Effects on Some Therapeutical, Biochemical, and Immunological Parameters of Honey Bee (Apis mellifera) Exposed to Probiotic Treatments, in Field and Laboratory Conditions

Several negative factors contribute to a decline in the number of insect pollinators. As a novel approach in therapy, we hypothesize that the EM® for bees could potentially have an important therapeutic and immunomodulatory e ect on honey bee colonies. The aim of our study was to evaluate its impact on honey bees at the individual and colony level. This is the first appliance of the commercial probiotic mix EM® PROBIOTIC FOR BEES in honey bees as economically important social insects. The sugar syrup with 10% of probiotic was administered by spraying or feeding the honey bee colonies in the field conditions, in order to evaluate the infection levels with spores of Nosema spp. and colonies’ strength. Moreover, in laboratory-controlled conditions, in the hoarding cages, adult workers have been fed with sugar syrup supplemented with 2.5, 5, and 10% of EM® for bees for biochemical and immunological analyses of hemolymph, and with 5 and 10% for measuring the size of hypopharyngeal glands. It was found that following the EM® for bees administration the Nosema spp. spore counts in colonies were significantly reduced, and colonies’ strength was increased. The results at the individual level showed significant positive physiological changes in treated groups of adult bees, revealing at the same time a higher mortality rate when feeding sugar syrup supplemented with the probiotic

Physiological and immunological status of adult honeybees (Apis mellifera) fed sugar syrup supplemented with Pentadecapeptide BPC 157

Various factors contribute to a decline in diversity and number of bees. Here, an integrated approach in experimental BPC 157 therapy was implemented, combining laboratory-controlled and field study results. The aim of a study was to assess the effects of BPC 157 additional feeding of newly emerged worker honeybees on few biochemical and immunological parameters in hemolymph (glucose, trehalose, lipids, proteins, vitellogenin, glucose-oxidase (GOX)), and hypopharyngeal gland (HPG), in laboratory-controlled conditions. Additionally, to examine the physiological status of protein digestion, the enzymatic activity of leucine aminopeptidase (LAP) in the mid-guts of worker honeybees was analyzed. It was found that individual honeybees, in hoarding cages, following BPC 157 administration through carbohydrate food, showed positive physiological changes when compared to the control groups. Those results were complemented by strong and visible LAP activity, particularly noticeable in the apical parts of the epithelial cells in the mid-guts of young worker honeybees originated from treated hives, suggesting a link between alternative oral therapy with BPC 157 and honeybees’ immunity

Likvidace Varroa destructor ve včelstvech včely medonosné pomocí Flumetrinu nebo Fluvalinátu

Mite mortality in two apiaries, one with 32 and the other with 15 honeybee (Apis mellifera carnica) colonies, was recorded prior to and after flumethrin or fluvalinate treatments and after a control, oxalic-acid application. During the 42- and 51-day pre-treatment periods, the average daily natural mite drop was 0.04 (± 0.04) and 2.82 (± 2.19), respectively, which represents 1.09% (± 1.06) and 3.84% (± 3.04) of the total number of mites found during the experiment. The flumethrin or fluvalinate applications resulted in an average mite mortality at the two apiaries of 214.46 (± 260.02) and 4,098.64 (± 2,508.31). The treatments resulted in a 19.11% (± 14.62) and a 39.28% (± 10.47) reduction in the number of mites in slightly infested colonies and 94.30% (± 4.26) and 96.24% (± 3.14) in highly infested colonies. The difference in treatment efficacy between both apiaries was significant (P < 0.001) and indicates that fluvalinate and flumethrin are highly efficacious in dealing with highly infested honeybee colonies with sealed brood. The importance of effective mite control in colonies with a high level of natural mite mortality is discussed in this study.Ve dvou včelínech, s 32 a s 15 včelstvy (Apis mellifera carnica), byla sledována mortalita roztoče před a po ošetření flumetrinem nebo fluvalinátem a po kontrolní aplikaci kyseliny šťavelové. Během doby před ošetřením (42 a 51 dní) byl průměrny přirozený spad roztočů 0,04 (± 0,04) a 2,82 (± 2,19), což bylo 1,09 % (± 1,06) a 3,84 % (± 3,04) z celkového počtu roztočů zachycených během experimentu. Po aplikaci flumetrinu nebo fluvalinátu ve dvou včelínech byla průměrná mortalita roztočů 214,46 (± 260,02) a 4 098,64 (± 2 508,31). Ošetření vedlo ke snížení počtu roztočů o 19,11 % (± 14,62) a 39,28 % (± 10,47) u lehce napadených včelstev a o 94,30 % (± 4,26) a 96,24 % (± 3,14) u silně napadených včelstev. Rozdíl v účinnosti ošetření mezi oběma včelíny byl statisticky významný (P < 0,001) a ukázalo se, že fluvalinát a flumetrin jsou vysoce účinné v boji se silně napadenými včelstvy se zavíčkovaným plodem. V této studii je pojednáno o významu/důležitosti efektivní kontroly roztočů ve včelstvech s jejich vysokou přirozenou mortalitou

