IN-SITU HYBRIDIZATION ANALYSIS OF LEUCOMYOSUPPRESSIN MESSENGER-RNA EXPRESSION IN THE COCKROACH, DIPLOPTERA-PUNCTATA

In the cockroach Diploptera punctata, sequencing of the cDNA for the i nsect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demons trated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-re lated peptide to be encoded by this gene (Donly et al. [1996] Insect B iochem. Mel. Biol, 26:627-637). However, in the present study, high pe rformance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-elut ed with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D, punctata. Immunohistochemi stry with a polyclonal antiserum generated against FMRFamide, but whic h recognizes extended RFamide peptides, demonstrated numerous RFamide- like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and process es of the brain and optic lobes, the stomatogastric nervous system, in cluding the frontal and ingluvial ganglia, and the suboesophageal gang lion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, p ositively stained processes were found in the crop, midgut, and hindgu t, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning th e entire LINTS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression wa s most abundant in the brain and optic lobes and in the frontal and su boesophageal ganglia. LMS mRNA was also apparent, although less intens ely, in all other ganglia of the ventral nerve cord. Within the alimen tary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of L MS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion. (C) 1998 Wiley-Liss, In c.