A peptide from the male accessory glands of the Colorado potato beetle

Research output: Thesisinternal PhD, WU

Abstract

<p>This thesis describes a study of the male accessory glands of the Colorado potato beetle, <em>Leptinotarsa decemlineata</em> (Say). These glands add various substances to the ejaculate. On mating, the ejaculate is transferred to the female, together with the substances from the male accessory glands. The function of these substances is unknown in the case of the Colorado potato beetle. From research on other insect species, we know that some of these substances stimulate the female to oviposit at a higher rate, and to refuse further matings for a certain period. These two effects are known to be evoked by the action of one peptide hormone in the case of <em>Drosophila melanogaster</em> , and this peptide is called sex-peptide. At least part of its activity is thought to be accomplished by stimulation of the corpus allatum (allatotropic) activity, as indicated by in vitro experiments. The activity of the corpora allata is normally under control of some neurons in the brain, the lateral neurosecretory cells, which innervate these glands. These neurons use as yet unidentified peptide hormones as messenger substance, and it is possible that these peptides are similar in structure and activity to sex-peptide.</p><p>Our immunohistochemical studies on the Colorado potato beetle give indirect support for the possible dual control of corpus allutm activity. We revealed that some glandular cells in the male accessory glands are labelled by a monoclonal antibody that was raised against the peptides in the lateral neurosecretory cells. The question arises whether the antigen in the accessory glands is indeed identical to the antigen in the lateral neurosecretory cells. In that case, both antigens are involved in stimulation of the corpus allatum activity. The aim of the present study is to compare the antigens in the accessory glands and the lateral neurosecretory cells, and to study the function of the former in more detail (chapter 1).</p><p>The antigen in each of the two accessory glands is present in a specific set of approximately 100 dispersed glandular cells. The immuno-reactive cells contain granules with crystalline contents, and these crystals have a rod-like appearance. Such rods are also immuno-labelled in the lumen of the gland (chapter 2).</p><p>The antigen in the accessory glands is a peptide of 8 kDa., designated Led-MAGP ( <em><u>Le</u> ptinotarsa <u>d</u> ecemlineata</em><u>m</u> ale <u>a</u> ccessory <u>g</u> land <u>p</u> eptide). Part of the amino acid sequence has been determined. Using this structural information the gene encoding this peptide has been identified and thereby the structure of the entire peptide. The peptide is expressed exclusively in the accessory glands, not in the brains. The peptide does not resemble any known peptide hormone, but it shows a considerable degree of similarity to the N-terminus of the chicken prion protein. Prion proteins are at present in the centre of interest since they are involved in certain fatal neurodegenerative diseases in man (Creutzfeldt-Jakob disease) and cattle (scrapie, bovine spongiformic encephalopathies, better known by its acronym BSE) (chapter 3).</p><p>Four antigens from the lateral neurosecretory cells have been characterized to determine their relatedness to Led-MAGP. However, the peptides isolated all differ in size and chromatographic properties from Led-MAGP. Our initial interpretation that the antigens from the lateral neurosecretory cells might be identical to Led-MAGP, had therefore to be rejected (chapter 4).</p><p>A recombinant baculovirus is constructed, equipped with the gene encoding Led-MAGP. This way large amounts of this peptide are produced in order to study its function. The recombinant Led-MAGP is produced by infection of insect cells cultures with the recombinant baculovirus. Large scale production is hampered by the formation of large aggregates of Led-MAGP. Nevertheless, sufficient peptide has been harvested to produce a new antibody against Led-MAGP. This antibody recognized the authentic peptide with superior specificity, compared with the monoclonal antibody used previously (chapter 5).</p><p>Microscopical analysis with the new antiserum reveals the fate of Led-MAGP during copulation. Male and female reproductive tracts were taken from mating couples for immunohistochemical analysis with the new antiserum. The route of the Led-MAGP could be analyzed in detail. Led-MAGP is transferred from the male accessory glands to the spermathecal duct in the female. Led-MAGP most probably diffuses to the hemolymph within minutes after its deposition (chapter 6).</p><p>A hypothesis is put forward as to the physiological function of Led-MAGP. This hypothesis is based on the homology of Led-MAGP with the chicken prion protein, and on the observation that it binds hemolymph protein. The N-terminus of the chicken prion protein namely contains 8 hexa-repeats, whereas Led-MAGP has and 7 hexa-repeats that are largely homlogous. Although the biological function of the prion protein itself is unknown, the section with the 8 hexarepeats serves as a signal that induces the uptake of the ch-prp in the endocytosis route. By analogy, Led-MAGP could, by binding to hemolymph proteins, induce the uptake of these proteins by the developing oocytes. This way led-MAGP stimulates the growth of the oocytes on the expense of female hemolymph proteins. In other words, the balance of protein use between maintenance and reproduction, is shifted more towards reproduction. This mechanism would at the same time explain the reduction of female receptivity for mating, as remating will lead to the acquisition of too much Led-MAGP, and thus to overstimulation of reproduction at the expense of other body functions (chapter 7).</p><p>A test of the hypothesis that is proposed in chapter 7 awaits methodological improvements. Detection of Led-MAGP in the mated female, by using the specific antiserum is hampered due to cross-reactivity of several female-derived proteins. The tendency of Led-MAGP to aggregate further complicates a functional analysis. Furthermore, the available inbred laboratory strain of the Colorado potato beetle is probably unsuitable for a bioassay on stimulation of oviposition (general discussion).</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Schoonhoven, L.M., Promotor, External person
  • Schooneveld, H., Promotor, External person
Award date22 Apr 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054858188
Publication statusPublished - 1998

Keywords

  • sex hormones
  • chrysomelidae
  • androgens
  • androsterone
  • proteins

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