Jerzy Paleolog

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.

Curcumin Stimulates Biochemical Mechanisms of Apis Mellifera Resistance and Extends the Apian Life-Span

Abstract We examined the influence of curcumin-supplemented feeding on worker lifespan, Nosema resistance, key enzyme activities, metabolic compound concentrations and percentage of the global DNA methylation. Two worker groups (Apis mellifera) were set up: 1) control group; workers were fed ad libitum with sucrose syrup; 2) workers were fed with the syrup with the addition of curcumin. Dead workers were removed every two days and the Nosema spp. infection levels were assessed. Hemolymph was taken from living workers for biochemical analyses. The global DNA methylation level was analysed using DNA from worker heads and thoraces. The bees that consumed curcumin lived longer and were less infested with Nosema spp. The curcumin-treated workers had higher concentrations of proteins, non-enzymatic biomarkers (triglycerides, glucose, cholesterol, Mg2+ and Ca2+), uric acid and creatinine, as well as elevated activities of antioxidant enzymes (SOD , GPx, CAT , GST ), neutral proteases, protease inhibitors, enzymatic biomarkers (AST , ALT , ALP ). The concentrations of albumin and urea, and the activities of acidic and alkaline proteases were higher in the control group. Curcumin decreased global DNA methylation levels especially in older bees in which the natural, age-related level increase was observed. Most of the parameters increased over the apian youth and adulthood, and decreased in older bees. The decrease was markedly delayed in the bees fed with curcumin. Curcumin appeared to be an unexpectedly effective natural bio-stimulator, improving apian health and vitality. This multifactorial effect is caused by the activation of many biochemical processes involved in the formation of apian resistance.

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

The aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-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.

Influence of Amitraz and Oxalic Acid on the Cuticle Proteolytic System of Apis mellifera L. Workers

This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments.

The influence of formic acid on the body surface proteolytic system at different developmental stages in Apis mellifera L. workers

Summary To verify the hypothesis that formic acid (FA) has a suppressive effect on the proteolytic system of Apis mellifera cuticle depending on the developmental stage of the insects, 1 to 4-day-old larvae, 8-day-old larvae, pupae, 1-day-old workers, and foragers were sampled from FA- treated and untreated colonies for seven weeks. Hydrophilic (H+) and hydrophobic protein (H-) solutions were washed out from the sampled individual body surfaces. Subsequently, protein concentration, protease activities and protease inhibitor activities were determined. Antifungal and antibacterial activities were also determined. Two-week exposure to FA increased H+ and H- protein concentration but when it was prolonged to three to seven weeks, the concentration fell below the level of untreated colonies. FA treatment decreased H+ protease activities in workers and larvae, but increased them in pupae. H- protease activities oscillated (destabilisation) up and down relative to the control level which was steady. Asparagine and serine proteases were present on apian cuticles independently of the developmental stage, while FA application additionally activated thiolic proteases. FA treatment considerably decreased both H+ and H- natural protease inhibitor activities in larvae and pupae but mostly increased them in workers. Antifungal and antibacterial activities of the body surface washings (in vivo microbiological tests) were suppressed in workers and larvae treated with FA. FA treatment may suppress proteolytic resistance of the bee cuticle but the responses of larvae and workers are different.

Nosema ceranae Infection Promotes Proliferation of Yeasts in Honey Bee Intestines

