Martín Javier Eguaras

Immune-related gene expression in nurse honey bees (Apis mellifera) exposed to synthetic acaricides.

The mite Varroa destructor is an ectoparasite affecting honey bees worldwide. Synthetic acaricides have been among the principal tools available to beekeepers for its control, although several studies have shown its negative effects on honey bee physiology. Recent research suggests that those molecules strongly impact on immune signaling cascades and cellular immunity. In the present work, LC(50) in six-day-old bees were determined for the following acaricides: tau-fluvalinate, flumethrin, amitraz and coumaphos. According to this obtained value, a group of individuals was treated with each acaricide and then processed for qPCR analysis. Transcript levels for genes encoding antimicrobial peptides and immune-related proteins were assessed. Flumethrin increased the expression of hymenoptaecin when comparing treated and control bees. Significant differences were recorded between coumaphos and flumethrin treatments, while the first one reduced the expression of hymenoptaecin and abaecin, the last one up-regulated their expressions. No significant statistically changes were recorded in the expression levels of vitellogenin, lysozyme or glucose dehydrogenase among bees treated with acaricides and control bees. This work constitutes the first report, under laboratory conditions, about induction of immune related genes in response to synthetic miticides.

Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American foulbrood (AFB)

Summary Five essential oils from Argentinan wild plants were tested in vitro for antimicrobial activity against different strains of Paenibacillus larvae larvae, causal agent of American foulbrood in honey bees, Apis mellifera L. The species selected from which to extract the essential oils were aguaribay (Schinus molle var. areira L), Andean thyme (Acantholippia seriphioides A. Gray), peperine (Mintosthachys mollis H.B.K. Gris), camomile (Tagetes minuta L.) and pennyroyal (Lippia turbinata Griseb.). Oils were extracted from different parts of the plants by steam distillation. Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) were evaluated on Mueller-Hinton broth and on MYPGP agar respectively, by the tube dilution method. MIC and MBC minimum and maximum values obtained were 200–250 mgL−1 and 200–300 mgL−1 for Andean thyme and 800–1000 mgL−1 and 850–1100 mgL−1 for pennyroyal, respectively. The other oils had intermediate MIC and MBC values between the formers mentioned. Highly significant differences between MIC and MBC values for the essential oils tested were found. Tukeys mean separation test indicated that Andean thyme (A. seriphioides) essential oil was different from the other four essential oils at α<0.01. Andean thyme proved to be the most effective in vitro essential oil against P. larvae larvae. Therefore, it may be a promising natural alternative to traditional antibiotics, meriting further studies on its field applicability for AFB control.

Antimicrobial activity of Pimpinella anisum and Foeniculum vulgare essential oils against Paenibacillus larvae.

Abstract The essential oil obtained by hydrodistillation from the fruits of Pimpinella anisum L. (green anise) and Foeniculum vulgare Miller (fennel) were analyzed by GC and GC/MS and physicochemical properties. The oils of P. anisum and F. vulgare were found to be especially rich in (E)-anethole, 96.3% and 92.7%, respectively. The MICs were determined by the tube dilution method against Paenibacillus larvae. The oils showed MICs values were 300 µg/mL and 250 μg/mL for P. anisum and F. vulgare, respectively. Both oils presented great similarity in physiochemical properties values and antimicrobial activity.

Efficacy of formic acid in gel for Varroa control in Apis mellifera L.: importance of the dispenser position inside the hive.

The efficacy of formic acid in a gel matrix was evaluated in two groups of honeybee colonies. In Group 1, a dispenser with 120 g of formic acid (70%) in gel was placed on the brood combs and another dispenser with the same dose was located on the hive bottom (total dose, 240 g). Group 2 received two doses of 240 g of formic acid (70%) in gel and each application was applied in two dispensers containing 120 g of the formic acid solution each and they were located over the brood chamber (total dose, 480 g). In Group 2, the period between both applications was 15 days, and the efficacies after the first and both applications were calculated. Significant differences were registered for final efficacy between both groups. When final efficacy of Group 1 was compared with efficacy after first application of Group 2, significant differences were found (P=0.0005). Same doses in different positions within the hive have different final efficacy. The higher efficacy was registered when the dispensers were placed over brood combs and on the hive bottom. It is suggested that efficacy is related to dispenser position within the hive.

