Identification of two new brain/midgut peptides in a mosquito

M. R. Brown 1 , D. A. Stanek 2 , & J. Pohl 3

1 Department of Entomology, 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA;
3 Microchemical Facility, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA

To identify peptides originating from the midgut endocrine system of the mosquito, Aedes aegypti, several hundred thousand adult abdomens were extracted in a boiling acetic acid solution. A radioimmunoassay for Drosophila neuropeptide F (NPF) was used to monitor purification of immunoreactive peptides from the extracted material. Solid-phase extraction and two steps of semi-preparative, reversed-phase HPLC yielded four groups of immunoreactive fractions. From these groups, a total of eight peptides were isolated individually after five or more steps of HPLC. Five of the peptides were purified in sufficient quantity for amino-terminal sequencing, mass spectroscopy, and amino acid composition. Based on sequence similarity, three of the peptides are “head peptides”: Aea-HP-1 and 3 and a new octapeptide, which is more closely related to the misnamed “neuropeptide F I and II” of the Colorado potato beetle. Isolation of the Aea-HP’s was expected, but the characterization of a new mosquito “head peptide”, not encoded by the Aea-HP gene, is significant. This lengthening list of related “head peptides” from Limulus to Drosophila suggests the existence of a subfamily of arthropod Arg-Phe-amide peptides. Sequences for the other two mosquito peptides show that they are members of the invertebrate neuropeptide F (NPF) family and the vertebrate NPY/peptide YY (PYY)/pancreatic polypeptide (PP) family. One of the mosquito peptides is an amino-terminus truncated form (3100 MW) of the other one (31 residues, 3600 MW). As revealed with an antiserum to the Drosophila NPF, the putative mosquito NPF is found in a few brain cells and in a hundred or more midgut endocrine cells. To date, only the mosquito and Drosophila NPF’s are known for arthropods, and comparison of these sequences to those identified in other invertebrates and the vertebrate NPY/PYY/PP family reveals important conserved domains. The amino acid sequences for the new “head peptide” and the mosquito NPF will allow us to identify the encoding cDNAs and to synthesize the peptides for antiserum production to develop immunoassays and for bioassays. The bioassays will determine whether the peptides directly affect digestion of a blood meal or interact with other peptides known to regulate aspects of reproduction in mosquitoes.

Index terms: Head peptides, neuropeptide F, radioimmunoassay, HPLC

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