at stage 2 of feeding of a naive rabbit. Giemsa’s stain. FIG. 18. Electronmicrograph to show excretory or secretory vesicles budding off from a sessile digestive cell in a nymph at stage 2 on a resistant calf. dg = dermal gland, exv = excretory or secretory vesicle, het = heterolysosome, lip = lipid droplet, lu = lumen, npm - non-pinocytotic plasma membrane of residual sessile digestive cells, pin = pinocytotic plasma membrane of sessile digestive cell, pol = polysaccharide material in peripheral cytoplasm of sessile digestive cetis, res = residual bodies, rsdc = residual sessile digestive cell, sect = secretory cell,sdc = sessile digestive cell, stc = stem cell.

FIG. 19. Electronmicrograph

to compare sessile and motile digestive cell m an engorged nymph on a reststant can. FIG. 20. Higher power view as in Fig. 19. FIG. 2 1. Electronmicrograph to show a motile digestive cell migrated away from the basal lamina but still attached to a sessile digestive cell; in a nymph engorged on a resistant calf. FIG. 22. Scanning electronmicrograph to compare lumenal plasma membranes of sessile and motile digestive cells in a nymph engorged on a naive rabbit. The sessile digestive cell had a regular felt of short microvilli associated with pinocytosis. The motile digestive cell had long irregular microvilli and folds of the plasma membrane involved in phagocytosis of the erythrocytes and leucocytes seen in the cell surface. er = erythrocyte, het = heterolysosomes either homogenous containing pinocytotic material in sessile digestive cells or heterogeneous containing phagocytosed material in motile digestive cells, mdc = motile digestive cell, neut = neutrophil, npm = non-pinocytotic plasma membrane, nu = nucleus, pin = pinocytotic plasma membrane, ppm = phagocytic plasma membrane, sdc = sessile digestive cell.

1406

A. R. WALKER and J. D. FLETCHER

Digestion

infestations the nymphs appeared to have access only to cells and liquid exudate which infiltrated between the bundles of collagen in dermis which was relatively intact. Excreta from nymphs during feeding

Table 4 shows the large amount of dry excreta produced during feeding on naive calves. There was less produced when feeding was on naive rabbits and much less when feeding was on either host species when resistant. The excreta resembled dried blood but appeared to have less haemoglobin than whole blood when reconstituted. DISCUSSION

gut of nymphs of R. appendiculatus is affected adversely by components of the meal from resistant hosts. The most severe effect is on the stem cells which are unable to proliferate or differentiate adequately. The resulting hypoplasia prevents the gut from accommodating a blood meal at final engorgment of the large size characteristic of ticks feeding on naive hosts. In nymphs fed on resistant cattle the reduction of stem cell activity results in significant reduction in the proportion of these cells in the gut epithelium. This is not the case in the nymphs fed on resistant rabbits where the stem cells divide sufficiently to maintain the same proportions but do not differentiate sufficiently to provide successive generations of digestive cells. Other pathological effects on the sessile digestive cells compound the inability of the gut to grow during the preparative stage 2 of feeding. We have no direct evidence of what these components harming the gut cells may be. Obvious candidates are the degranulation products from neutrophils, eosinophils or basophils; or antibodies. We observed no interaction between intact granulocytes and tick gut except in the heterolysosomes of the motile digestive cells where it appeared that the tick cell usually demolished the granulocytes. This occurred at the later stages of feeding but the stem cells were inhibited at the early stages of feeding. At day 3 of the infestations on resistant hosts the attachment sites contained numerous granulocytes of all types, many of them degranulated. Thus the The

