Aust. J. Bioi. Sci., 1976, 29, 487-97 Boophilus microplus: Characterization of Enzymes Introduced into the Host A. V. Schleger and D. T. Lincoln Division of Animal Production, CSIRO, Long Pocket Laboratories, Private Bag No.3, Indooroopilly, Qld 4068. Abstract A number of enzymes, presumably secreted by larvae of B. microplus under natural feeding conditions, have been investigated in the skin of previously unexposed calves 4 h after infestation at the attachment site. Carboxylic ester hydrolase activity was demonstrated in the dermis, immediately adjacent to the mouthparts, or in the attachment cone, depending on substrate and reaction pH. The carboxylic ester hydrolase acting on naphthol AS-D acetate (2-acetoxy-3-naphthoic-O-toluidide) at pH 7·1 was characteristically found in the dermis and not in the attachment cone. The use of specific inhibitors showed that this enzyme was primarily a B-esterase or carboxylesterase with possibly a small portion of C-esterase or acetylesterase. It is postulated that carboxylic ester hydrolase could contribute to the dilation observed in the subepidermal capillaries adjacent to the attachment sites of unexposed animals, through the formation of plasma kinins. Other enzymes demonstrated in the dermis, adjacent to the mouthparts, were triacylglycerol lipase, as an aggregated deposit, and small amounts of aminopeptidase (microsomal) and monophenol monooxygenase. Aminopeptidase (microsomal) was also demonstrated in the attachment cone or adjacent epidermis, according to the substrate used. No activity was found in the host tissue, in association with the attachment site, for either alkaline or acid phosphatase, acetylcholinesterase or cholinesterase, peroxidase or amine oxidase (flavin-containing), despite the intense histochemical reaction for the latter in the tissues of larvae. Introduction Tick resistance in cattle, known to be immunologically mediated (Roberts 1968a), is especially manifest during the first 24 h of larval attachment (Roberts 1968b). The saliva secreted by larvae therefore probably contains the antigens against which the host has mounted an immunological response. No information is available on the secretions by tick larvae soon after attachment, studies so far having been restricted to saliva obtained from adult females which have completed the parasitic life cycle and have been stimulated with pilocarpine. Tatchell (1971) obtained a number of esterase and acid phosphatase bands in electrophoretic studies on saliva, whilst Geczy et al. (1971) showed that the saliva of adult ticks contains esterase. Tatchell also demonstrated three enzyme types in the haemolymph, namely esterases, phosphatases and aminopeptidase. If there are enzymes in the saliva of larvae it should be possible to detect them at attachment sites by histochemical techniques. Such enzymes might also be antigens and this could be important in studies of host response and mechanisms of the resistance of cattle to ticks. In the present study enzyme activity at larval attachment sites was investigated on previously unexposed hosts, using histochemical procedures 488 A. V. Schleger and D. T. Lincoln for carboxylic ester hydrolase, acid and alkaline phosphatase, acetylcholinesterase and cholinesterase, triacylgJyceroJ lipase and three oxidoreductases. Materials and Methods Animals In each investigation a Shorthorn calf, previously unexposed to tick larvae, was used. The unexposed calf was clipped along the loin and a number of chambers, 1· 5 cm in diameter, were fixed to the skin. Approximately 2000 larvae of the Yeerongpilly strain (Stone 1957) were placed in each chamber and confined with nylon gauze. After 4 h the skin under each chamber site was infiltrated with 2 % xylocaine (Astra Chemicals Pty Ltd, North Ryde, N.S.W.) free of adrenaline, and a tissue biopsy was taken with a 1 cm diameter trephine. Table 1. Commercial and systematic names of substrates, diazonium salts and inhibitors used in histochemical studies Commercial name Systematic name Substrates Naphthol AS-D acetate 2-Acetoxy-3-naphthoic-o-toluidide Naphthol AS-TR phosphate 3(4-Chloro-2-methyl anilide)-2-naphthyl phosphate Diazonium salts Fast blue RR 4-Benzoylamino-2,5-dimethoxy aniline Fast blue B 0-Dianisidine Fast red TR 5-Chloro-o-toluidine Fast garnet GBC 4-Amino-3,I-dimethyl azobenzene Hexazonium PR Hexazotized fuchsin (p-rosanilin) Inhibitors Coroxon 3-Chloro-7-hydroxy-4-methyl coumarin diethylphosphate Iso-OMPA Tetra-isopropylpyrophosphoramide 284C51 1,5-Bis-(4-allyl dimethyl ammonium phenyl) pentan-3-1-diiodide PCMB p-Chloromercuribenzoate Tissue Processing The biopsies were quenched in a tube of liquid propane surrounded by a jacket of liquid nitrogen, and transferred to a liquid nitrogen cabinet until required. Some tissues were fixed for 4-6 h in 10% neutral formalin at 4°C, and, after washing, held in the cryostat at -20°C. Control data on the enzyme activities of unfed, freshly hatched larvae were obtained by immersing larvae in 20 % gelatin, with 1 % Triton X-I00 added, in an aluminium foil thimble, and the