A standardised protocol for measuring phenoloxidase and prophenoloxidase in the honey bee, Apis mellifera

Abstract

-The prophenoloxidase activating system (ProPO-AS) is an integral part of the constitutive innate immune response in insects, the products of which are commonly assayed to assess an individual's ability to respond to immune challenges. However, there is considerable variation in the methodologies associated with these assays, and as such, it is not always clear how to interpret results. We have optimised assays for measuring phenoloxidase in its active (PO) and zymogen (ProPO) forms in the honey bee, Apis mellifera. Contrary to results for other insects, we found that the activator α-chymotrypsin, when used at a low concentration (0.5 mg mL −1 ), combined with a minimal activation time (5 min), provided optimal conditions for assaying ProPO. In addition, a saturated L-dopa solution was required for assaying both PO and ProPO. The results highlight the importance of defining the working parameters of each assay to be species-specific. honey bee / Apis mellifera / phenoloxidase / innate immunity / activator INTRODUCTIONThe innate immune system of insects consists of two broad categories of effectors: constitutive and induced (Schmid-Hempel, 2005). Constitutive responses generally comprise the humoral prophenoloxidase activating system (ProPO-AS) and cellular responses including coagulation, phagocytosis, nodule formation and encapsulation (Gillespie et al., 1997).Whilst not maintained at a constant maximum, constitutive immune responses are always present Cerenius et al., 2008), are non-specific but immediate in effect, and are responsible for clearing most of a microbial challenge very rapidly after infection (Haine et al., 2008). By contrast the induced responses, such as antimicrobial pepCorresponding author: A.M. Laughton, alice.laughton@emory.edu * Manuscript editor: Bernd Grünewald tides, occur only once the invading pathogen has been recognised. They are specific and take longer to produce, but are longer lasting in their effect (Boman and Hultmark, 1987).We concentrate on one important arm of the constitutive immune responses. The ProPO-AS is a series of complex interactions between enzymes and their zymogens, leading to the production the enzyme phenoloxidase (PO; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1). PO activity ultimately results in the formation of melanin, which is subsequently involved in cuticle sclerotisation, wound healing and cellular defence responses (Mason, 1955;Ratcliffe et al., 1984;Cerenius et al., 2008). Due to the cytotoxic nature of the by-products of this process, PO is commonly stored as its inactive precursor prophenoloxidase (ProPO), and activated as part of the cascade following the recognition of foreign compounds (Ratcliffe et al., 1984;Riley, 1988; Gillespie et al., 1997; Söderhäll and Apidologie (2011) 42:1 -1

Reconfiguration of the immune system network during food limitation in the caterpillarManduca sexta

“…This difference may be due, in part, to species differences, but may also be due to methodological issues, especially regarding how PO activity is measured (e.g. Laughton and Siva-Jothy, 2011;Moreno-Garcia et al, 2013;Kohlmeier et al, 2015). Lysozyme appears to regulate PO activity during an immune response (e.g.…”

Ant sperm storage organs do not have phenoloxidase constitutive immune activity

“…For the three species sampled, male seminal vesicles and female spermathecae and bursae copulatrix were dissected in deionized water at room temperature, carefully opening them in a 1 ll drop of sodium cacodylate buffer (0.01 M Na-Cac, 0.005 M CaCl 2 , pH 6.5 (Laughton and Siva-Jothy, 2010)) and removing the envelope tissue afterwards. Each 1 ll sample was diluted in 19 ll of sodium cacodylate buffer after which 10 ll was used to measure free PO activity and another 10 ll to measure proPO activity.…”

“…It is a constitutive, non-specific and immediate response involved in wound healing and the immobilization and eradication of foreign agents (Sugumaran, 2002;Schmid-Hempel, 2005). The immune response is activated by the recognition of pathogen-associated molecular patterns or cell injury (Schmid-Hempel, 2005), which triggers the conversion of zymogenic prophenoloxidase (proPO) into active phenoloxidase (PO) through enzymatic interactions (Söderhäll and Cerenius, 1998;Laughton and Siva-Jothy, 2010). PO catalyzes the oxidation of phenols into quinones, which results in melanin synthesis and deposition around wounds or pathogens (González-Santoyo and Córdoba-Aguilar, 2011).…”

Expression of the Prophenoloxidase Gene and Phenoloxidase Activity, During the Development of Apis Mellifera Brood Infected with Varroa Destructor

“…Our experimental design was adopted to identify varroasisrelated changes and to evaluate transcript levels and the active form of the gene product. The enzymatic activity of the bee hemolymph was determined in earlier studies (Zufelato et al, 2004;Laughton and Siva-Jothy, 2010). The activity of PO sampled from the entire body can be an indicator of the overall status of the immune system, including the cuticle which provides the first structural barrier of resistance (Brey et al, 1993;Grzywnowicz et al, 2009).…”