Innate immunity in insects: surface‐associated dopa decarboxylase‐dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes

Abstract

Summary Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte‐surface‐associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine‐derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.

Development and evolution of insect pigmentation: Genetic mechanisms and the potential consequences of pleiotropy

“…Mutations in pale (encoding Tyrosine Hydroxylase) and Dopa-decarboxylase affect pigmentation and the melanic immune response [59,62,70,71]; and genes encoding phenoloxidases polymerize dopa and dopamine into melanins during pigmentation development as well as wound healing and encapsulation [69,[72][73][74][75]. Despite the production of similar pigments, melaniza-tion that occurs as part of the immune response and melanization that is used for body pigmentation, does not require all of the same genes.…”

The Global Transcription Factor Lrp Is both Essential for and Inhibitory to Xenorhabdus nematophila Insecticidal Activity

“…The bacterium Xenorhabdus nematophila is a pathogen of several insect groups and during infection overcomes insect immunity (3)(4)(5)(6)(7)(8)(9), which is comprised of two central tissues, hemocytes and fat body. Hemocytes include several types of blood cells that mediate cellular responses, such as phagocytosis and aggregation (nodulation) around a microbial invader (10). Fat body is the major site of synthesis for macromolecular immune factors (comprising humoral immunity), such as antimicrobial peptides (AMPs), small cationic peptides that bind to and disrupt microbial cell membranes (11).…”

An immune-induced Reeler protein is involved in the Bombyx mori melanization cascade

“…Dopamine present in hemolymph is also a significant substrate for PO. PO-based oxidation of dopamine produces dopaminequinone and consequently the cross-linking and melanization of proteins (Sideri et al, 2008). A relationship between Reeler protein and the proPO activation system has not yet been reported in insects; therefore, a functional analysis of Reeler is of considerable interest.…”

“…Further study is needed to understand how this reeler domain-containing protein participates in the nodulation response. Melanization is of central importance in the nodulation and capsulation responses in some lepidopteran and dipteran insects, such as Pseudoplusia and Drosophila, and results in melanin formation Strand, 2002, 2003;Sideri et al, 2008). Prophenoloxidase (proPO) activation is an important step in the insect melanization cascade; proPO is activated by microbial infection and leads to the proteolytic conversion of proPO into phenoloxidase (PO) in the hemolymph (Ji et al, 2004).…”