A modified method for obtaining tick oral secretion.

This paper describes a technique modified from one previously used for Dermacentor andersoni, to obtain salivary secretions of Boophilus microplus by the application of capillary tubes of critical diameter to mouthparts. Hypodermic injections of pilocarpine in saline were found to cause sixfold increase in secretion over that obtainable from tactile stimulus alone, while the constituents of the secretion were unchanged except for pilocarpine being present. It was not possible to determine whether secretions obtained by these techniques represent the entire range of salivary secretions. The properties of the salivary secretion of ticks are of obvious interest for studies on feeding mechanisms, pathogenicity to host, and disease transmission. Gregson (1960), demonstrated that production of salivary (or oral) secretion can be stimulated in Dermacentor andersoni by an appropriate tactile stimulus to the chelicerae applied by fitting a capillary tube of critical internal diameter over the mouthparts. We have confirmed that saliva can be obtained by this method from females of Boophilus microplus recently removed from the host, using a micropipette with an internal diameter of 0.23 mm at the tip. Capillaries that did not compress the chelicerae failed to elicit any secretion. The most satisfactory stage of B. microplus was found to be a female weighing 50 to 150 mg (i.e., a tick that had started the final period of approximately 12 hr rapid engorgement before detaching). However, even with this stage it was only successful in 10% to 25% of cases, with a maximum of 5 /0l saliva/tick, and the saliva was liable to be quickly sucked back by the tick. The results of Day (1951) with the cockroach suggested the injection of the parasympathetic stimulant pilocarpine in order to obtain larger amounts of salivary secretion. This induced the production of up to 30 ul fluid in the majority of ticks. For convenience, recently detached fully engorged adult females were used and the production of secretion was enhanced if the ticks were maintained for 3 to 4 hr after injection at 37 C and relative humidity higher than 90%. Figure 1 shows a row of ticks stuck to double-sided cellulose tape on an inclined ledge (the cuticle of some ticks, e.g., Ixodes holocyclus, does not adhere to cellulose tape). Received for publication 24 May 1967. A further strip of clear cellulose tape is stuck over the row of ticks. The injection is given hrough the tape and under the cuticle of the tick using 26or 27-gauge hypodermic needles and a syringe mounted in an Agla micrometer syringe holder. The needle can be observed during the operation and excessive trauma prevented. On removal of the needle the puncture usually seals itself with a small cecal extrusion, although some hemolymph and injected fluid may escape. Capillary tubes are then brought in contact with the mouthparts of the tick and held in place with plasticine; the tubes fill under capillarity and gravity. The injection consists of 5 to 10 1J 10% pilocarpine in 1.75 x diluted Levy stock saline (Levy, 1928). Greater yields were obtained with this strong saline or with NaCl solutions of equivalent concentration than with normal solutions (Fig. 2). It is likely that a certain amount of ad hoc adjustment to these concentrations might be necessary with different arthropods. The use of pilocarpine has also been reported by Howell (1966), who found with Ornithodoros savignyi, an argasid tick, that an injection of 30 /J1 0.2% to 0.3% pilocarpine (the solvent is not stated) produced 5 to 9 1d of secretion. Howell also found that the effect of the stimulant tended to dissipate after 45 min; this was not noted with B. microplus over 3 to 4 hr, possibly because of the greater concentration of stimulant used. The secretion collected by this method has been found to be virtually identical with that obtained more naturally by Gregson's (1960) technique. Thus the same enzymes, amino acids, electrolytes, etc., have been found, although the pharmacological activity may be less. Caution must be used in interpreting these aspects of salivary activity and relating them