Isolation and Identification of a Plasmatocyte-spreading Peptide from the Hemolymph of the Lepidopteran InsectPseudoplusia includens

Abstract

Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and identification of a peptide from the moth Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte-spreading peptide (PSP1), contains 23 amino acid residues in the following sequence: H-ENFNGGCLAGYMRTADGRCK-PTF-OH. In vitro assays using the synthetic peptide at concentrations >2 nM induced plasmatocytes from P. includens to spread on the surface of culture dishes. Injection of this peptide into P. includens larvae caused a transient depletion of plasmatocytes from circulation. Labeling studies indicated that this peptide induced 75% of plasmatocytes that were double-labeled by the monoclonal antibodies 49G3A3 and 43E9A8 to spread, whereas plasma induced significantly more plasmatocytes to spread. This suggests that only a certain subpopulation of plasmatocytes responds to the peptide and that other peptidyl factors mediate plasmatocyte adhesion responses.

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine

“…Plasma was collected by first narcotizing final (fifth) instar (day 5) B. mori with CO 2 and then surface-rinsing them with ethanol. After rinsing in sterile water, larvae were bled by cutting a proleg and collecting hemolymph into a 1.5-ml Eppendorf tube on ice (29). Hemolymph was immediately centrifuged at 16,000 ϫ g for 30 s to pellet hemocytes, and the resulting plasma supernatant was transferred to a new centrifuge tube for use in experiments.…”

Specific Residues in Plasmatocyte-spreading Peptide Are Required for Receptor Binding and Functional Antagonism of Insect Immune Cells

“…A third possibility is that much lower concentrations of peptide (100 pM 125 I-M12A) were used for binding than were needed to induce rapid spreading of plasmatocytes (3 M PSP). As previously noted, the IC 50 for binding of M12A is 0.7 nM, whereas the ED 50 for PSP-induced spreading of plasmatocytes is 10 -100 nM (6). The reason for this difference could be that large numbers of receptors per plasmatocyte must bind ligand before the signaling pathway regulating adhesion and spreading is activated.…”

“…In Lepidoptera (moths and butterflies) like Pseudoplusia includens, many intruders are recognized as foreign by a class of hemocytes called granular cells, which release cytokines that induce a second class of hemocytes, plasmatocytes, to bind to the target and form a capsule (4,5). The most potent known activator of plasmatocytes is plasmatocyte-spreading peptide (PSP) 1 (6). Natural and synthetic PSP induce plasmatocytes to adhere and spread on foreign surfaces within minutes at concentrations Ն100 pM.…”

“…The percentage of plasmatocytes that spread in an assay was scored 30 min later by counting 100 cells from a randomly selected field of view. Plasmatocytes were scored as spread if they assumed a flattened morphology and were Ն35 m along their longest axis (6,19). Unspread plasmatocytes remained nonadhesive and spheroidal in shape.…”

Cellular immune response of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae), to infection by the entomopathogenic fungus, Beauveria bassiana

“…The possible reason might be formation of a nodule requires that circulating haemocytes change from non-adhesive to adhesive cells that are able to bind to the target and one another and this change requires the involvement of plasma (Clark et al, 1997;Choi et al, 2002;Nardi et al, 2005;Shu et al, 2016). Some molecules have been reported to be involved in this process, such as extracellular matrix (ECM) proteins lacunin and the ligand for peanut agglutinin (PNA) lectin in M. sexta (Nardi et al, 2005), calrecticulin in Galleria melonella (Choi et al, 2002), and plasmatocyte spreading peptide (PSP) in the soybean looper moth, Pseudoplusia includens Walker (Lepidoptera: Noctuidae) (Clark et al, 1997) etc. Therefore, we hypothesize that Asian corn borer plasma supplies similar molecules for the nodule formation upon the challenge of B. bassiana conidia.…”