High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae).

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for a rapid identification of African mitotypes in Apis mellifera samples.

Laurel leaf extracts for honeybee pest and disease management: antimicrobial, microsporicidal, and acaricidal activity.

A diverse set of parasites and pathogens affects productivity and survival of Apis mellifera honeybees. In beekeeping, traditional control by antibiotics and molecules of synthesis has caused problems with contamination and resistant pathogens. In this research, different Laurus nobilis extracts are tested against the main honeybee pests through an integrated point of view. In vivo effects on bee survival are also evaluated. The ethanol extract showed minimal inhibitory concentration (MIC) values of 208 to 416 µg/mL, having the best antimicrobial effect on Paenibacillus larvae among all substances tested. Similarly, this leaf extract showed a significant antiparasitic activity on Varroa destructor, killing 50 % of mites 24 h after a 30-s exposure, and on Nosema ceranae, inhibiting the spore development in the midgut of adult bees ingesting 1 × 10(4) µg/mL of extract solution. Both ethanol extract and volatile extracts (essential oil, hydrolate, and its main component) did not cause lethal effects on adult honeybees. Thus, the absence of topical and oral toxicity of the ethanol extract on bees and the strong antimicrobial, microsporicidal, and miticidal effects registered in this study place this laurel extract as a promising integrated treatment of bee diseases and stimulates the search for other bioactive phytochemicals from plants.

Anthelmintic effect of Mentha spp. essential oils on Echinococcus granulosus protoscoleces and metacestodes.

The aim of the present work was to determine the in vitro effect of Mentha piperita and Mentha pulegium essential oils against Echinococcus granulosus and to compare the effectiveness of both oils according to the exposure time and concentration. Although both treatments had a protoscolicidal effect, M. pulegium had a considerably stronger effect than M. piperita. Essential oil of M. pulegium produced dose- and time-dependent effects. Maximal protoscolicidal effect was observed after 12 days of incubation and reached 0% after 18 days. This lack of viability was proved during the determination of infectivity into mice. Essential oil of M. piperita produced only a time-dependent effect. At 24 days p.i., the viability of protoscoleces decreased to approximately 50%. Scanning and transmission electron microscopy (SEM and TEM) demonstrated the drug-induced ultrastructural damage. On the other hand, a loss of turgidity was detected in all M. pulegium-treated cysts respective of the drug concentration. There was a correlation between the intensity of damage and the concentration of the essential oil assayed. Studies by SEM revealed that the germinal layer of treated cysts lost the feature multicellular structure. M. pulegium essential oil showed piperitone oxide as main compound in their composition, and we suggest that this component could be responsible of the markedly anthelmintic effect detected. Our data suggest that essential oils of Mentha spp. can be a promising source of potential protoscolicidal agents. The isolation of active anthelmintic constituents is in progress and may lead to the discovery of compounds with improved therapeutic value.

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.

Resistance phenomena to amitraz from populations of the ectoparasitic mite Varroa destructor of Argentina.

In Argentina, Varroa destructor resistance to coumaphos has been previously reported. However, the status of mite susceptibility to other hard acaricides is still unknown. At present, high infestation levels of V. destructor are being detected in colonies of Apis mellifera after treatment with amitraz. The aim of the present study was to determine the LC(50) of amitraz in V. destructor from three apiaries with high mite density after treatment with the acaricide. The LC(50) values were 3.9, 3.5, and 3.7 µg/Petri dish for mites from three different apiaries. Significant LC(50) differences were detected between resistant and susceptible mites. LC(50) increased 35-39-fold when compared to the corresponding baseline, suggesting the development of resistance. These results are the first report of resistance to amitraz in V. destructor in Argentina and extend the knowledge according to the status of acaricides resistance in the country.

Effect of bacterial metabolites on microsporidian Nosema ceranae and on its host Apis mellifera.

Nosemosis, a disease caused by a microsporidian infection, is one of the most frequently observed parasitic pathologies affecting adult honeybees. Presently, Nosema ceranae seems to be the main microsporidian infection in Apis mellifera. The antibiotic fumagillin is the only compound available to treat Nosema diseases; however, it is no longer licensed in most EU member states; therefore, the need to identify new molecules/substances prevails. The intent of this paper is to test bacterial metabolites by Bacillus and Enterococcus strains, isolated from bee midgut and honey. The toxicity on bees and the antiparasitic activity on N. ceranae were assessed under laboratory conditions. Results did not yield toxicity for the administered surfactin or bacteriocin concentrations. Spores exposed to direct contact with a particular surfactin revealed a significant infectivity reduction when inoculated on bees. This surfactin, administered ad libitum from the individuals' emergence, led to a significant reduction in parasitosis development when bees were infected with untreated spores 7 days postemergence. Based on the results obtained, one of the surfactins is herein postulated as a molecule capable of reducing N. ceranae development, acting either by direct exposure to purified spores or incorporated into the digestive tract of the bee.

