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Microsporidian infections are dangerous to honeybees due to the absence of an efficient treatment for nosemosis. In the present work, the abilities of several porphyrins to directly inactivate microsporidia derived from Nosema-infected honeybees were studied in vitro. Amide derivatives of protoporphyrin IX (PPIX) conjugated with one and two amino acid moieties were synthesized, and their activities were compared with those of two cationic porphyrins, TMePyP and TTMePP. The most active porphyrins, PP[Lys-Asp] 2 , PP[Lys-TFA] 2 , PP[Asp(ONa) 2 ] 2 and PP[Lys-Lys] 2 at concentrations as low as 10-50 µM exerted significant effects on microsporidia, reducing the number of spores by 67-80% compared to the control. Live-cell imaging of the spores treated with porphyrins showed that only 1.6% and 3.0% of spores remained alive after 24 h-incubation with 50 µM PP[Asp(ONa) 2 ] 2 and PP[Lys-Asp] 2 , respectively. The length of the amino acid side chains and their identity in the PPIX molecules affected the bioactivity of the porphyrin. Importantly, the irradiation of the porphyrins did not enhance their potency in destroying Nosema spores. We showed that the porphyrins accumulated inside the living spores but not inside dead spores, thus the destruction of the microsporidia by non-metallated porphyrins is not dependent on photosensitization, but is associated with their active transport into the spore cell. When administered to honeybees in vivo, PPIX[Lys-TFA] 2 and PPIX[Lys-Lys] 2 reduced spore loads by 69-76% in infected individuals. They both had no toxic effect on honeybees, in contrast to zinc-coordinated porphyrin.

It has been demonstrated in numerous studies that bee pollen supplementation shows numerous positive effects on health. However, its impact on bones is largely unknown. The purpose of this study was to investigate the effect of bee pollen supplementation on the tibia biomechanical properties and bone morphometric measures using Japanese quail as an animal model. The experiment was arranged in a 2x2x2 factorial design, with sex, quail line (meat-type or egg-lying type), and bee pollen inclusion (0 or 10 g/kg of feed) as factors. The quails were one-day-old at the beginning of the experiment, they were euthanized after 42 days. Our study showed for the first time unfavorable effects of bee pollen on bones properties. Bee pollen supplementation negatively affected bone structure, irrespective of quails' sex or line type. Bone length (P < 0.001), weight (P < 0.01), and mean relative wall thickness (P < 0.01) and mineralization (P < 0.05) were reduced by bee pollen treatment. For female quails, irrespective of line type, the decrease of yield load (P < 0.001), ultimate load (P < 0.01), yield stress (P < 0.001) and ultimate stress (P < 0.05) was noted. Analysis of growth plate in bone metaphysis showed that bee pollen supplementation slowed the process of bone maturation irrespective of sex (P < 0.05). On contrary, dietary bee pollen positively affected bone homeostasis of trabecular bone in bone metaphysis as bone mineral density increased in experimental groups (P < 0.05). In males, this was the result of the increase of trabecular thickness (P < 0.01), in females due to the reduction of trabecular space (P < 0.001). In conclusion, our results demonstrate that bee pollen (1.0%, 10 g/kg of feed) supplementation caused significant negative effects on the mechanical endurance of the tibia of quails, while showed beneficial effects on trabecular bone histomorphometry., Competing Interests: The authors have declared that no competing interests exist.

Microsporidia Nosema are transferred among bees via the faecal-oral route. Nosema spp. spores have been detected on flowers and transferred to hives along with the bee pollen. The aim of the present study was to determine whether Nosema microsporidia are transferred by air in an apiary, in a control area (without the presence of bee colonies), and/or in a laboratory during cage experiments with artificially infected bees. The novel way of transmission by air was investigated by the volumetric method using a Hirst-type aerobiological sampler located on the ground in the apiary, in the Botanical Garden and on the laboratory floor. Concurrently, the mean rate of Nosema infections in the foragers in the apiary was estimated with the Bürker haemocytometer method. Spore-trapping tapes were imaged by means of light microscopy, Nomarski interference contrast microscopy and scanning electron microscopy. The highest concentration of Nosema spores per 1m 3 of air (4.65) was recorded in August, while the lowest concentration (2.89) was noted in July. This was confirmed by a Real-Time PCR analysis. The presence of N. apis as well as N. ceranae was detected in each of the tested tapes from the apiary. The average copy number of N. apis was estimated at 14.4 × 10 4 copies per 1 cm 2 of the tape; whereas the number of N. ceranae was 2.24 × 10 4 copies per tape per 1 cm 2 . The results indicate that Nosema microsporidia were transferred by the wind in the apiary, but not in the Botanical Garden and laboratory by air. This was confirmed by genetic analyses. DNA from immobilised biological material was isolated and subjected to a PCR to detect the Nosema species. A fragment of the 16S rRNA gene, characteristic of Nosema apis and N. ceranae, was detected. Our research adds knowledge about the transfer of Nosema spp. microsporidia in the natural environment and indicates the season associated with the greatest risk of a bee colony infection with Nosema spp.

