Background

Phagocytosis, defined as the cellular uptake of particles bigger than 0.5 μm through formation of a membrane derived phagosome, is an ancient and evolutionarily conserved mechanism of insects cellular response (Lemaitre and Hoffmann, 2007; Melcarne et al., 2019a and 2019b). Phagocytosis is mediated by phagocytes, the dedicated cells, which can digest both “altered-self” particles and pathogens (Hillyer and Strand, 2014; Melcarne et al., 2019b). The phagocytes can be not only circulating hemocytes in hemocoel but also sessile hemocytes on tissues (Hillyer and Strand, 2014; Hillyer, 2016; Sigle and Hillyer, 2016). When pathogens enter hemocoel of insects, the phagocytes rapidly phagocytose pathogens and phagocytosis generally finishes in hours (Hillyer et al., 2003; King and Hillyer, 2012; Sigle and Hillyer, 2016).

There are mainly four different methods to perform hemocyte phagocytosis assay:
In vivo phagocytosis: (1) The insects are injected with fluorescently labeled latex beads or fluorescein-labeled dead bacteria. After incubation, trypan blue is injected to quench extracellular fluorescence. Then, the fluorescence is detected on a fluorescence microscope and the fluorescence intensity from dorsal vessel-associated hemocytes is quantified. The detailed methods were described in literature (Elrod-Erickson et al., 2000; Gonzalez et al., 2013; Garg and Wu, 2014; Nazario-Toole et al., 2018). For some insects, the fluorescent background in the hemolymph and tissues are strong and this may influence the results. (2) The insects are injected with fluorescently labeled latex beads or fluorescein-labeled bacteria. After incubation, the hemocytes are collected by ripping the larval in PBS solution containing trypan blue to quench extracellular fluorescence. The hemocytes are transferred and attached to a glass slide, and then the cells are fixed with formaldehyde. The phagocytosis is observed under microscope and the fluorescence intensity is quantified. The details were described previously (Kocks et al., 2005; Hao et al., 2018). For some insects, such as pea aphid, when inject bacteria into the body cavity, a lot of bacteria are centralized around the wound. Therefore, in vivo phagocytosis is not suitable for these insects.

Ex vivo phagocytosis: (1) After collected in insect medium in low binding tubes, hemocytes are mixed with fluorescein-labeled bacteria. Samples are incubated to enable phagocytosis, and then placed on ice to stop the process. Phagocytosis is quantified using a flow cytometer after quenching fluorescence of extracellular particles. The detailed method is described as previously (Melcarne et al., 2019b). (2) Drops of hemolymph are collected into insect medium and then mixed well with fluorescein-labeled bacteria. The samples are transferred to tissue culture round coverslips in a cell culture plate. After settled and adhered to the coverslips, the cells are fixed with paraformaldehyde. Then, F-actin of cells is stained with Phalloidin after permeabilized with Triton-100. Phagocytosis is observed under a laser scanning confocal microscope. The details of hemolymph collection and phagocytosis methods were described before (Schmitz et al., 2012; Ma et al., 2020).

The second method of ex vivo phagocytosis overcomes the difficulty due to small size of some insects for clean hemolymph collection. An accurate assay was described as previously: the fluorescence intensity in phagocytosing hemocytes is calculated and phagocytic index is represented as the capacity of hemocytes (Melcarne et al., 2019b). Combining these advantages, we provide a practicable method for pea aphid hemocyte phagocytosis assay in this study. Meanwhile, we provide protocols for pea aphid rearing and bacterial infection, which offer referential methods for related research.