Distinguishing Manduca sexta haemocyte types by cytometric methods

Abstract

Manduca sexta larvae possess haemocytes which are classified according to their appearance into five distinct types typical for Lepidoptera: plasmatocytes (PLs), granular cells (GRs), spherule cells (SPs), oenocytoids (OEs), and prohaemocytes (ProHCs). Some haemocytes do not fit morphologically into either class, i.e. sharing morphological criteria of two cell types, obscuring their classification. We characterised larval haemocytes of M. sexta, whose identity was confirmed by specific markers, based on morphometric and cytometric methods by light microscopy and fluorescence microscopy. For each haemocyte class, the size, the length-to-width-ratio, the nuclear morphology, the relative DNA-content and the nucleus-to-cytoplasm-ratio were measured. PLs and GRs occur in either round or spread form, the latter resulting in a high length-to-width-ratio. The shape of the nuclei also differed between haemocyte types, with almost round nuclei in GRs, ProHCs and OEs, and oval to irregularly formed nuclei in SPs and PLs. The nucleus-to-cytoplasm-ratio was shown to differ significantly between each type. PLs and OEs exhibited polyploidy, while granular cells, spherule cells and putative prohaemocytes were presumably 2n (G0/G1) or 4n (G2/M). Finally, a dichotomous guide allowing determination of the haemocyte types by means of light microscopy and DNA labelling was created.