FIG. 23. Electronmicrograph FIG. 24. Electronmicrograph

in ticks

degranulation products would be at the plasma membrane of the stem cells in the lumen and could be taken up and damage the cells internally. Possible the sessile digestive cells are less affected because their plasma membranes at the lumen are dedicated to extremely active selective pinocytosis coupled to lysosomal digestion. For antibodies to be produced by the host against tick gut antigens during the normal feeding processes, these antigens must occur in the lumen of the gut and be regurgitated into the attachment site. Potential antigens include surface glycoproteins on the plasma membranes around the vesicles which are budded off from the digestive cells, and the glycoprotein acid phosphatases from the secretory cells. Evidence for regurgitation is equivocal. In the attachment sites of adult R. appendiculatus we have seen, mixed with the cement, patches of the small dense residual bodies that are voided into the lumen from the sessile residual cells at the start of feeding (unpublished observations). Regurgitation during tick feeding has been observed by Gregson (1967) but the structure of the mouthparts of hard ticks (Gregson, 1960; Kemp & Tatchell, 1971) appears to be adapted to prevent regurgitation. It is just possible that saliva and the gut lumen share some common antigens; extracts of salivary glands and gut of R. appendiculatus have been shown to contain common antigens by Shapiro, Voigt & Fuji&i (1986). Potential antibodies to glycoproteins on the plasma membranes of digestive cells do not account well for the differentially adverse effect of resistant hosts on the stem cells. A toxin which prevents adequate cell proliferation or differentation seems a more likely explanation. On numerical grounds the predominant source of such a toxin is the extra eosinophils at the attachment sites on the resistant hosts. The effect of resistant hosts on the gut of R. appendiculatus has little effect on subsequent moulting as long as the tick engorges sufficiently to enter into moult. However the resultant adults are small and contain sparse reserves of lipid and protein in their guts. It has been demonstrated by Chiera et al. (1985) that there is a cumulative effect of the harm done by resistant hosts from one instar to the next instar in

to show cell types in gut of an adult female at completion feeding on a naive calf. to show heterolysosomes completion of post-moult

1407

of post moult development

with extruded vesicles in a motile digestive development after feeding on a naive calf.

after

cell of an adult female at

FIG. 25. Photomicrograph of gut caecum of a nymph fed to stage 2 on a resistant rabbit. In comparison with Figs. 13 & 17 the pathological effects in the gut are not striking by light microscopy except for an appearance of lesser development of the epithelium. The lumen typically contained cell debris in comparison with the homogenous liquid ingesta from a naive host. Giemsa’s stain. FIG. 26. Electromnicrograph from a nymph at stage 2 of feeding on a resistant calf to show a pair of moribund stem cells with poorly defined nuclei in comparison with nuclei of the digestive cells, and the paucity of convolutions of all epithelial cells at the basal lamina. bl = basal lamina, daut = dense autolysosome, het = heterolysosome, laut = lucent autolysosome, lip = lipid droplet, lu = lumen, mdc = motile digestive cell, must = muscle cells, rmdc = residual motile digestive cell, sdc = sessile digestive cell, stc = stem cell.

1408

A. R. WALKERand J. D. FLETCHER

Digestion

R. appendiculatus .

It must be stressed that the resistance effects on the gut are only one component occurring towards the end of a sequence of events in the feeding lesion and gut. It does mean however that even ticks which survive earlier avoidance, grooming, intra-epidermal pustulation and congestion of the feeding site by pus and fibrin, or adverse effects on gustatory receptors, will have their viability reduced by a specific effect on the gut. Very strong expression of resistance at early stages of potential infestation could of course prevent attachment and feeding before any effect on the gut was possible. The relationship between what was seen in the attachment sites and in the gut was puzzling. Although there was little haemorrhage at any stage of feeding the nature of the excreta indicates that much material derived from erythrocytes is passed through the gut. Haematin has low solubility in water and although TABLE ~-COUNTS

