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.

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.

Bio-analysis of insects is increasingly dependent on highly sensitive methods that require high quality biological material, such as hemolymph. However, it is difficult to collect fresh and uncontaminated hemolymph from adult bees since they are very active and have the potential to sting, and because hemolymph is rapidly melanized. Here we aimed to develop and test a quick and easy method for sterile and contamination-free hemolymph sampling from adult Apidae. Our novel antennae method for hemolymph sampling (AMHS), entailed the detachment of an antenna, followed by application of delicate pressure to the bee's abdomen. This resulted in the appearance of a drop of hemolymph at the base of the detached antenna, which was then aspirated using an automatic pipetter. Larger insect size corresponded to easier and faster hemolymph sampling, and to a greater sample volume. We obtained 80–100 μL of sterile non-melanized hemolymph in 1 minute from one Bombus terrestris worker, in 6 minutes from 10 Apis mellifera workers, and in 15 minutes from 18 Apis cerana workers (+/−0.5 minutes). Compared to the most popular method of hemolymph collection, in which hemolymph is sampled by puncturing the dorsal sinus of the thorax with a capillary (TCHS), significantly fewer bees were required to collect 80–100 μL hemolymph using our novel AMHS method. Moreover, the time required for hemolymph collection was significantly shorter using the AMHS compared to the TCHS, which protects the acquired hemolymph against melanization, thus providing the highest quality material for biological analysis.

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.