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics.

Proper bioelement content is crucial for the health and wellness of all organisms, including honeybees. However, the situation is more complicated in these important pollinators due to the fact that they change their physiology during winter in order to survive the relatively harsh climatic conditions. Additionally, honeybees are susceptible to many diseases such as nosemosis, which during winter can depopulate an entire colony. Here we show that summer bees have a markedly higher content of important bioelements such as: Al, Cu, P, V, (physiologically essential); Ca, K, Mg, (electrolytic); Cr, Se, Zn, (enzymatic); As, Hg, (toxic). In contrast, a markedly higher content of: Fe (physiologically essential); Mn, Ni, (enzymatic); Cd (exclusively toxic) were present in winter bees. Importantly, N. ceranae infection resulted in an increased honeybee bioelement content of: S, Sr (physiologically essential) and Pb (exclusively toxic), whereas the Nosema-free worker-bees had higher amounts of B and Si (physiologically essential). We propose that the shortages of Fe, Mn, Ni, and Na observed in Nosema-infected bees, could be the reason for the higher mortality of Nosema-infected bees throughout overwintering. In addition, a shortage of bioelements such as B and Si may be a reason for accelerated aging in foragers that is observed following N. ceranae infection. Therefore, in winter, bioelement content was more strongly affected by N. ceranae infection than during summer. We found a strong correlation between the bioelement content of bees and seasons (summer or winter) and also with Nosema infection. We conclude that the balance of bioelements in the honeybee is altered by both seasonal affects and by Nosema infection., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

The study was conducted to investigate the effect of Lactobacillus rhamnosus (a commercial probiotic) and inulin (a prebiotic) on the survival rates of honeybees infected and uninfected with Nosema ceranae, the level of phenoloxidase (PO) activity, the course of nosemosis, and the effect on the prevention of nosemosis development in bees. The cells of L. rhamnosus exhibited a high rate of survival in 56.56 % sugar syrup, which was used to feed the honeybees. Surprisingly, honeybees fed with sugar syrup supplemented with a commercial probiotic and a probiotic + prebiotic were more susceptible to N. ceranae infection, and their lifespan was much shorter. The number of microsporidian spores in the honeybees fed for 9 days prior to N. ceranae infection with a sugar syrup supplemented with a commercial probiotic was 25 times higher (970 million spores per one honeybee) than in a control group fed with pure sucrose syrup (38 million spores per one honeybee). PO activity reached its highest level in the hemolymph of this honeybee control group uninfected with N. ceranae. The addition of probiotics or both probiotics and prebiotics to the food of uninfected bees led to the ~2-fold decrease in the PO activity. The infection of honeybees with N. ceranae accompanied an almost 20-fold decrease in the PO level. The inulin supplemented solely at a concentration of 2 μg/mL was the only administrated factor which did not significantly affect honeybees' survival, the PO activity, or the nosemosis infection level. In conclusion, the supplementation of honeybees' diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality.

Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

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., (© 2014 Wiley Periodicals, Inc.)

Lotmaria passim (L. passim) is a single-celled flagellate which colonises the bee gastrointestinal tract and is highly prevalent in honey bees. This parasite is associated with colony losses. Honey bee (Apis mellifera) colonies were sampled from five apiaries in the north-eastern part of Poland for the phylogenetic analysis of L. passim. Each apiary consisted of approximately 60 bee colonies, of which 20 were randomly selected. Samples of 60 differently aged worker bees were collected from each colony and pooled. A total of 100 bee colonies from five apiaries were examined. Protozoa of the Trypanosomatidae family were identified by PCR. L. passim was detected in 47 (47%) of the samples. The 18S ribosomal (r) RNA amplicons of L. passim were sequenced by a commercial service. Their sequences were analysed with BLASTN and noted to be compatible with the GenBank sequences of this region of the organism's genome. A sequence analysis was performed using the BioEdit Sequence Alignment Editor and Clustal W software. The amplicon sequences of L. passim were 100% homologous with the sequences deposited in GenBank under accession numbers KM066243.1., KJ684964.1 and KM980181.1. This is the first study to perform a phylogenetic analysis of L. passim in Polish honey bees. The analysis demonstrated high levels of genetic similarity between isolates of L. passim colonising apiaries in the north-eastern region of Poland. [ABSTRACT FROM AUTHOR]

