Immunocytochemical Evidence of a Tubulin Reserve at the Tip of Growing Flagella in Spermatogenesis of the Mediterranean Mealmoth,Ephestia kuehniella Z. (Pyralidae,Lepidoptera, Insecta)

Abstract The distal swellings of growing flagella in spermatocytes of Ephestia kuehniella Z. contain dense material associated with the ends of axonemal microtubules. In order to define the nature of this material, spermatocytes were lysed under microtubule-stabilizing conditions, spun onto cover-slips, probed with an antibody against β-tubulin and processed for indirect immunofluorescence. Whereas the dense material was lost from the cells when untreated spermatocytes were used, a block of stained material was visible in cold-treated spermatocytes. Most probably, cold-treatment alters the dense material and guarantees its survival during preparation of the cells for anti-tubulin immunofluorescence. The positive reaction with the antibody indicates the presence of β-tubulin. Flagellar outgrowth in spermatogenesis of the moth starts in late prophase I and continues throughout both meiotic divisions. Therefore, spindles and flagella compete for tubulin monomers. A tubulin reserve, deposited early in development at the elongating tip of axonemes, may ensure their uninterrupted growth, independent of tubulin-consuming cytoplasmic events. In order to test this hypothesis, flagellar outgrowth was studied in the spermatocytes of a long-horned beetle, Agapanthia villosoviridescens de Geer (Cerambycidae, Coleoptera) using electron microscopy. In this species, flagella begin to elongate only in telophase II, when the second meiotic spindle is just disassembling. The absence of dense material at the tip of flagellar stubs in the beetle corroborates the hypothesis formulated above.