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.

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.

S u m m a r y 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.

The principal mechanism of gene activation/silencing is DNA 5-methylcytosine methylation. This study was aimed at determining global DNA methylation levels in larvae, prepupae, pupae, and 1-day-old adults of Apis mellifera queens, workers and drones. The Imprint Methylated DNA Quantification Kit MDQ1 was used. Percentages of DNA 5-methylcytosine were low and relatively similar in the larvae of all the castes until 4th day of larval development (3-5%). However, they were higher in the drone and worker larvae than in the queen larvae. Generally, the developmental patterns of changes in the DNA methylation levels were different in the queens in comparison with the drones and workers. While methylation increased in the queens, it decreased in the drones and workers. Methylated DNA methylcytosine percentages and weights in the queen prepupae (15%, 9.18 ng) and pupae (21%, 10.74 ng) were, respectively, three and four times higher than in the worker/drone brood of the same age (2.5-4%, 0.03-0.07 ng). Only in the queens, after a substantial increase, did DNA methylation decrease almost twice between the pupal stage and queen emergence (from 21% and 10.74 ng to 12% and 6.78 ng). This finding seems very interesting, particularly for experimental gerontology.

a b s t r a c t 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.

Metalloproteases (metalloproteinases, MMP) digest extracellular matrix proteins. They have zinc ions (Zn2+) in their active site. They are synthesized within cells as proenzymes, to be subsequently activated in the extracellular environment. MMP are active in a neutral or slightly alkaline pH in the presence of Ca2+ ions. Cells that synthesize metalloproteases also produce metalloprotease inhibitors. Until now, 4 of MMP inhibitors as well as 28 endometalloproteases have been discovered, out of which 22 occur in humans. On the other hand, these enzymes have not been well explored in insects, in which only 2 metalloproteases were identified (Ance and ECE). Their optimal activity ranges between pH 7.0 and 9.4. MMP inhibitors control the concentration of metalloproteases in physiological conditions. They fall within two types: specific tissue MMP inhibitors and non-specific plasma MMP inhibitors. Metalloproteases and their inhibitors play an important role in both physiological and pathological processes in the organism. Metalloproteases are cell growth promotors. They inhibit/induce apoptosis, stimulate the development of healthy cells and control the activity of neoplastic cells both in people and in insects. Their activity is increased in skin and periodontal diseases, in arthritis, arteriosclerosis or in the period following myocardial infarction. In insects the activity of MMPs is also increased by environmental pollution, by the use of antibiotics and varroacides. The insect MMPs participate in digestion, biosynthesis of peptide hormones and neurotransmitters, and melanisation. They also affect the development of the reproductive system and the development of larvae and pupae, as well as prevent pathogen invasions. Worthy of special attention is the insect cuticle defensive barrier associated with MMP. Activation of metalloproteases is dependent on the physiological state of the organism, as well as on environmental pressure. Analyzing activities of metalloproteases and their inhibitors enables better monitoring of the pathological conditions in both insects and mammals.

Xylocopa valga, commonly called the carpenter bee and the largest bee with metallicviolet hair cover, is extremely rarely observed in Poland. We hypothesize that a stable and possibly long-term population of X. valga can be maintained in Poland through the creation of suitable nesting conditions. X. valga has been observed since the spring of 2014 in Wisznice (south-eastern Poland). A nesting box made out of 25 wooden blocks with drilled holes was hung about 2.5 meters above the ground. X. valga were interested in the blocks made of willow wood but did not nest in the beech, alder and pine. The carpenter bees chose holes made with drill bits of 10, 15, 20 mm in diameter and a length of 10, 15 and 20 cm. X. valga flying in the same direction most often visited the flora taxa: Aquilegia vulgaris, Ballota nigra, Consolida ajacis, Delphinium consolida, Deutzia scabra, Catalpa spp., Wisteria spp., Robinia ambigua, Stachys spp. and Trifolium pretense. X. valga is a solitary bee, but unlike most other solitary bees it demonstrates aspects of social behavior. It was observed to display cohabitative behavior involving the use of a single hole by more than one female. The females showed aggressive defensive behavior and if approached too closely started buzzing loudly. The information obtained during the long-term observation shows that X. valga can be maintained in partly artificial conditions to increase and stabilize the bee population.