Muscle and nerve are commonly thought of as functioning optimally in an extracellular environment whose potassium concentration is low compared to that iiitracellularly. There is, however, evidence that in certain insects the tissue environment may contain very large amounts of potassium. Thus, it has been claimed that in insect haemolymph in general "sodium seems always to be replaced largely by potassium" (Wigglesworth, '39), and recently it has been shown that while the Na:K ratio in the haeinolymph of carnivorous insects is greater than unity, it is less than unity in phytophagous insects (Bon6, '44). For ariyoiie interested in the physiology of muscle and nerve such findings can act as adequate stimuli for quantitativc chemical studies beyond those presently available in the literature. I n response, this report concerns itself with studies on an omnivorous insect ; preliminary work on phytophagus insects is reported in the paper follofving this one (Tobias, '48).
Toward the end of understanding the situation in Periplaneta americana measurements have been made of water, sodium and potassium in haemolymph, serum, nerve cord and
The present investigation was aided in part by a grant from the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago, and in part under contract between the Medical Thision, Chemical Corps, U. S. Army and the University of Chicago Toxicity Laboratory. Under the terms of the contract the authors are free t o draw their own conclusions.
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JULIAN 31. TOBIAS muscle with the insects on a mixed diet (Purina dog chow, apple and water), on a high potassium diet (lettuce and water) and at fixed times after the ingestion (oral intubation) of measured volumes of known concentration KC1 solutions.
Methods
The roaches, Periplaneta americana, which have been used throughout, were collected at random from the basements of 2 University buildings. Precapture history and nutrition are therefore unknown, but all the animals were kept f o r at least 2 weeks on a diet of Purina dog chow checkers, apple and water before beinw used.
Oral administration of measured solutions is readily accomplished, after pinning the supine insect, through the wings, to cardboard, by gently inserting the blunted tip of a 27 gauge hypodermic needle through the mouth into the gut. Volumes up to at least 0.2cm3 per gram of body weight can then be injected from a 0.25 em3 tuberculin syringe with relatively rare regurgitations.
To obtain tissues for analysis the supine insect is fixed in position by pins passing through the pronotum and posterior abdomen. The antennae are amputated and the head and legs immobilized with pins. Kext the ventral surface of the neck and thorax is cleaned with a bit of distilled water on a swab and dried. A small incision (0.5-1mm long) is then made through the thin exoskeleton where the prothoracic leg joins the thorax. The haemolymph which wells up is drawn by capillarity into melting point tubes (0.5 mm i.d.). Bleeding can be speeded and increased by gentle massage of the abdomen, and, on the average, 5-20mg of haemolymph are obtained from each insect. If whole haemolymph is to be analyzed it is expelled from the tubes into previously weighed, small, platinum combustion boats, reweighed, dried and analyzed as described below. If serum only is wanted, the capillary tube is sealed at one end, with care not to warm the contents, and centrifuged. The cells go to the bottom which can be broken off with a file, and the clear serum is then blown into a boat.