Richard R. Mills

Hormonal control of tanning in the American cockroach: Changes in blood cell permeability during ecdysis

Abstract The content of diuretic hormone (DH), antidiuretic hormone (ADH), and tanning hormone (bursicon) in the haemolymph have been determined for several periods during the moulting process in Periplaneta americana. ADH builds up immediately prior to the ecdysis and causes the blood volume to increase. DH and bursicon rise simultaneously after the ecdysis which results in a dramatic decrease in blood volume. Further investigations have shown that blood cell permeability to tyrosine is enhanced by post-ecdysis blood and by partially purified hormone preparations. From these data (and from separation procedures which indicate a similar molecular weight for DH and bursicon) it is postulated that DH and tanning hormone may be analogous. Tanning hormone or DH may serve the function of increasing the blood cell permeability to tyrosine and thus allowing the subsequent formation of N-acetyl dopamine.

Studies on vitellogenesis in the American cockroach

Abstract An ootheca is produced approximately every 6 days by the female American cockroach. Feeding takes place on the first 2 or 3 days of this 6 day cycle and the blood protein level peaks on the fourth day. Most of the yolk protein is deposited on the last 2 days of the cycle. Total RNA concentrations have been determined for both the midgut and fat body during the cycle, and the results of these experiments suggest that both organs take part in blood protein synthesis. Ultrastructural examination of these two tissues support this contention. Cauterization of the medial neurosecretory cells results in a decrease of blood protein levels. Allatectomy causes a rise in blood protein and prevents vitellogenesis. Implantation of three active corpora allata restores both the normal blood protein level and vitellogenesis. A humoral control of blood protein synthesis is postulated and the hormonal and/or nervous control of vitellogenesis and subsequent ootheca production is discussed.

Biosynthesis of N-acetyl dopamine by the American cockroach

Abstract Tyrosine is transaminated to para-hydroxyphenylpyruvic acid, decarboxylated to para-hydroxyphenethylamine (tyramine), and hydroxylated to dihydroxyphenylalanine (dopa) by crude homogenates of freshly ecdysed American cockroaches. Dopa is further decarboxylated to dihydroxyphenethylamine (dopamine) which is subsequently acetylated to N-acetyldopamine. Tyramine is acetylated to N-acetyltyramine, and these two N-acetyl derivatives are postulated to be the precursors of the sclerotization quinones. The addition of purified bursicon preparations to each of the reaction mixtures fails to accelerate or inhibit. This suggests that cockroach bursicon does not exert a direct effect on the synthesis of the two N-acetyl derivatives.

Correlation of phenoloxidase activity with ecdysis and tanning hormone release in the American cockroach

Abstract Phenoloxidases from the cuticle and haemolymph of the American cockroach have been followed during the process of ecdysis. The cuticle enzyme is most active immediately before the moult and becomes progressively less active during and after ecdysis. The lowest point of activity occurs during the postmoult period. The blood phenoloxidase, on the other hand, peaks during the moult and is slightly less active on either side. Since bursicon release occurs after ecdysis there appears to be no correlation between the tanning hormone and phenoloxidase activation. The cuticle diphenoloxidase is active against N-acetyldopamine, dopamine, and dopa whereas the haemolymph component will act on a variety of mono- and diphenols. The cuticle enzyme has been localized histochemically in the exocuticle, and the phenoloxidase of the blood is confined to the haemocytes.

The midgut epithelium of the American cockroach: Acid phosphomonoesterase activity during the formation of autophagic vacuoles

Abstract Ultrastructural examination of the midgut epithelium of Periplaneta americana (L.) has shown that starvation causes a cyclic change in the number of autophagic vacuoles. A peak in number of vacuoles occurs 4 days after starvation is initiated. Correlated with these changes is a rise and fall of acid phosphatase activity with a peak identical to that of the vacuoles on day 4. Histochemical localization of the enzymatic activity within the vacuoles suggest that they are autophagic in nature. The midgut acid phosphatase has been partially purified and characterized.

Cuticle sclerotization by the American cockroach: Immunological evidence for the incorporation of blood proteins into the cuticle

Abstract Partially purified proteins from the cuticle of the American cockroach were injected into mature rabbits. Immunodiffusion techniques using the rabbit antibody show that there are at least two cockroach blood proteins which are antigenically similar to the cuticle protein. These blood proteins appear to be present throughout the life of the insect but preliminary results indicate that a higher concentration occurs during the inter-ecdysial stage. The significance of these findings in regard to cuticle synthesis and protein aggregation are discussed.

Hormonal control of tanning in the American cockroach—IV. Preliminary purification of the hormone

Abstract The tanning hormone ‘bursicon’ has been partially purified from the central nerve cord by centrifugation, ammonium sulphate fractionation, and gel filtration. An eightyfold purification was achieved but the limited protein concentration prohibited further isolation. Subsequent purification procedures utilized intact newly moulted cockroaches and a 1700-fold purification was realized. Separation involved the use of differential centrifugation, ammonium sulphate fractionation, gel filtration on P-30 and P-60 polyacrylamide gels, and column chromatography on DEAE cellulose.

Changes in electrophoretic properties of haemolymph and terminal oöcyte proteins during vitellogenesis in the American cockroach

Abstract Fifteen negatively charged protein bands were found by acrylamide gel electrophoresis to be present in whole blood of the American cockroach during the vitellogenic cycle. Six of the blood proteins appear to be present in the soluble fraction of the terminal oocytes. Most of the blood proteins were found to be cyclic in nature reaching their greatest concentration on days 3 and 4 of the cycle. Most of the soluble proteins in the terminal oocyte exhibited a maximum concentration corresponding to that found for whole blood. Day 5 or 6 was characterized by a decrease in the concentration of oocyte proteins. Mechanisms accounting for the cyclic changes of protein concentrations in blood and oocyte are hypothesized.

Hormonal control of excretion in the american cockroach, II. preliminary purification of a diuretic and antidiuretic hormone

a n1. n1. An in vitro rectal preparation has been devised which allows the measurement of lumen volume changes by the isotope dilution technique. n n2. n2. This sensitive assay provided the necessary accuracy to determine small changes incurred when dilute endocrine samples from column chromatography were usedas the hormone. n n3. n3. A diuretic hormone of a molecular weight greater than 30,000 and an antidiuretic hormone of about 8000 have been partially purified by gel filtration on columns of P-30 polyacrylamide. n n4. n4. The purification scheme indicates that contaminates of low molecular weight are present in the terminal abdominal ganglion which could be released in the intact animal and also modify rectal water absorption.

Hormonal control of tanning in the American cockroach—II. Assay for the hormone and the effect of wound healing

Abstract Previous investigations have indicated that an apparent inhibitory centre exists in the mesothorax of the American cockroach. In an effort to elucidate the reason for its presence, various organs were tested for hormone activity, and the effect of wound healing was determined. Neither appear to influence darkening by the thorax. An alternate hypothesis involving the circulation of the blood has been suggested. An assay for tanning hormone revealed that all parts of the nervous system were active, but only the terminal abdominal ganglion released hormone into the blood. This confirms previous observations that tanning is controlled by the ganglion.