The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.
The effect of sublethal doses of imidacloprid on protein content and activity of proteases on honey bees was analyzed. The study was conducted in three experimental groups: colonies from groups BE-5 and BE-200 were contaminated with 5 and 200 ppb of imidacloprid, respectively, via their food supply (syrup and pollen), while group BE was used as control (untreated). Bee samples were collected 3 and 10 weeks after feeding started. Protein concentration in bee tissue extracts was analyzed with reference: (a) to the dose of imidacloprid; and (b) duration of exposure to the chemical. The average quantity of protein content was significantly higher at the 3-week interval than in the 10-week interval and the bees from control colonies (BE) had significantly higher protein contents than contaminated bees (BE-5 and BE-200), even 3 weeks after feeding with imidacloprid started. Similarly, the activity of proteolytic enzymes (proteases) was found to be dependent on the dose of imidacloprid used, compared to bees from control colonies showing significantly higher activity.Influencia de las dosis subletales de Imidacloprid en el contenido de proteína y la actividad proteolítica en las abejas melíferas (Apis mellifera L.)Se ha analizado el efecto de las dosis subletales de imidacloprid en el contenido de proteínas y la actividad de las proteasas en las abejas melíferas. El estudio se realizó en tres grupos experimentales: las colonias de los grupos de BE-5 y BE-200 se contaminaron con 5 y 200 ppb de imidacloprid, respectivamente, a través del alimento suministrado (jarabe y polen), mientras que el grupo SER se utilizó como control (sin tratar). Se recogieron muestras de abejas en las semanas 3 y 10 después del comienzo de la alimentació n. La concentració n de proteína en extractos de tejido de abejas se analizó en relació n con: a) la dosis de imidacloprid; y b) la duració n de la exposició n a la sustancia química. La cantidad promedio del contenido de proteína fue significativamente mayor en el intervalo de 3 semanas que en el intervalo de 10 semanas y las abejas de las colonias control (BE) tuvieron un contenido de proteína significativamente más alto que las abejas contaminadas (BE-5 y BE-200), incluso 3 semanas después de que comenzara la alimentació n con imidacloprid. Del mismo modo se encontró que la actividad de las enzimas proteolíticas (proteasas) depende de la dosis de imidacloprid usada, en comparació n con las abejas de colonias de control que muestran una actividad significativamente más alta.
Varroa destructor is an ectoparasite that causes serious damage to the population of the honeybee. Increasing resistance of the parasite to acaricides is related, among others, to metabolic adaptations of its esterases to facilitate decomposition of the chemicals used. Esterases are a large heterogeneous group of enzymes that metabolize a number of endogenous and exogenous substrates with ester binding. The aim of the present study was to determine the activity of esterases in the body extracts (BE) and excretion/secretion products (E/SP) of the mite. The enzymes contained in the E/SP should originate mainly from the salivary glands and the alimentary system and they may play a particularly important role in the first line of defence of the mite against acaricides. Activity of cholinesterases (ChEs) [acetylcholinesterase (AChE) and butyrylcholinesterase], carboxylesterases (CEs) and phosphatases [alkaline phosphatase (AP) and acid phosphatase (AcP)] was investigated. The activity of all the enzymes except AChE was higher in the E/SP than in the BE. ChEs from the BE and from the E/SP reacted differently on eserine, a ChE inhibitor. Eserine inhibited both enzymes from the BE, increased decomposition of acetylcholine, but did not influence hydrolysis of butyrylcholine by the E/SP. Activity of the CEs from the BE in relation to the esters of carboxylic acids can be presented in the following series: C10 > C12 > C14 > C8 > C2 > C4 = C16, while activity of the CEs from the E/SP was: C4 > C8 > C2 > C14 > C10 > C12 > C16. The inhibitor of CEs, triphenyl phosphate, reduced the activity of esterases C2–C8 and C14–C16; however, it acted in the opposite way to CEs C10 and C12. The activity of both phosphatases was higher in the E/SP than in the BE (AcP about twofold and AP about 2.6-fold); the activities of AP and AcP in the same material were similar. Given the role of esterases in resistance to pesticides, further studies are necessary to obtain complete biochemical characteristics of the enzymes currently present in V. destructor.