OF CELLS IN ATTACHMENTsrrss

in ticks

1409

presumably present in the excreta during feeding it is considered unlikely to have confused the assessment of haemoglobin. Similar erythrocyte derived material has been detected in the excreta of other hard ticks by Hamdy (1977). Possibly many erythrocytes are rapidly lysed in the attachment site before ingestion. On resistant hosts some nymphs had such reduced access to blood that they engorged mainly on liquid exudate and appeared white. Rhipicephalus appendiculatus usually engorge to a slightly heavier weight at all instars when fed on cattle rather than rabbits and this improved feeding performance may relate to the greater passage of ingesta through the ticks during the pre-engorgement feeding. Willadsen, Kemp & McKemia (1984) have noted for B. microplus a more extreme difference in engorgement weights when adults were fed on rabbit blood in vitro, despite readiness of the ticks to commence feeding. The differences noted by these authors was

OF R. appendiculatus NYMPHS

OF NAIVE

AND

RESISTANT

RABBITS

AND

CATTLE

Neutrophils

Mononuclear cells

Eosinophils

0 45 (8) 147 (92) 538 (182)

0 0 10 (22) 41 (44)

488 708 684

Basophils

Mast cells

Rabbits 1:

Controls day -3 1st infestation day 1 1st infestation day 3 1st infestation day 5

132 414

(19) (145) (210)

3rd infestation 3rd infestation 3rd infestation

816 570 664

(303) (325) (376)

day 1 day 3 day 5

(102) (240) (188)

102 218 269

(56) (89) (199)

9 (6) 6 (7) 51 (24)

8 8 7 4

(4) (5) (4) (2)

49 (28) 89 (35) 84 (50)

2 (1) 2 (2) 2 (2)

Cattle Controls day -3 1st infestation day 1 1st infestation day 3 1st infestation day 5 4th infestation 4th infestation 4th infestation

day 1 day 3 day 5

0 3 (4) 68 (96) 29 (31) 100 219 181

(120) (144) (134)

1 11 52 94 226 246 330

(1) (6) (44) (30) (112) (58) (112)

2 1 4 10 206 254 170

(1) (2) (5) (12) (196) (204) (120)

1 2 6 3

(1) (2) (12) (3)

39 (17) 43 (27) 52 (41)

8 11 10 11

(5) (5) (3) (6)

8 (4) 7 (4) 7 (3)

Means of two biopsies each from four rabbits and four calves for each day; total recognizable infiltrating cells and mast cells counted in each biopsy for each animal so that each count in the Table represents a mean of the totals for 20 fields of view counted with a total area 0.48 mm*. Standard deviations in parentheses calculated from totals for each biopsy.

FIG. 27. Electronmicrograph

to show a pair of normal stem cells in a nymph comparison with Fig. 28.

as stage 2 of feeding

on a naive rabbit,

for

FIG. 28. Electronmicrograph to show a pair of moribund stem cells in a nymph at stage 2 of feeding on a resistantrabbit. The nuclei, rough endoplasmic reticulum and convolutions of the peripheral plasma membrane (asterisked) are all poorly defined. The adjacent sessile digestive cells are less affected and of more normal appearance. FIG. 29. Electronmicrograph to compare nuclei of a stem cell and a sessile digestive cell in a nymph at stage 2 of feeding on a resistant calf. The stem cell nucleus has a poorly defined nuclear membrane and poorly staining heterochromatin. FIG. 30. Electronmicrograph to show a heterolysosome in a motile digestive cell from a nymph at engorgement on a resistant rabbit. The heterolysosome has lost the typical simple rounded shape and contains an intact host leucocyte. The crystalline inclusions in the leucocyte indicate it may be an eosinophil. The membrane of the heterolysosome appears to have disintegrated in contact with the eosinophil. asterisks = areas of convoluted peripheral plasma membrane, bl = basal lamina, het = heterolysosome, lip = lipid droplet, lu = lumen, nu = nucleus, phi = phagocytosed leucocyte, ppm = phagocytic plasma membrane, sdc = sessile digestive cell, stc = stem cell.

