A simple, computer-assisted method for obtaining and counting hemocytes in milkweed bugs

Insect hemocytes have been the focus of researchers for decades (e.g., Jones, 1962; Ravindranath, 1978; Hall, 1983; Ribeiro & Brehelin, 2006). Most research has sought to increase our understanding of what factors influence hemocytes numbers, especially in response to various stressors (e.g., Silva et al., 2002; Andrade et al., 2003; Mochiah et al., 2003). Moreover, the recent increase in questions related to invertebrate ecological immunology (Rolff & Siva-Jothy, 2003) have further stimulated interest in these factors. To carry out such studies, it is often necessary for the researcher to quantify the numbers of hemocytes in their study subjects, and several techniques have been traditionally employed for this purpose. One method relies on estimates of overall hemocyte volume after centrifugation of hemolymph in hematocrit tubes (Clark & Jones, 1980). Electronic particle counters have been used to count marine arthropod hemocytes (Stewart et al., 1967). Liquid nitrogen fixation has been used to compare the numbers of hemocytes in tissue sections to counts made of hemolymph from severed appendages (Feir & O’Connor, 1969). However, the most common method by far involves placing a diluted amount of hemolymph in a hemocytometer and counting the number of cells that fall randomly within specified areas of the hemocytometer grid (e.g., Feir, 1964; Armitage & Siva-Jothy, 2005). The hemocytometer technique is simple and provides data that can be compared between species or within a species. However, this method is also tedious, especially if large numbers of individuals are examined in a study, and because many hundreds of cells must often be quantified for each. In this article, I describe a computer-assisted (i.e., using image analysis software) technique for counting hemocytes in milkweed bugs that is both objective and automated and that yields data within seconds. Materials and methods