A quantitative peptidomics approach to unravel immunological functions of angiotensin converting enzyme in Locusta migratoria.

Locusta migratoria angiotensin converting enzyme (LmACE) is encoded by multiple exons displaying variable number of genomic duplications. Treatments of lipopolysaccharide (LPS) as well as peptidoglycan but not ß-1-3 glucan resulted in enhanced expression of angiotensin converting enzyme in hemocytes of Locusta migratoria. No such effect was observed in fat body cells. Differential peptidomics using locust plasma samples post infection with LPS in combination with both an LmACE transcript knockdown by RNAi and a functional knockdown using captopril allowed the identification of 5 circulating LPS induced peptides which only appear in the hemolymph of locust having full LmACE functionality. As these peptides originate from larger precursor proteins such as locust hemocyanin-like protein, having known antimicrobial properties, the obtained results suggest a possible direct or indirect role of LmACE in the release of these peptides from their precursors. Additionally, this experimental setup confirmed the role of LmACE in the clearance of multiple peptides from the hemolymph.

Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

Locust cellular defense against infections: sites of pathogen clearance and hemocyte proliferation.

The locust cellular defense is mediated by hemocytes and hematopoietic tissue. In Locusta migratoria, the hemocytes and hematopoietic tissue mutually assist each other in clearing invading pathogens from circulation. A ß-1, 3-glucan infection induces nodule formation and apoptotic, TUNEL positive, cells in the hematopoietic tissue and massive loss of hemocytes in the circulation, calling for instant proliferation of hemocytes and hematopoietic tissue cells to assure continued host cellular defense. As the locust hematopoietic tissue persists at the adult stage, it was originally designated as being the major source for the replenishment process. Revisiting post infection hemocyte proliferation, using immunofluorescence based tests for DNA synthesis and mitosis, evidenced the lack of ß-1, 3-glucan induced cell proliferation in the hematopoietic tissue. Instead these tests identified the circulating hemocytes as the major source for hemocyte replenishment in the circulation. The hematopoietic tissue, however, undergoes a continuous, slow and infection independent regeneration, thereby accumulating potential phagocytes despite infection, and might serve a prophylactic role in containing pathogens in this swarming insect.

Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides.

Growth blocking peptides (GBPs) are recognized as insect cytokines that take part in multifaceted functions including immune system activation and growth retardation. The peptides induce hemocyte spreading in vitro, which is considered as the initial step in hemocyte activation against infection in many insect species. Therefore, in this study, we carried out a series of in vitro bioassay driven fractionations of Locusta migratoria hemolymph combined with mass spectrometry to identify locust hemocyte activation factors belonging to the family of insect GBPs. We identified the locust hemocyte spreading peptide (locust GBP) as a 28-mer peptide encoded at the C-terminus of a 64 amino acid long precursor polypeptide. As demonstrated by QRT-PCR, the gene encoding the locust GBP precursor (proGBP) was expressed in large quantities in diverse locust tissues including fat body, endocrine glands, central nervous system, reproductive tissues and flight muscles. In contrary, hemocytes, gut tissues and Malpighian tubules displayed little expression of the proGBP transcript. The bioactive peptide induces transient depletion of hemocytes in vivo and when injected in last instar nymphs it extends the larval growth phase and postpones adult molting. In addition, we identified a functional homologous hemocyte spreading peptide in Schistocerca gregaria.