A. R. WALKERand J. D. FLETCHER

1410 TABLET-WEIGHT AND

HAEM~GLOB~N

DURlNGFEEDlNGON

R.appendiculatus

CONTENTOFEXCRETAFROM

NYMPHS

NAlVEANDRESlSTANTRABBlTSANDcATTLE

Excreta (mg/nymph)

Haemoglobin

Haemoglobin

in tick excreta (g/ 100 ml)

in host blood (g/100 ml)

Rabbits 1st infestation

1.5

6.1

3rd infestation

>0.4*

ND

ND 12.3

4.2 0.4

6.8 2.8

ND 10.3

Cattle 1st infestation 4th infestation

* Excreta was seen but too little to collect and weigh accurately, estimate given by visual comparison with excreta from nymphs on calves. ND = not done. Values for excreta represent the total produced per nymph over its entire feeding period.

also in the ability of the ticks to convert the blood meal to eggs which may relate more the ability of the digestive cells to absorb the blood meal. Host specificity may thus relate to the ability of the sessile digestive cells to absorb, by selective pinocytosis, sufficient nutrients to prepare the tick for full engorgement. At engorgement the gut was seen packed with intact erythrocytes but the contents remained fluid. No sources of anticoagulant were apparent in the gut of R. appendiculatus nymphs. Other authors have suggested that anticoagulants or haemolysins are secreted by gut cells (Agbede & Kemp, 1985; Hughes, 1954; Chinery, 1964). In R. appendiculatus nymphs there are the Cz cells of the salivary glands (closely similar to those seen in adults as described by Walker, Fletcher & Gill, 198.5) which actively secrete a glycoprotein with the metachromatic staining characteristic of anticoagulant proteoglycans. In the gut the acid phosphatase secreted may act as a haemolysin but is most active at the pre-engorgment stage of feeding. Proteolytic enzymes in other species of hard tick have been found active only during feeding by Araman (1979) and by Bogin & Hadani (1973). In R. appendiculatus the stem cells correspond with those described for other species and the residual sessile cell corresponds with the spent digestive cell described by Agbede & Kemp (1985). However the secretory cell appears to have unique characteristics of rarity, transience and dedication to secretion of acid phosphatase into the lumen. It may however perform similar functions to the S, secretory cell described by Agbede & Kemp (1985). Our descriptions of the phases of activity of the two types of digestive cell are contrary to those of other authors. The pinocytotic type and the phagocytic types described by other authors presumably have similar functions to the sessile and motile cells respectively as described in this paper. In contrast to previous descriptions we maintain that the phagocytic cell is derived from the pinocytotic cell when some of the latter detach from the basal lamina at and after engorgement. The relationship of these two cell types to the basal lamina

seems more fundamental than the activity of their plasma membranes. The phagocytic cells are derived from pinocytotic cells and are capable of reverting to pinocytotic activity after the moult. Thus the terms sessile and motile with respect to the basal lamina have been introduced to describe the pinocytotic digestive cell and the phagocytic digestive cell respectively. It is difficult to prove that the motile digestive cells are not attached to the basal lamina by slender peduncles but we have never seen such connections. Large cells containing dark masses of residual bodies are seen free in the lumen when the gut is torn open after engorgement and such cells when isolated and prepared histologically can be seen to be motile digestive cells. It remains difficult to explain how the digestion products reach the haemolymph from these motile digestive cells during moulting, but after moulting these cells contain the protein reserves in the gut for the questing stage of the life cycle. Residual bodies were difficult to classify but all were membrane bound and thus seemed derived from auto- or heterolysosomes. Siderosomes may be expected but were not detected according to the ultrastructural criteria of Richter (1957) for mammalian siderosomes. We achieved no useful results for the haemosiderin test but this may have been due to technical difficulties with blue/black granules embedded in methacrylate. Haematin containing residual bodies may also contain melanin as suggested for I. ricks by Hughes (1954). The main mode of digestion in R. appendiculatus appears to be intracellular by the lysosomal acid phosphatase system as demonstrated for H. asiaticum by Raikhel(l975). But the other sources of acid phosphatases in the tick feeding system, the neutrophil granules from the host and the secretory cells in the gut, may pre-digest the ingesta in such a way that selective pinocytosis is more efficient. This may be particularly important during the early preparative stages of feeding when the tick has the opportunity of selecting the most suitable nutrients for rapid growth whilst voiding large volumes of less suitable blood derivatives.