Histology basics and cell death detection in honeybee tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed

MORPHOLOGICAL AND HISTOLOGICAL CHANGES IN DEVELOPMENT OF HYPOPHARYNGEAL GLANDS IN HONEYBEE (Apis mellifera carnica)

Hipofaringealne žleze proizvajajo in izločajo sestavine matičnega mlečka, ki je pomembna hrana za zalego in matico. Mlade delavke imajo razvite žleze z velikimi acinusi, ki aktivno izločajo sekret. Med seboj smo primerjali delavke iz rednika in delavke iste starosti iz čebelje družine. Ugotovili smo, da so imele krmilke iz rednika v starosti nad 15 dni večji premer žleznih acinusov. Aktivno izločanje sekreta se podaljšuje s starostjo (do 27 dni) pri krmilkah iz rednika, ki ostajajo v družini in intenzivno oskrbujejo ličinke v matičnih lončkih. Opisali smo morfološke in histološke značilnosti žlez pri delavkah v razvojni stopnji bube, pri dolgoživih, zimskih čebelah in pri delavkah krmilkah v starosti 1 do 27 dni. V drugem delu poskusa smo različno stare delavke v kletkah tretirali z imidaklopridom, diazinonom ali kumafosom v časovnem obdobju za 24, 48 ali 72 ur. Po zaključenem tretiranju smo izmerili velikost premera žleznih acinusov in ugotovili značilen vpliv tretiranja na zmanjšano velikost žlez (p < 0,05). Delavke, ki so bile dlje časa (48 in 72 ur) izpostavljene tretiranju, so imele manjše žleze v primerjavi s kontrolno skupino netretiranih delavk. V žleznih acinusih smo lokalizirali stresne proteine (Hsp 70 ali Hsp 90) in določali stopnjo celične smrti. Pri delavkah, ki so bile tretirane s kumafosom ali diazinonom, smo pogosteje lokalizirali Hsp v celičnem jedru in citoplazmi, kot pri delavkah, ki so bile tretirane z imidaklopridom. Ugotovili smo, da se Hsp lokalizirajo različno glede na vrsto pesticida in starost čebel. Pri delavkah, ki so bile tretirane s kumafosom, smo ugotovili povečano stopnjo apoptotske celične smrti, po tretiranju z imidaklopridom je bila v žleznih celicah nakazana tudi nekroza. V drugi starostni skupini (7 do 12 dni) delavk je bila stopnja celične smrti po 48 urah tretiranja z imidaklopridom okoli 50-odstotna in se je po 72 urah pojavila v jedrih vseh celic. Hsp smo določili tudi v žleznih celicah netretiranih delavk in ugotovili, da je celična smrt ostala na stopnji običajne obnove celic (do 10 odstotkov) pri delavkah iz vseh štirih starostnih skupin. Pesticidi, ki smo jih uporabili v poskusu, so pri delavkah medonosne čebele povzročili povečano stopnjo odmiranja celic žleznih acinusov.Hypopharyngeal glands produce and secrete the components of rojal jelly which is the most important food for brood and queen. In young workers, the glands are well developed with large actively secreting acini. The workers from the breeder colony were compared to workers from the honeybee colony. We found that nurses from the breeder colony aged over 15 days had larger acini diameter. Active gland secretion is extended with age (until 27 days) in the nurse workers which seem to stay in the hive and intensivelly feed the brood in the queen cells. The morphological and histological characteristics of the hypopharyngeal glands were described in worker pupa, winter bees and in nurse workers aged 1 to 27 days. In the second part of our research, the workers were treated with imidaclopride, diazinon, or coumaphos for 24, 48 and 72 hours. After the treatment we measured the size of the acinar diameter and found significant effect of treatment on the decreased size of the acini (p < 0.05). In the workers that were exposed to the treatments for longer time periods (48 and 72 h), the glands were smaller comparing to the glands in control group of untreated workers. Hsps (Hsp 70 or Hsp 90) were localised in the glandular acini and the level of cell death was determined. In the workers treated with coumaphos or diazinon, the Hsps were more frequently localised in the cell nuclei and cytoplasm in comparison to imidacloprid treated workers. Hsps seem to localise differently, depending on a pesticide and age of bees. In coumaphos-treated workers we detected increased apoptotic cell death and in imidacloprid-treated workers the necrosis was indicated. In the second age-group (7–12 days) the level of cell death was found 50 % after 48 h of treatment with imidacloprid, and encreased after 72 h of treatment when all of the cell nuceli were found positive. In the untreated workers, the Hsps were found in the glandular cells and the normal cell renewal stayed at the low level (< 10 %) in workers of all four age-groups. The pesticides used in our experiments caused a high level of the cell death in the glands cells of the treated honeybee workers

