Immunolocalization of ubiquitin in degenerating insect flight muscle

Ubiquitin was localized by immunofluorescence microscopy during post-mating histolysis of fibrillar flight muscle in female fire ants, Solenopsis spp. Normal muscles, as well as histolysing muscles from artificially inseminated and haemolymph-injected females contained ubiquitin in association with nuclei, Z-lines, myofilaments and mitochondria. However, the density of the ubiquitin immunoreaction was markedly increased in the nuclei, Z-lines and mitochondria of degenerating tissues 6, 12 and 24 h posttreatment. At these times the heaviest immunoreactivity for ubiquitin was seen in association with the nuclei, Z-lines and mitochondria. Immuno-controls, incubated in the absence of the primary antibody, showed no similar immunostaining. When insemination was preceded by the injection of actinomycin D, muscle degradation was significantly depressed after a 24-h period. Also, ubiquitin immunofluorescence was markedly reduced in tissues pre-treated with actinomycin D. These observations suggest that insemination increases the ubiquitination of specific myofibrillar proteins destined for degradation.