Martín J. Eguaras

LD50 and Repellent Effects of Essential Oils from Argentinian Wild Plant Species on Varroa destructor

Abstract The repellent and acaricidal effects of some essential oils from the most typical wild plant species of northern Patagonia, Argentina, on Varroa destructor Anderson & Trueman were evaluated using a complete exposure test. Honey bees, Apis mellifera L., and mites (five specimens of each per dish) were introduced in petri dishes having different oil concentrations (from 0.1 to 25 μl per cage). Survival of bees and mites was registered after 24, 48, and 72 h. An attraction/repellence test was performed using a wax tube impregnated with essential oil and another tube containing wax only. The lowest LD50 values for mites were registered for Acantholippia seriphioides (A. Gray) Mold. (1.27 μl per cage) and Schinus molle L. (2.65 μl per cage) after 24 h, and for Wedelia glauca (Ortega) O. Hoffm. ex Hicken (0.59 μl per cage) and A. seriphioides (1.09 μl per cage) after 72 h of treatment. The oil with the highest selectivity ratio (A. mellifera LD50/V. destructor LD50) was the one extracted from S. molle (>16). Oils of Lippia junelliana (Mold.) Troncoso, Minthostachys mollis (HBK) Grieseb., and Lippia turbinata Grieseb. mixed with wax had repellent properties. None of the oils tested had attractive effects on Varroa mites.

Efficacy of natural propolis extract in the control of American Foulbrood

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease that affects larvae of the honeybees. Due to the serious effects associated with AFB and the problems related to the use of antibiotics, it is necessary to develop alternative strategies for the control of the disease. The aim of the present work was to evaluate the effect of a propolis ethanolic extract (PEE) against P. larvae and its potential for the control of AFB. In vitro activity of PEE against P. larvae isolates was evaluated by the disk diffusion method and the minimum inhibitory concentration (MIC) was determined. Toxicity for honeybees was evaluated by oral administration of PEE and its lethal concentration was assessed. Lastly, colonies from an apiary with episodes of AFB on previous years were divided into different groups and treated with sugar syrup supplemented with PEE by aspersion (group one), sugar syrup by aspersion (group two), fed with sugar syrup supplemented with PEE (group three) and fed with sugar syrup only (group four). All isolates were sensitive to PEE and the MIC median was 0.52% (range 0.32-0.64). PEE was not toxic for bees at least at 50%. Field assays showed that 21 and 42 days after the application of the treatments, the number of P. larvae spores/g of honey was significantly lower in colonies treated with PEE compared to the colonies that were not treated with PEE. To our knowledge, this is the first report about the use of propolis for the treatment of beehives affected with P. larvae spores.

Genetic variation and widespread dispersal of Nosema ceranae in Apis mellifera apiaries from Argentina

Using molecular techniques, we documented the presence of Nosema ceranae in honeybees (Apis mellífera) from Argentina. Samples were collected from A. mellifera colonies in 38 districts of Buenos Aires province, Argentina. Molecular characterization was achieved with a multiplex PCR-based method, which allows parallel diagnosis of N. ceranae and N. osema apis. N. ceranae was identified in all the samples analyzed. Moreover, coinfections with N. apis were detected in Balcarce and Maipú districts. We identified three rRNA sequence variants of N. ceranae, which may represent diverse sources of bee importation. The results suggest that N. ceranae is widely distributed in Argentina and that the genetic variation observed between the different isolates could be related with the difference in the symptomatology found previously by our work group. Our results highlight the need to re-assess the health protocols currently in force so that they recognize N. ceranae as the main causal agent of Nosemosis in this country.

An in vitro Evaluation of Tagetes minuta Essential Oil for the Control of the Honeybee Pathogens Paenibacillus larvae and Ascosphaera apis, and the Parasitic Mite Varroa destructor

Abstract Biological activity was evaluated of the Tagetes minuta essential oil in different in vitro laboratory experiments on the mite (Varroa destructor), honeybees (Apis mellifera), the bacterium that causes the American Foulbrood (Paenibacillus larvae), and the fungus that produces chalkbrood (Ascosphaera apis). Two methods of complete exposure were used for mite lethality test: by spraying in Burgerjons tower with 10 mg of active ingredient in solution with distilled water and emulsion, and in unmodified Petri dishes (60 x 20 mm) with oil (different concentrations) diluted in 1 mL of ethanol. Ratio selection was obtained as: LD50 of Apis mellifera/LD50 of V. destructor. Determination of Minimal Inhibitory Concentrations (MIC): to P. larvae was tested at concentrations of 25, 50, 100, 150, 200, 250, 350, 450, 500, 600, 650, 700, 800 and 1000 ppm. An A. apis strain was grown on agar MY20 supplemented with variable concentrations (between 0–800 ppm) of T. minuta oil was evaluated. Results obtained in tests of total exposure showed that the concentration was able to kill 50% of mites in 24 h (DL50) and was estimated to be 4.37 mg/cage. The efficacy after spray treatment reached 56%. The ratio selection was 3:11. Against P. larvae, the oil showed MIC values ranging from 700–800 μL/L depending on the tested bacterial strains. Tagetes minuta oil in agar MY20 inhibited mycelial growth of A. apis above concentrations of 200 ppm (p = 0.0001). Oil concentrations of 700 and 800 ppm achieved maximum growth inhibition of A. apis (67% of growth inhibition on average). Tagetes minuta oil demonstrated in vitro antibacterial, antifungical and miticide activity, although this oil shows a moderate inhibitor effect compared with other essential oils of native plants from Argentina. However, this oil presents a ratio selection that would allow it to be used in field conditions with a good safety margin. It is possible that this oil can be used in combination with others, in integrated pest management strategies in bee colonies.

Efficacy of thymol against Echinococcus granulosus protoscoleces.