Digestion Acknowledgemenrs-We wish to thank Prof. D. W. Brocklesby and Mr. C. G. D. Brown of this Centre for their advice and encouragement and Mr. D. W. Penman and Mr. S. R. Mitchell of the electron microscopy unit of the Royal (Dick) School of Veterinary Studies, Edinburgh, for their photographic assistance and preparation of the SEM specimens. This study was supported by the Overseas Development Administration of the United Kingdom. REFERENCES ACBEDE R. I. S. & KEMP D. H. 1985. Digestion in the cattle .tick Boophilus microplus: light microscope study on the gut cells in nymphs and females. ~nternat~onalJournalfor Par~~olff~ 15: 147-157. AGBEDE R. I. S. &KEMP D. H. 1986. Immunisationof cattle against Boophilus microplus using extracts derived from adult female ticks: histopathology of ticks feeding on vaccinated cattle. International Journal for Parasitology 16: 35-41. ARAMAN S. F. 1979. Protein digestion and synthesis in ixodid females. IX Recent Advances in Acarology, Vol. I (Edited by RODRIGUEZ J. D.), pp. 385-395. Academic Press, New York. BALASHOV Y. S. 1972. Bloodsucking ticks (Ixodoidea)vectors of diseases of man and animals. Miscellaneous Publications of the Entomological Socieiy of America 8: 159-376. BALASHOV Y. S. 1983. An Atlas of Ixodid Tick Ultrastructure. Special Publication of the Ent~moio~cai

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GREGSONJ. D. 1960. Morphology and functioning of the mouthparts of Dermacentor andersoni. Acta Tropica 17: 48-79.

in ticks

GREGSONJ. D. 1967. Observations

1411

on the movement of

fluids in the vicinity of the mouthparts of naturally feeding Dermacentor andersani. Parasitoloav 51: l-8. HAMDY B. H. 1977. Biochemical and>hvsiolotical studies of certain ticks. Excretion during ^fekding.- Journal of Medical Emomolo~ 14: 15-18. HUGHES T. E. 1954. sbme histobgical changes which occur in the gut epithelium of Zxodes ricinus femrlles during and up to oviposition. Annals of Tropical Medicine and Parasitology 48: 397-404. JOHNSTON L. A. Y., KEMP D. H. & PEARSONR. D. 1986. Immunisation of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced i~u~ty on tick ~pulations. ~ntemational Journal for Parasitolo~ 16: 27-34. KEMP D. H. & TATCHELL R. J. 1971. The mechanism of feeding and salivation in Boophilus microplus. Zeitschrifi fiir Parasitenkunde 37: 55-69. KEMP D. H., AGBEDE R. I. S., JOHNSTON L. A. Y. & GOUGH J. M. 1986. Immunisation of cattle against Boophilus microplus using extracts derived from adult female ticks:

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TILL W. M. 1961. A contribution to the anatomy and histology of the brown ear tick Rhipicephalus appendiculatus. Memoirs of the Entomological Sociery of Southern Africa 6: 1-124. WALKER A.R., FLETCHER J. D. & GILL H. S. 1985. Structural and histochemic~ changes in the salivary glands of Rhip~cephalus appe~~culat~ during feeding. international Journalfor Parasitology 15: 8 l-100. WALKER A. R. & FLETCHER J. D. 1986. Histological study of the attachment sites of adult Rhipicephalus appendiculatus on rabbits and cattle. International Journal for Parasitology 16: 399-413. WILLADSEN P., KEMPD. H. & MCKENNA R. V. 1984.Blood meal ingestion and utilisation as a component of host specificity in the tick Boophilus microplus. Zeitschri~~r Parasitenkunde 70: 4 15-420.