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NPF Immunolocalization in Cockroachesand LocustsComparison of Antisera to Beetle, Tapeworm,and Pig NPY/NPF-Type Peptides

WEI ZHU,a PETER VERHAERT,a CHRIS SHAW,b AARON MAULE,b

ARNOLD DE LOOF,a AND HUBERT VAUDRYc

aDepartment of Biology, University of Leuven, Naamsestraat 59, B-3000 Leuven, BelgiumbDepartment of Medicine, University of Belfast, Belfast BT7 1NN, United KingdomcINSERM U413, University of Rouen, F-76821 Mont-Saint-Aignan, France

We recently identified two neuropeptides from the beetle Leptinotarsa decemlineataby virtue of their immunoreactivity for antibodies to the invertebrate prototype ofthe neuropeptide Y (NPY)/pancreatic polypeptide (PP) superfamily of peptides, viz.,flatworm (Moniezia expansa) neuropeptide F (Moe-NPF).1 These first insect “NPF-like” neuropeptides, designated Led-NPF-I (a decapeptide) and Led-NPF-II (an oc-tapeptide), are substantially shorter than all invertebrate NPFs sequenced so far: tur-bellarian, cestodan (Plathyhelminthes), and gastropod (Mollusca) NPFs varybetween 36 and 39 amino acid residues in length.

ANIMALS, ANTISERA, AND IMMUNOCHEMICAL STRATEGY

Serial paraffin sections of cockroach (Periplaneta americana) and locust (Schis-tocerca gregaria) central nervous systems and midguts were used in immunoperox-idase histochemistry. Antisera used were all produced in rabbits and includedantibodies highly specific (1) to the common Led-NPF carboxyl-end (both peptidesshare a LeuArgLeuArgPhe-amide sequence), and (2) to the C-terminus of Moe-NPF.In differential cross-absorption tests, the above antibodies were found not to bind toeither NPY/PP-like peptides (ArgXxxArgTyr-amides) or “typical” FaRPs (XxxYy-yArgPhe-amides, with Xxx being different from Arg (typically an aromatic residue).The third antiserum included in this study was one raised against porcine NPY,which had previously been employed in immunochemical studies of other insectspecies.2 In order to find out whether hitherto described “vertebrate-type” NPY/PPimmunoreactivities in insects may have been due to the immunological cross-reac-tion of the sera with “invertebrate-type” (NPF-like) peptides, that is., ArgXxxArg-Phe-amide-terminating ones, instead of NPY-like, alternating sections were treatedwith the different antibodies.

RESULTS AND DISCUSSION

In both insect species, positive immunostaining obtained with NPF- and NPY-an-tibodies was often found in the vary same neuronal perikarya and their tracts, partic-

626 ANNALS NEW YORK ACADEMY OF SCIENCES

ularly throughout the cerebral neuropile areas, in the optic lamina, medulla andlobula, in the pars intercerebralis (pi), and in the subesophageal ganglion. Also in thegut (partial) overlappings were detected. Invariably, however, more “insect NPF”-like material was apparent than “flatworm NPF”-related material, which, in its turnincluded all (vertebrate) NPY staining (FIG. 1). Moreover, all NPY immunolabel-ings could be blocked by liquid phase absorption with either beetle or tapeworm(synthetic) NPF.

Our results show that most, if not all, insect structures stained by NPY-type anti-bodies are also visualized by NPF antisera, indicating that the former are cross-re-acting with NPF-like peptides. The opposite is not true, in view of the specificity ofthe NPF antibodies employed. Furthermore, our immunohistochemical results alsopoint to the presence of multiple insect NPF-type peptides.

FIGURE 1. Cockroach brain labeled for NPY and NPF. Abbreviations: ag, antennalglomeruli; cp, corpora pedunculata; pi, pars intercerebralis. Arrowheads point to markeddifferences in the number of immunostained cell somata.

627ZHU et al.: NPF IMMUNOLOCALIZATION

REFERENCES

1. SPITTAELS K., VERHAERT P., SHAW C., JOHNSTON R.N., DEVREESE B., VAN BEEU-MEN J. & DE LOOF A. 1996. Insect neuropeptide F (NPF)-related peptides: Isola-tion from Colorado potato beetle (Leptinotarsa decemlineata) brain. InsectBiochem. Mol. Biol. 26: 375–382

2. SCHOOFS L., DANGER J.M., JÉGOU S., PELLETIER G., HUYBRECHTS R., VAUDRY H.& DE LOOF A. 1988. NPY-like peptides occur in the nervous system and midgut ofthe migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sar-cophaga bullata. Peptides 9: 1027–1036.