Background Nosema ceranae infection not only damages honey bee (Apis melifera) intestines, but we believe it may also affect intestinal yeast development and its seasonal pattern. In order to check our hypothesis, infection intensity versus intestinal yeast colony forming units (CFU) both in field and cage experiments were studied. Methods/Findings Field tests were carried out from March to October in 2014 and 2015. N. ceranae infection intensity decreased more than 100 times from 7.6 x 108 in March to 5.8 x 106 in October 2014. A similar tendency was observed in 2015. Therefore, in the European eastern limit of its range, N. ceranae infection intensity showed seasonality (spring peak and subsequent decline in the summer and fall), however, with an additional mid-summer peak that had not been recorded in other studies. Due to seasonal changes in the N. ceranae infection intensity observed in honey bee colonies, we recommend performing studies on new therapeutics during two consecutive years, including colony overwintering. A natural decrease in N. ceranae spore numbers observed from March to October might be misinterpreted as an effect of Nosema spp. treatment with new compounds. A similar seasonal pattern was observed for intestinal yeast population size in field experiments. Furthermore, cage experiments confirmed the size of intestinal yeast population to increase markedly together with the increase in the N. ceranae infection intensity. Yeast CFUs amounted to respectively 2,025 (CV = 13.04) and 11,150 (CV = 14.06) in uninfected and N. ceranae-infected workers at the end of cage experiments. Therefore, honey bee infection with N. ceranae supported additional opportunistic yeast infections, which may have resulted in faster colony depopulations.

Evaluation of Anthelmintic Activity and Composition of Pumpkin (Cucurbita pepo L.) Seed Extracts—In Vitro and in Vivo Studies

A significant number of studies report growing resistance in nematodes thriving in both humans and livestock. This study was conducted to evaluate the in vitro and in vivo anthelmintic efficiency of Curcubita pepo (C. pepo) L. hot water extract (HWE), cold water extract (CWE) or ethanol extract (ETE) on two model nematodes: Caenorhabditis elegans (C. elegans) and Heligmosoides bakeri (H. bakeri). Methods: Raman, IR and LC-MS spectroscopy analyses were performed on the studied plant material to deliver qualitative and quantitative data on the composition of the obtained extracts: ETE, HWE and CWE. The in vitro activity evaluation showed an impact of C. pepo extracts on C. elegans and different developmental stages of H. bakeri. The following in vivo experiments on mice infected with H. bakeri confirmed inhibitory properties of the most active pumpkin extract selected by the in vitro study. All of the extracts were found to contain cucurbitine, aminoacids, fatty acids, and-for the first time-berberine and palmatine were identified. All C. pepo seed extracts exhibited a nematidicidal potential in vitro, affecting the survival of L1 and L2 H. bakeri larvae. The ETE was the strongest and demonstrated a positive effect on H. bakeri eggs hatching and marked inhibitory properties against worm motility, compared to a PBS control. No significant effects of pumpkin seed extracts on C. elegans integrity or motility were found. The EtOH extract in the in vivo studies showed anthelmintic properties against both H. bakeri fecal egg counts and adult worm burdens. The highest egg counts reduction was observed for the 8 g/kg dose (IC50 against H. bakeri = 2.43; 95% Cl = 2.01–2.94). A decrease in faecal egg counts (FEC) was accompanied by a significant reduction in worm burden of the treated mice compared to the control group. Conclusions: Pumpkin seed extracts may be used to control of Gastrointestinal (G.I.) nematode infections. This relatively inexpensive alternative to the currently available chemotherapeutic should be considered as a novel drug candidate in the nearest future.

A new detection method for a newly revealed mechanism of pyrethroid resistance development in Varroa destructor.

The Varroa destructor mite has recently displayed an ever increasing resistance to new drugs, contributing to CCD proliferation. This work was aimed at determining new viable methods for identifying the pyrethroid resistance of V. destructor and DNA methylation in resistant and sensitive mites. DNA was extracted from Varroa mites. Nucleotide changes in the DNA of pyrethroid-resistant, pyrethroid-sensitive, and control mites were identified with polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in the case of five mitochondrial gene fragments. More bands were observed in the drug-resistant mites than in the other two groups. Sequencing confirmed these observations. Decreased global DNA methylation levels were observed in the pyrethroid-resistant mites. There exists a previously undescribed mechanism of pyrethroid resistance development in Varroa mites. The PCR-SSCP methods can be considered and further developed as useful tools for detecting V. destructor resistance.

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.