Heat shock proteins and cell death in situ localisation in hypopharyngeal glands of honeybee (

Worker honeybees (Apis mellifera carnica Polm.) were treated with imidacloprid or coumaphos. Significant effects of treatment and treatment duration were found on hypopharyngeal glands (HPG) acinus diameter (P < 0.05). Differences in the size of acini were evident in all long term (48 h and 72 h) treatments. Short term (24 h) imidacloprid treatment induced heat shock protein 70 (Hsp 70) localisation in nuclei and cytoplasm and Hsp 90 activity was found in most cell cytoplasm. Coumaphos triggered an increased level of programmed cell death, and imidacloprid induced extended necrosis in comparison to coumaphos. In 7–12 day old workers, the level of cell death after 48 hours of imidacloprid treatment was approximately 50% and increased to all cells after 72 hours. Programmed cell death remained at the normal level of approximately 10%. Our results suggest that both pesticide treatments have an influence on the reduced size of HPG and also on the extended expression of cell death

Characteristics of Honey Bee (Apis Mellifera Carnica, Pollman 1879) Queens Reared in Slovenian Commercial Breeding Stations

In this three-year-trial study, we examined the quality of mated queens based on morphological and physiology traits. At each location, sister queen bees were reared each year from one Apis mellifera carnica breeder queen. Queens were also reared and mated in different locations. Altogether, we sampled and analysed 324 queens from 27 apiaries in 2006, 288 queens from 24 apiaries in 2008, and 276 queens from 23 apiaries in 2010. Nine queens from each apiary were sampled and dissected for morphological analyses and Nosema ceranae (N. ceranae) spores, if present, were quantified. Three queens from each apiary were prepared and tested for four viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), deformed wing virus (DWV), and sacbrood virus (SBV). The highest average queen weight of 209.49 ± 9.82 mg was detected in 2008. The highest average ovary weight of 78.67 ± 11.86 mg was detected in 2010, and the highest number of ovarioles was 161.59 ± 8.70 in 2006. The average number of spermatozoa in queens ranged from 3.30 x 106 in 2006 to 5.23 x 106 in 2010. Nosema ceranae spores were found in queens sampled in 2008 and 2010. Viruses were discovered sporadically during the queen testing periods from 2006 - 2010. This study importantly demonstrates that queens from rearing stations require regular evaluation for morphological and physiological changes as well as for infection from harmful pathogens. These results could also be used in establishing relevant commercial standards for rearing quality queens

Toxicological and immunohistochemical testing of honeybees after oxalic acid and rotenone treatments

Bees removed capped brood and young larvae from combs at a greater rate after a rotenone treatment than after an oxalic acid (OA)/sucrose treatment. Rotenone (1%) caused 75.2% of capped brood to be removed, OA (3%) 18.7% and a control treatment, 13.3%. Caged worker bees treated with a 1% rotenone powder, a 3% OA or with a control solution had mortality rates of 10.9%, 5.1% and 1.9% respectively. Rotenone (1%) significantly affected the mortality of brood and adult bees whereas OA (3%), did not. Solutions of 3% OA/32% sucrose, 3.4% OA/47.6% sucrose, 3.7% OA/27.1% sucrose (w/w) and a 32% sugar solution applied to adult bees resulted in death rates of 11%, 14%, 11.2% and 6.5% respectively. Individually treated bees consumed more of a 3% OA solution than solutions with higher OA concentrations. A TUNEL assay detected necrotic cell death in 69% of bee midgut cells 24 h after an OA treatment. Normal cell turnover is approximately 8%