Entomopathogenic nematodes (EPNs) have been a useful model for studying wound healing in insects due to their natural mechanism of entering an insect host either through the cuticle or an orifice. While many experiments have shed light on nematode and host behavior, as well as the host immune response, details regarding early nematode entry and proliferative events have been limited. Using high-resolution microscopy, we provide data on the early infection kinetics of Heterorhabditis bacteriophora and its symbiotic bacteria, Photorhabdus luminescens. EPNs appendage themselves to the host and enter through the host cuticle with a drill-like mechanism while leaving their outer sheath behind. EPNs immediately release their symbiotic bacteria in the host which leads to changes in host behavior and septicemia within 6 h while EPNs travel through the host in a predictable manner, congregating in the anterior end of the host. This paper sheds light on the entry and proliferative events of EPN infection, which will further aid in our understanding of wound healing and host immune activation at a high spatiotemporal resolution.

In most insects, hemolymph coagulation, which is analogous to mammalian blood clotting, involves close collaboration between humoral and cellular components. To gain insights into the secretion of cellular clotting factors, we created tagged versions of three different clotting factors. Our focus was on factors which are released in a non-classical manner and to characterize them in comparison to a protein that is classically released, namely Glutactin (Glt). Transglutaminase-A (Tg) and Prophenoloxidase 2 (PPO2), both of which lack signal peptide sequences, have been previously demonstrated to be released from plasmatocytes and crystal cells (CCs) respectively, the two hemocyte classes in naive larvae. We found that at the molecular level, Tg secretion resembles the release of tissue transglutaminase in mammals. Specifically, Drosophila Tg is associated with vesicular membranes and remains membrane-bound after release, in contrast to Glt, which we found localizes to a different class of vesicles and is integrated into clot fibers. PPO2 on the other hand, is set free from CCs through cytolysis. We confirm that PPO2 is a central component of the cytosolic crystals and find that the distribution of PPO2 appears to vary across crystals and cells. We propose a tentative scheme for the secretory events during early and late hemolymph coagulation.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors Schmid et al., 2019. The linked article demonstrates the localization of two secretory proteins in Drosophila melanogaster, Prophenoloxidase (PPO2) and Transglutaminase-A (Tg) in hemocytes as well the clot with different tissue-specific drivers. Here we provide further data for the usefulness of the GFP-tagged version of the two crosslinking enzymes that are involved in clot hardening. The morphology of crystal cells is described using GFP-tagged PPO2 rather than with the use of antibodies in ex vivo hemolymph preparations. The use of the GFP-tagged proteins PPO2 and Tg is shown in additional contexts.

Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines upon cell bursting. This form of cell death has been increasingly well-characterized in mammals but there has been little evidence for the existence of pyroptosis across phyla. Here we provide evidence for convergent evolution of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to the wound and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects. Further, we unveil key players upstream in the activation of cell death in these cells., Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.