Carbonic anhydrase and bicarbonate regulation during the larval-pupal transformation of the tobacco hornworm (Manduca sexta) and the silkworm (Hyalophora cecropia)

This study represents an effort to answer basic questions concerning physiological mechanisms involved in the larval-pupal transformation of lepidopterous insects. More specifically it examines the role of bicarbonate in the process of moulting in the tobacco hornworm, Manduca sexta, and the silkmoth, Hyalophora cecropia. This role has been evaluated through an examination of:

1. the levels of bicarbonate in hemolymph and moulting fluid, 2. the mode and sites of bicarbonate production, 3. the regulation of this production, and 4. the physiological mechanisms of bicarbonate involvement in the secretion and resorption of moulting fluid.

In order to effectively quantitate levels of bicarbonate in the extremely small fluid volumes associated with insects, a new technique for assaying carbon dioxide under conditions of small fluid volumes has been devised. By this technique it is now possible to cheaply and conveniently assay micromolar quantities of carbon dioxide in fluid volumes as small as ten microliters.

The mode and sites of bicarbonate production were evaluated by assaying for the major enzyme involved, carbonic anhydrase. This study represents the first time that carbonic anhydrase has been reported in insect fat body and integument. In addition, important questions dealing with the regulatory effects of halide anions and alkali metal cations on carbonic anhydrase activity in a variety of insect tissues have been answered. In M. Sexta all three enzymes (midgut, fat body and integument) are activated by physiological levels of K ⁺ and either inhibited or unaffected by physiological levels of Cl". In foliage reared H. Cecropia both K⁺ and Cl^- are inhibitory to the three tissue enzymes, while lab reared feeding fifth midgut carbonic anhydrase is activated by Cl l^-

These regulatory effects differ from the anion and cation effects on mammalian carbonic anhydrase. In fact, Cl is reported to always be inhibitory to mammalian carbonic anhydrase, while the effects of cations have not been reported. In addition, a major controversy has arisen regarding the kinetics of this Cl l^- inhibition. Cl l^- inhibition is

to the insect enzyme (especially in M. Sexta, in which K⁺; activates and Cl l^- inhibits), the controversy regarding the inhibitory mechanism of Cl on mammalian carbonic anhydrase has been resolved. The kinetics of Cl^- inhibition are different because of the presence (noncompetive) or absence (competitive) of K ⁺

Finally, through the use of radioisotopic flux determinations and tissue voltage clamping techniques, the physiological involvement of bicarbonate movement in the processes of secretion and resorption of moulting fluid has been ascertained. Bicarbonate accounts for electroneutrality, pH optium and osmotic equivalency during moulting fluid secretion, while the active movement of bicarbonate back into hemolymph from the exuvial side during resorption of moulting fluid provides the driving force for this resorptive process.