Jacek Chobotow

COENZYME Q10 TREATMENTS INFLUENCE THE LIFESPAN AND KEY BIOCHEMICAL RESISTANCE SYSTEMS IN THE HONEYBEE, Apis mellifera

Natural bioactive preparations that will boost apian resistance, aid body detoxification, or fight crucial bee diseases are in demand. Therefore, we examined the influence of coenzyme Q10 (CoQ10, 2,3-dimethoxy, 5-methyl, 6-decaprenyl benzoquinone) treatment on honeybee lifespan, Nosema resistance, the activity/concentration of antioxidants, proteases and protease inhibitors, and biomarkers. CoQ10 slows age-related metabolic processes. Workers that consumed CoQ10 lived longer than untreated controls and were less infested with Nosema spp. Relative to controls, the CoQ10-treated workers had higher protein concentrations that increased with age but then they decreased in older bees. CoQ10 treatments increased the activities of antioxidant enzymes (superoxide dismutase, GPx, catalase, glutathione S-transferase), protease inhibitors, biomarkers (aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase), the total antioxidant potential level, and concentrations of uric acid and creatinine. The activities of acidic, neutral, and alkaline proteases, and concentrations of albumin and urea were lower in the bees that were administered CoQ10. CoQ10 could be taken into consideration as a natural diet supplement in early spring before pollen sources become available in the temperate Central European climate. A response to CoQ10 administration that is similar to mammals supports our view that Apis mellifera is a model organism for biochemical gerontology.

Unexpectedly strong effect of caffeine on the vitality of western honeybees (Apis mellifera).

We examined the influence of caffeine on honeybee lifespan, Nosema resistance, key enzyme activities, metabolic compound concentrations, and total DNA methylation levels. Caffeine slowed age-related metabolic tendencies. Bees that consumed caffeine lived longer and were not infested with Nosema spp. Caffeine-treated workers had higher protein concentrations. The levels increased with aging but they then decreased in older bees. Caffeine increased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), AST, ALT, ALP, neutral proteases, and protease inhibitors, and the concentrations of uric acid, triglycerides, cholesterol, glucose, and Ca2+. Acidic and alkaline protease activities were lower in the bees treated with caffeine. Creatinine and Mg2+ concentrations were higher in the caffeine-treated workers but only up to 14 days of age. Caffeine significantly decreased DNA methylation levels in older bees. The compound could be considered as a natural diet supplement increasing apian resistance to stress factors. Our studies will enhance possibilities of using Apis mellifera as a model organism in gerontological studies.

THE EFFECT OF AMPHOTERICIN B ON THE LIFESPAN, BODY-SURFACE PROTEIN CONCENTRATIONS, AND DNA METHYLATION LEVELS OF HONEY BEES (Apis mellifera)

Abstract Three groups of caged bees were fed with sugar syrup (the control), sugar syrup supplemented with amphotericin B (AmB) in a dose of 0.5 mg/ml, and sugar syrup with AmB in a dose of 0.25 mg/ ml. Amphotericin B shortened the life span of the bees and reduced the level of global DNA methylation compared to the control, however, it increased the body-surface protein concentrations. In the hindguts of the bees, there were found AmB deposits. Honeybees appear to be a useful model for studying the side effects of anti-fungal AmB therapy. Among other things, epigenetic changes and senescence acceleration are considered to be the side effects of the therapy. Streszczenie Trzem grupom pszczół w klatkach podawano syrop cukrowy (grupa kontrolna), syrop cukrowy z dodatkiem amfoterycyny B (AmB) w dawce 0,5 mg/ml oraz syrop cukrowy z AmB w dawce 0,25 mg/ml. AmB wpływała na skrócenie długości życia pszczół oraz obniżała poziom globalnej metylacji DNA w porównaniu do grupy kontrolnej, jednocześnie zwiększała stężenie białek na powierzchni ciała. AmB odkładała się w jelicie tylnym pszczół. Pszczoła miodna okazała się użytecznym modelem dla badań efektów ubocznych terapii antygrzybiczej z zastosowaniem AmB. Między innymi, za efekty uboczne takiej terapii uważa się zmiany epigenetyczne i przyspieszenie procesów starzenia.

Insights into the biochemical defence and methylation of the solitary bee Osmia rufa L: A foundation for examining eusociality development

We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June. Concentrations/activities of (enzymatic and non-enzymatic) antioxidant and proteolytic systems, as well as phenoloxidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were constant during the diapause, usually lower in the males than the females. Prior to the diapause/overwintering, the concentrations/activities of all the compounds were higher in the fat bodies than in the hemolymph. Later in the spring and in the summer, they increased in the hemolymph and on the body surfaces, while decreasing in the fat bodies. The global DNA methylation levels increased with age. Higher levels were always observed in the males than in the females. The study will promote better understanding of bee evolution and will be useful for the protection and management of solitary bees, with benefits to the environment and agriculture.

Body-Surface Compounds in Buckfast and Caucasian Honey Bee Workers (Apis Mellifera)

Abstract Body-surface chemical compounds were studied in 1-day-old nest workers and foragers both in Buckfast and Caucasian bees. The workers of these two age-castes were sampled twice in each of two consecutive years. Body-surface lipids were determined by means of gas chromatography, with a GCQ mass spectrometer. Protein concentrations and activities on the body surface were examined in bee cuticle rinsings obtained from worker bees according to the methods of Lowry, of Anson, and of Lee and Lin. Protease and protease inhibitor activities were determined. Polyacrylamide gel electrophoresis was performed. Caucasian bees, particularly foragers, had more lipids, but Buckfast bees (two age-castes) had more proteins on their body surfaces. A total of 17 alkane types (C17 - C33), 13 alkene types (C21 - C33), 21 esters (C12 - C32), and a phenol (C14) were detected in both races. Alkene C33 was detected only in Caucasian bees. More alkanes, esters, and phenols were found in Caucasian 1-day-old nest workers and foragers than in these age-castes of Buckfast bees. The protein concentration and protease inhibitor activities were lower in Caucasian bees that had higher protease activities. These values corresponded with specific numbers and widths of the electrophoretic bands.

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.