LC50 baseline levels of amitraz, coumaphos, fluvalinate and flumethrin in populations of Varroa destructor from Buenos Aires Province, Argentina

Summary This study estimates the LC50 baseline levels for amitraz, coumaphos, fluvalinate, and flumethrin in susceptible Varroa destructor populations in Argentina. Concentration response bioassays were conducted with each acaricide. Laboratory results of lethal concentrations (LC50) were: 0.1 μg/dish for amitraz; 0.29 μg/dish for fluvalinate; 0.34 μg/dish for flumethrin; and 0.57 μg/dish for coumaphos, respectively. All tests guaranteed 100 % bee survival. LC50 references for mite populations susceptible to the most commonly used acaricides were thus established for Argentina, which will aid the establishment of integrated pest management programmes for V. destructor.

The concentration effect of selected acaricides present in beeswax foundation on the survival of Apis mellifera colonies

Summary Acaricides used for the control of Varroa destructor are a major source of pollution in a honey bee (Apis mellifera) hive. Because they involve a slow release, they must be present for a period of up to 45 days inside a hive to be effective. The aim of this study was to determine whether the presence of acaricides in beeswax affects the survival of breeding bees, and if this effect is greater at higher concentrations. Three types of recycled beeswax foundation containing paraffin wax in different proportions (0%, 20% and 40%) were used. Brood survival rate was calculated in each treatment (N°. pupae/N°. Eggs * 100). Acaricide content in wax sheets was determined by multi-residue analysis by GC-ECD. Survival rate was higher when using beeswax adulterated with paraffin. Recycled beeswax without added paraffin wax (0%) had high levels of coumaphos and fluvalinate contamination, and when paraffin wax was added in different percentages (20%, 40%) the concentration of these components was lower. The presence of acaricides in beeswax adversely affected brood survival. When the pesticide concentration decreased, an improvement in the survival rate was found. Larvae developed in beeswax foundation without paraffin wax, exposed at higher concentration of pollutant residues were more vulnerable to the toxic effects of the acaricides.

Bioactivity of Rosmarinus officinalis essential oils against Apis mellifera, Varroa destructor and Paenibacillus larvae related to the drying treatment of the plant material

In this study, chemical composition, physicochemical properties and bioactivity of two essential oils of Rosmarinus officinalis extracted from plant material with different drying treatments against Apis mellifera, Varroa destructor and Paenibacillus larvae were assessed. The lethal concentration 50 (LC50) for mites and bees was estimated using a complete exposure method test. The broth microdilution method was followed in order to determine the minimum inhibitory concentrations (MICs) of the essential oils against P. larvae. Physicochemical properties were similar in both the essential oils, but the percentage of components showed certain differences according to their drying treatment. β-Myrcene and 1,8-cineole were the main constituents in the oils. The LC50 for complete exposure method at 24, 48 and 72 h was minor for mites exposed to R. officinalis essential oil dried in oven conditions. MIC values were 700–800 µg mL−1 and 1200 µg mL−1 for R. officinalis dried in air and oven conditions, respectively. The results reported in this research show that oil toxicity against V. destructor and P. larvae differed depending on the drying treatment of the plant material before the distillation of essential oil.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Evaluation of the toxicity of a propolis extract on Varroa destructor (Acari: Varroidae) and Apis mellifera (Hymenoptera: Apidae)

Summary The effects of a propolis extract on Varroa destructor and Apis mellifera were evaluated by three different application methods: topical, spraying and oral. A propolis sample was extracted and its organoleptic and physic-chemically traits characterized. These analyses showed that it was a typical propolis from the Pampean region in Argentina, with elevated contents of biologically active compounds. Topical application was carried out by subjecting mites to contact with various propolis concentrations for different periods of time, which resulted in mortality and narcosis. Acaricidal effects were stronger with increasing concentrations of the propolis extracts. Spraying infested bees with a 10% propolis solution was harmless for bees but killed 78% of mites. Feeding infested bees with propolis extract in sugar syrup was not toxic to the mites but caused the death of bees treated with the highest concentration. Our results suggest that the propolis extracts from the Pampean Region could be incorporated into bee colonies by spraying, although the appropriate doses and concentrations to be administered, and the mechanism of action of the extracts on the mites are still to be elucidated.

In vitro antibacterial and antiparasitic effect of citrus fruit essential oils on the honey bee pathogen Paenibacillus larvae and the parasitic mite Varroa destructor.

Sandra Rosa Fuselli, Matías Maggi, Susana Beatriz García de la Rosa, Judith Principal, Martín Javier Eguaras and Rosalía Fritz Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Exactas y Naturales, Mar del Plata, Argentina. Comisión de Investigaciones Científicas (CIC), La Plata, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. Estación de Apicultura, Decanato de Ciencias Veterinarias, Universidad Centroccidental “Lisandro Alvarado” (UCLA), Tarabana, Estado Lara, Venezuela.