a b s t r a c t osmia rufa is a solitary bee that is used commercially for pollinating crops. The bee enters obligatory diapause as an imago. The activity of proteolytic enzymes during diapause has not been investigated. we studied the proteinase activity on four substrates -casein, haemoglobin, bovine serum albumin (bsa), and gelatine -during diapause (from october to march) and in newly hatched males and females in april. during diapause, greater fluctuations in enzyme activity levels were noted in males than in females, and a significant decrease in male enzyme activity was observed in January and march. male enzymes were most effective in decomposing gelatine; whereas, female enzymes were equally effective in hydrolysing gelatine and bsa. The differences in substrate preferences between male and female enzymes were particularly pronounced in october and in the newly hatched individuals. The levels of gelatinolytic activity likely indicate that a high proportion of proteinases in o. rufa are elastase-like enzymes. They are involved in the digestion and remodelling of proteins with numerous peptide bonds formed by amino acids with short side-chains.
The study was undertaken to examine the immunogenic potential of pea protein of Polish variety Rodan and its trypsin hydrolysates differing in degree of hydrolysis. The physicochemical characteristic of pea protein extract and its hydrolysates, DH 2.0 and 5.0, were determined by SDS-PAGE electrophoresis, chromatofocusing, affinity chromatography, and sequential analysis. The immunogenic properties of pea protein and its trypsin hydrolysates, DH 2.0 and 5.0, were investigated by direct and competitive ELISA methods. The results confirmed that protein extract is a stronger immunogen than hydrolysates, while hydrolysate DH 2.0 was a stronger immunogen than DH 5.0. The dominant antigen isolated from pea protein extract and both trypsin hydrolysates had a molecular weight of about 20 kDa and was in the glycoprotein fraction. The N-terminal sequence of this antigen was determined to be: Thr-Glu-Thr-Thr-Ser-Phe-Leu-Ile-Thr-Lys-.
Lymnaea stagnalis is an intermediate host of many Digenea. The infestation affects host metabolism. The aim of the work was to investigate hemolymph biochemical indicators of L. stagnalis infected with four species of trematodes: Diplostomum pseudospathaceum, Paryphostomum radiatum, Plagiorchis elegans or Opisthioglyphe ranae. The protein profiles and proteinase activity in the hemolymph of sexually mature individuals of Lymnaea stagnalis maintained at 19 • C were tested. As the carbohydrates are main substrates for energetic metabolism of the great pond snail their content and disaccharidase activity were also studied. Hemolymph samples were collected during weeks 3 and 4 of rearing. No significant differences in the total protein content between uninfected individuals and snails infected with the first three trematode species were detected. In the snails infected with O. ranae the quantity of total proteins was near twice higher than in those uninfected. A higher share of 70 kDa proteins in infected than in uninfected snails as well as reduction of the low molecular weight fractions of proteins for snails infected with D. pseudospathaceum and P. radiatum were detected. During week 3, carbohydrate content in the infected snails did not differ from that in the controls while during week 4 it was significantly lower in snails infected with P. elegans or O. ranae. The content of the major soluble carbohydrate in the hemolymphsaccharose -changed in a similar way. No activity of trypsin or pepsin in the hemolymph sample was detected while the activity of chymotrypsin was lower in infected snails vs. controls. On the other hand, saccharase and maltase activities were higher in infected than in uninfected snails. The biochemical hemolymph indicators in naturally infected host-snails show some differences depending on the parasite species but they are not sufficiently species-specific to offer the basis for establishing the model unique for a particular parasitosis. Our results from the field did not always coincide with those from the laboratory.
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