Simple Summary: Honeybees live in two different environments that are affected by multiple factors, e.g., pathogens or weather conditions. Thanks to the construction of the nests and behavioral cooperation honeybees became independent from the influence of external conditions. Moreover, a single bee has barriers that maintain homeostasis in its body and protect it from harmful factors, such as pesticides, air pollutants, or pathogens, through a system of proteases and their inhibitors. In feral bee colonies, bee comb cells vary in their width, while in Europe, colonies are kept only on standard-cell combs (5.50 mm cell width) or small-cell combs (4.90 mm cell width). In apiary conditions, the activities of proteases and their inhibitors in the 1-day-old workers were always higher in workers reared in standard-cell combs than in workers reared in small-cell combs, and opposite results were observed in groups of older workers (aged 7, 14, and 21 d). However, evaluating the influence of the nest factor is complex and difficult in field tests. Therefore, we assessed the proteolytic system activity in the hemolymph of colonies kept simultaneously on standard and small-cell combs in laboratory cage tests. The use of two types of combs in bee colony nests is an innovative approach to the use of small-cell combs in beekeeping. Irrespective of the age of the bees, the workers reared in the small-cell combs had higher protein concentrations. In turn, the activities of proteases and their inhibitors in the hemolymph of 1-day-old workers were higher in the group reared in the standard-cell combs. In older workers (aged 7, 14, and 21 days), higher protease and inhibitor activities were detected in the group reared in small-cell combs. The comparison of the results of apiary studies with those of laboratory cage tests shows that functions performed in the nest have a greater influence than age on the activity of proteases and their inhibitors in the hemolymph of worker bees. The aim of the study was to compare the activities of proteases and their inhibitors in the hemolymph of honeybee workers reared in small-cell combs (SMC) and standard-cell combs (STC) in laboratory cage tests. The analyses conducted in laboratory conditions facilitated assessment of the impact of the comb cell width (small vs. standard) along with eliminating the influence of many environmental factors on the results. The width of the comb cells in which the workers were reared had a significant effect on the protein concentrations and proteolytic system activities in the hemolymph. Irrespective of the age of the workers, higher protein concentrations were found in the hemolymph of the SMC workers. In turn, the activities of proteases and their inhibitors in the hemolymph of 1-day-old bees were higher in the STC workers. In older bees, aged 7–21 days, activity was higher in the SMC workers. The role of the considerable cell width variability in natural combs that were built without the use of an artificially produced wax foundation is worth investigating. It is highly probable that the impact of the comb cell width on the features of workers reared in these combs modifies the age polyethism in the worker caste as well. The investigation results of one-season studies of honeybees could be seriously affected by random factors. To reduce the risk of these effects, it is advisable to continue experiments over a few consecutive years. [ABSTRACT FROM AUTHOR]

The aim of the study was to analyse the intestinal microbiota of honey bees (Apis mellifera) treated with amitraz. In the present study, the microbiological profile of the honey bee intestines showed minor changes in the microbiota following the application of amitraz. A comparison of the numbers of bacteria and fungi revealed a positive downward trend in the number of fungi. The number of decreased bacteria, included E. coli and especially Clostridium spp., which were not isolated from the intestines of bees treated with amitraz. The number of lactic acid bacteria before and after treatment was at a similar level of 8.3 log cfu/g of intestinal contents. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the impact of the combination of the colony type (kept on small-cell or standard-cell combs) and the width of worker comb cells (small-cell or standard-cell combs) on the body weight and morphometric traits of worker bees. The values of morphometric parameters of worker bees changed within a substantially lower range than the width of their rearing cells. This indicates that the worker body size is relatively constant, and manipulation with the cell width is not a good method for modeling the body size of workers. The reduction in the thorax weight was proportional to the decrease in the comb cell width, and this part of the body proved to be most susceptible to weight reduction caused by the use of small-cell combs. The rearing of workers in small-cell combs in the colony kept on standard-cell combs resulted in an increase in the value of the fill factor (thorax width to cell width ratio). The relatively constant body size of workers in combination with the use of small-cell combs resulting in an increase in the fill factor may be one of the determinants of increased resistance of the insects to Varroa destructor. The values of the morphometric traits commonly used for identification of honeybee subspecies, i.e., the length of the fore wing, the sum of the widths of 3rd and 4thth tergites, and the proboscis length, were inconsiderably altered vs. the changes in the comb cell width, which confirms their high suitability for identification of honeybee subspecies. [ABSTRACT FROM AUTHOR]

For more than forty years, laboratory cage tests (LCTs) have been used in honeybee research. These tests facilitate the control of elements of the environment present in the laboratory, which allows the minimization of the impact of the changing environmental conditions and conditions in the bee colony nest on the studied features. Therefore, it is possible to estimate the content of the genetic component of a particular trait and to assess the impact of a specific substance or tested agent on the traits of bees. An advantage of LCTs is the possibility of conducting easier and cheaper research than in the case of apiary experiments. LCTs allow a significant reduction in the number of required bees/colonies, investigations of a winter bee generation and individual treatment of a single bee. One of the first research directions in which LCTs were used was the instantaneous assessment of the honey yield. Due to discrepancies between LCT assessment and results of apiary productivity, LCTs are currently not a standard tool for the assessment and improvement of bee functional traits despite such undoubted advantages as reducing the time and cost of the evaluation. Moreover, LCTs are being used to assess the genetic and environmental conditions of workers' lifespan and are a standard method for assessing the impact of pesticides on bees. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]