Bioactivity of propolis from different geographical origins on Varroa destructor (Acari: Varroidae).

Varroa destructor is an ectoparasitic mite that affects colonies of honey bee Apis mellifera worldwide. In the last years, substances of botanical origin have emerged as natural alternative acaricides to diminish the population levels of the mite. In the present work, the bioactivity of propolis from different geographical locations of Pampean region from Argentina on V. destructor was evaluated. Fourteen propolis samples were organoleptic and physicochemically characterized and, by means topical applications, their activity was tested on mites. All propolis had a homogeneous composition and the bioactivity levels against mites were comparable among the different propolis samples. The percentage of mites killed by the treatments ranged between 60.5% and 90% after 30 s of exposure. Thus, V. destructor was highly susceptible to propolis. Moreover, the mites remained anesthetized during the first hours after topical treatment. The results suggest that propolis from Argentinean pampas could be incorporated in honey bee colonies as acaricidal treatment by spraying.

Acaricidal and insecticidal activity of essential oils on Varroa destructor (Acari: Varroidae) and Apis mellifera (Hymenoptera: Apidae).

Varroa destructor is an external parasitic mite that is a serious pest of honeybees and has caused severe losses of colonies worldwide. One of the feasible alternative treatments being used for their control is essential oils. The aim of this work was to evaluate the bioactivity of some essential oils on V. destructor and Apis mellifera in relation with their chemical composition and physicochemical properties. Lavender, lavendin and laurel essential oils showed linalool as main compound in their composition. 1,8-Cineole was also present as a predominant component in the laurel essential oil. However, thyme oil was characterized by a high concentration of thymol. Mites and bees toxicity was tested by means of complete exposure method. For mites, LC(50) values for laurel, lavender and lavendin essential oil did not show significant variation throughout all observation times. However, the LC(50) values for thyme oil at 48 and 72 h were lower than at 24 h. Bee mortality was evident only in treatment with thyme oil. At 48 and 72 h, lavender essential oil presented better selectivity indexes. In this research, all essential oils caused mite mortality without severe harmful effects on adult bees. The simultaneous evaluation of the physicochemical analysis of the essential oils, the characterization of the dosage response relationships among them, and the mortality effects on mite and bees, give us the possibility to obtain comparative results for future research in Varroa control.

Morphotypes of Varroa destructor collected in Apis mellifera colonies from different geographic locations of Argentina.

Parasites display considerable phenotypic plasticity in life-history traits such as, body size. Varroa destructor is an ectoparasitic mite of the western honey bee Apis mellifera. Several studies have reported that in V. destructor, there is a wide phenotypic plasticity within a population of mites. However, it is unknown if there are morphologic variations in V. destructor populations affecting different A. mellifera populations. A morphometric study of V. destructor populations was conducted to provide information concerned to the relationships among parasite populations found in different geographic locations from A. mellifera colonies of Argentina. The hypothesis tested was different morphotypes of V. destructor populations parasitizing different A. mellifera populations from Argentina exist. A discriminant analysis employing eight morphologic variables revealed that it is possible to differentiate morphotypes of mites in Argentina. However, the level of discrimination detected among mites population varied according to the grouping of mite's population. Possible causes explaining the morphometric variability in the V. destructor populations were discussed.

First detection of Varroa destructor resistance to coumaphos in Argentina.

In Argentina, studies on Varroa destructor resistance to coumaphos are still unknown. At present, high infestation levels of V. destructor are being detected in colonies of Apis mellifera after treatment with this acaricide. The aim of the present study was to determine the LC50 of coumaphos in V. destructor from four apiaries with high mite density after treatment with coumaphos. The LC50's were 112, 319, 127 and 133 microg/Petri dish for mites from the four apiaries. Significant LC50 differences were detected between resistant and susceptible mites. LC50 increased 197-559-fold when compared to the corresponding baseline, suggesting the development of resistance. These results are the first report of resistance to coumaphos in V. destructor in Argentina.