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 mug/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 microg/ml, viability reduced to 53.5+/-11.9% after 12 days of incubation. At day 42, viability was 11.5+/-15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 microg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.

Nosema ceranae development in Apis mellifera: influence of diet and infective inoculum

Summary To investigate the effect of nutritional condition of the honey bee Apis mellifera on the development of the microsporidian parasite Nosema ceranae under laboratory conditions, newly emerged bees were confined and fed on three ad libitum diets: high fructose corn syrup (HFCS) + fresh bee bread; HFCS + a commercial mixture of amino acid and vitamin, and HFCS. On day 7 post-emergence, bees from each diet treatment were individually infected with 4.60 × 104, 2.30 × 105 or 1.15 × 106 spores of N. ceranae, keeping later on the same diet. On days 3, 6, 9, and 12, post-infection bee midguts were removed to individually quantify the spores developed. The results indicate that this parasite multiplies successfully regardless of the inoculum given or the nutritional status of its host. When bees are fed on pollen, however, the parasite develops quickly, exhibiting significantly higher intensities than under other treatments. The longevity of infected bees fed on the same diet was not affected by the degree of parasitism, but by the quality of the ad libitum diet administered. The data demonstrate a parasite development that depends on host-condition. This should be considered when designing experiments to evaluate the development and virulence of this pathogen.

In Vitro Activity of Essential Oils from San Luis-Argentina Against Ascosphaera apis

Abstract Chalkbrood is an invasive mycosis produced by Ascosphaera apis affecting exclusively the larvae growth of Apis mellifera L. There exists no pharmacological treatment and the chemical products used are not able to control the disease generating resistances and residues in the apicultural production. An ecological alternative is the use of essential oils as natural products to control this mycosis. Eight oils were screened against A. apis for fungicidal activity. The oils of Baccharis coridifolia and Eupatorium patens did not possess any activity while the oils of Tessaria absinthioides, Aloysia gratissima, Heterotheca latifolia, Lippia juneliana, L. integrifolia and L. turbinata exhibited varying levels of fungicidal activity.

Laboratory Evaluations of Syzygium aromaticum (L.) Merr. et Perry Essential Oil Against Varroa destructor

Abstract The oil obtained by hydrodistillation of the foral bottom of Syzygium aromaticum (L.) Merr. et Perry was analyzed by GC and GC/MS. Eugenol was the main constituent in the oil (86.7%). The biological activity of the oil applied to Varroa destructor and Apis mellifera was evaluated in two laboratory tests. Mite lethality was estimated using a complete exposure method test with the oil at different concentrations, and a systemic administration method of oil at different concentrations diluted in syrup was placed in feeders for bees. The LC50 for complete exposure method at 24 h was 0.59 μL/dish. The inferior and superior limits obtained were 0.47 x 10−6 μL/dish and 1.22 μL/dish, respectively. LC50 estimated at 48 h showed a slight decrease as compared to that recorded at 24 h. Ratio selection (LC50 of A. mellifera/LC50 of V. destructor) for complete exposure method was 26.46 and 13.35 for 24 h and 48 h, respectively. Regarding the systemic administration method, mites LC50 at 24 h was 12,300 ppm. The inferior and superior limits calculated were 9,214 ppm and 15,178 ppm, respectively. LC50 estimated at 48 h showed a slight decrease as compared to that recorded at 24 h. Ratio selection for systemic administration method was 3.05 and 2.22 for 24 h and 48 h, respectively. Syzygium aromaticum oil was found to be an attractant for V. destructor at 4.8% (w/w) concentration. The results showed that oil toxicity against V. destructor differed depending upon its administration. Nevertheless, the ratio selection calculated by this oil is expected to enable its application under field conditions with a good safety margin. This oil could also be used in combination with other oils in integrated pest management strategies in bee colonies.

Effects of the organic acids produced by a lactic acid bacterium in Apis mellifera colony development, Nosema ceranae control and fumagillin efficiency.

The European honey bee Apis mellifera is known to be affected by many parasites and pathogens that have great impact over the insect development. Among parasites affecting bee health, Nosema ceranae is one of the main biotic factors affecting colony populations. As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. The main goal of the current study was to assess the effect of the oral administration of the metabolites produced by Lactobacillus johnsonii CRL1647 (mainly organic acids) supplemented in syrup, on: (I) N. ceranae sporulation dynamics before and after fumagillin application, and (II) performance of A. mellifera colonies. Different experiments were conducted to evaluate the effects of these bacterial metabolites on bees: in vitro administration revealed no toxic effects against bees. Colonies fed with the lactic acids incremented their beehive population and also the amount of fat bodies per bee. Finally, the organic acids reduced the intensity of the pathogen after the second application of treatment as well as enhanced the fumagillin efficiency. This study provides important information for the development of new control substances against nosemosis.

Repellent and acaricidal effects of botanical extracts on Varroa destructor

Extracts of indigenous plants from South America have shown a broad spectrum of bioactivities. No-contaminant and natural substances have recently resurged as control treatment options for varroosis in honey bee colonies from Argentina. The aim of this work was to evaluate the biological activity of botanical extracts from Baccharis flabellata and Minthostachys verticillata on Varroa destructor and Apis mellifera. The acaricidal and insecticidal activities were assessed by the spraying application method. Both ethanolic extracts showed high levels of toxicity against the mites and were harmless to their host, A. mellifera. During the attractive-repellent test, the olfactory stimulus evoked for the extract from B. flabellata resulted as a repellent for mites. The aromatic stimulus of these extracts would be strong enough to cause disturbance on the behavior of V. destructor. Thus, the repellent effect of these substances plus the toxicity on mites postulate these botanical extracts like promising natural compound to be incorporated for the control of varroosis.