thimble was quenched in liquid propane. The gelatin block was then transferred to the liquid nitrogen cabinet. Tissues were transferred from the liquid nitrogen cabinet to the cryostat in a container cooled with dry ice. Histochemical Procedure Sections 12.um thick were cut in a cryostat and, for the typing of the esterases, about 20 sections were taken up on each slide. Two slides provided sections through approximately 100 lesions, although different sections might come from the same lesion. After 20 min air drying of the sections at room temperature, the slides were held at 4°C for a minimum time before the reactions were carried out. The commercial and systematic names of substrates, diazonium salts and inhibitors used in this investigation are shown in Table 1. (i) Carboxylic ester hydrolase (EC 3.1.1) The histochemical procedures used for demonstrating esterase activity involved the use of naphthol AS-D acetate substrate (2-acetoxy-3-naphthoic-O-toluidide) at pH 7·1 with Fast blue RR Larval Enzymes of Boophilus microplus 489 coupling agent (Burstone 1962), O-acetyl-5-bromoindoxyl substrate at pH 8· 8 with Fast blue RR coupling agent (Delellis and Fishman 1965; Pearse 1972), and a-naphthyl acetate substrate at pH 7·4 with Fast blue B as coupling agent (Pearse 1972). When naphthol AS-D acetate was used as substrate, the carboxylic ester hydrolases were classified in this study as A, Band C types as suggested by Pearse (1972), approximately synonymous names being arylesterase (EC 3.1.1.2), carboxylesterase (EC 3.1.1.1) and acetylesterase (EC 3.1.1.6), respectively. The inhibitors used and their effect on the three types of esterase and cholinesterase are shown in Table 2. The inhibitors and their concentrations are as recommended by Pearse (1972) with the addition of the organophosphate Coroxon which was used at 0·001 M concentration. Table 2. Inhibitors used in the study of carboxylic ester hydrolases in lesions produced in previously unexposed animals by larvae of B. micropius Treatment Inhibitors Enzymes affected Enzymes unaffected 1 10 11M eserine Cholinesterases Carboxylic ester hydro lases 2 0·001 M CUS04 A-, C-esterase B-esterase 3 0·001 M CUS04 A-, C-esterase, B-esterase + 10 11M eserine cholinesterases 4 O' 001 M Coroxon B-esterase A-, C-esterase 5 0·001 M Coroxon B-, A-esterase C-esterase +0·001 M PCMB Sections were preincubated for 30 min in 0·2 M tris buffer (pH 7·1) at 37°C, with the appropriate inhibitor, before being transferred to the substrate with the inhibitor again added. Different combinations of inhibitors were used, and different comparisons of inhibitor effects were made, to deduce the contribution of each type of esterase activity. Because of the varying number of lesions appearing in the same number of sections, inactive lesions were expressed as a percentage of the total. The rationale of the esterase typing is shown in Table 3. Table 3. Carboxylic ester hydrolase activity deduced from the increase in inactive lesions through the use of specific inhibitors according to Table 2 Inactive lesions showed no enzyme activity Difference between treatments (Table 2) Carboxylic ester hydrolase activity 1-(Treatment 2+treatment 3)-treatment 1 A- and C-esterase Treatment 5-treatment 4 A-esterase Treatment 5-treatment 1 B- and A-esterase Treatment 4-treatment 1 B-esterase Lesions were assessed as inactive (-), i.e. showing no enzyme activity, or as having three degrees of intensity of histochemical reaction: +, + + and + + +. It was assumed that the loss of activity through the selective inhibition of one or more esterase components resulted in an increase in the number of inactive lesions, and the percentage of inactive lesions was used as an index of inhibitor effects. For that reason, intensity of reaction in the positive lesions was not considered in the data analysis. (ii) Acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) The activity of acetylcholinesterase and cholinesterase was Idemonstrated by the method of Koelle and Friedenwald (1949), using acetylthiocholine iodide and butyrylthiocholine iodide as substrates. The inhibitors used in the distinction between acetylcholinesterase and cholinesterase activity were as recommended by Pearse (1972). 490 A. V. Schleger and D. T. Lincoln (iii) Triacylglycerollipase (EC 3.1.1.3) The method of Gomori (1952), using Tween 80 or Polysorbate 80 (a complex mixture of polyoxyethylene ethers of mixed partial oleic esters of sorbitol anhydrides) at pH 7·3 as substrate, was used to demonstrate lipase. (iv) Aminopeptidase (microsomal) (EC 3.4.11.2) The demonstration of aminopeptidase (microsomal) was based on the method of Ackerman (1960). Two substrates were used~alanyl-;9-naphthylamide and l-leucyl-4-methoxy-;9-naphthylamide~at pH 6· 7 and two coupling agents~Fast garnet GBC and Fast blue B (Table 1). (v) Acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) Acid phosphatase activity was demonstrated by the method of Barka and Anderson (1962), using naphthol AS-TR phosphate at pH 5·0 as substrate and hexazonium PR as the coupling agent.