Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

Abstract

The aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of taufluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method -as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.

Proteomic analysis of Bayvarol® resistance mechanisms in the honey bee parasite Varroa destructor

“…Pyrethroids are metabolized by hydrolysis of ester bonds, oxidation at the acid and alcohol moieties, and several conjugation reactions such as hydrophilic and lipophilic conjugates (Mikata, Osobe, & Kaneko, 2011;Strachecka, Borsuk, Olszewski, Paleolog, & Lipiński, 2013). Here, a number of proteins involved in hydrolysis (processing peptidase beta subunit putative, acetyl-CoA hydrolase putative, aminoimidazole-4-carboxamide ribonucleotidetransformylase/IMP were present in higher levels in pyrethroid resistant mites (Table 2) and these could be potential effectors of the more efficient breakdown of the chemical in resistant mites.…”

Venom allergen 5is Associated With Deltamethrin Resistance inCulex pipiens pallens(Diptera: Culicidae)

“…In addition, insecticides induce physiological changes in mosquitoes such as a thickening of the epidermis, which subsequently lowers their permeability to insecticides (Wood et al 2010). Studies investigating the mechanisms of pyrethroid resistance have identified other resistance-related genes, such as UDP-glycosyltransferases , Bozzolan et al 2014) and serine proteases (Reid et al 2012, Strachecka et al 2013.…”

Mammalian and insect metalloproteases

“…Wiadomo, że metaloproteazy produkowane przez każdą z grup patogenów wpływają na indukcję humoralnej reakcji obronnej u owadów. Wydzielane są wówczas u owadów inhibitory skierowane przeciwko proteazom bakterii, grzybów i pasożytów (14,35). Z kolei poziom ekspresji mRNA metaloproteaz regulowany jest poziomem lipopolisacharydu (LPS) po inwazji bakteryjnej.…”

“…Interakcja enzym MMP-inhibitor MMP jest uzależniona od warunków atmosferycznych (sezonowa zmiana metabolizmu) oraz od wydzielania feromonów pszczelich (7,39). Najprawdopodobniej u pszczół, podobnie jak u mrówek (8, 9) metaloproteazy tworzą wraz z innymi elementami systemu proteolitycznego ochronny biofilm na powierzchni ciała (15,35,36). U Manduca sexta po infekcji patogenami, głównie bakteriami, następuje wzmożona synteza metaloproteaz, które włączane są również w mechanizmy melanizacji (17).…”