Embrace the dark side: Identification of the cellular and kinetic characteristics of melanization in the American Cockroach

Abstract

Insect immune systems are comprised of both cellular and humoral mechanisms that protect these organisms from injury and infection. Of these immune mechanisms, the hemolymphatic melanization reaction is a unique immunological process that is ubiquitous across insect species. Using prophenoloxidases (PPOs) expressed in specific hemocytes, insect species can synthesize cytotoxic melanin. This reaction involves the production of reactive oxygen species to kill infectious microbes. While the mechanism of this cascade is well described, the hemocyte populations participating in have not been standardized across Insecta. Another knowledge gap is that these hemocyte have been incompletely identified via microscopy. Additionally, the kinetic characteristics of this phenomenon are poorly understood with most studies evaluating melanin production as a reaction endpoint. Furthermore, a study correlating the in vivo and in vitro kinetics of melanization with an organism has not been documented. Therefore, thesis has attempted to identify PPO-containing hemocyte via a robust assay and elucidate the kinetics of melanization under various conditions within Periplaneta americana. Using cell sorting and an assay to induce melanization, we found that PPO containing hemocytes in P. americana primarily are characterized by a high affinity for the LEA lectin and high intracellular complexity. Furthermore, we found that this population exhibited a large amount of hemocyte clusters which may be indicative of nodulation. Evaluating the kinetics of melanization using spectrophotometry, we found that the addition of L-DOPA and ethanol to whole hemolymph resulted in the fastest rate and endpoint of melanization in comparison to samples containing only whole hemolymph or whole hemolymph and L-DOPA after about 17 hours. Observing the effects of PAMPs in vivo and in vitro, we found that unmethylated cytosine–guanine dinucleotide (CpG) motifs caused a high degree of melanization. Additionally, we found that supernatant derived from a stationary Serratia marcescens culture resulted in a high number of melanized nodules formed in vivo. Overall, my thesis supports that the previously stated methods can be used for a more robust characterization of PPO-containing cells in contrast to microscopy alone. Additionally, my thesis supports that different immunostimulatory PAMPs may cause a wide range of reactivity in the melanization reaction within P. americana. This thesis may be used to inform future research on germ-free insects, which are known to possess truncated immune systems.