First large-scale study reveals important losses of managed honey bee and stingless bee colonies in Latin America.

Over the last quarter century, increasing honey bee colony losses motivated standardized large-scale surveys of managed honey bees (Apis mellifera), particularly in Europe and the United States. Here we present the first large-scale standardized survey of colony losses of managed honey bees and stingless bees across Latin America. Overall, 1736 beekeepers and 165 meliponiculturists participated in the 2-year survey (2016-2017 and 2017-2018). On average, 30.4% of honey bee colonies and 39.6% of stingless bee colonies were lost per year across the region. Summer losses were higher than winter losses in stingless bees (30.9% and 22.2%, respectively) but not in honey bees (18.8% and 20.6%, respectively). Colony loss increased with operation size during the summer in both honey bees and stingless bees and decreased with operation size during the winter in stingless bees. Furthermore, losses differed significantly between countries and across years for both beekeepers and meliponiculturists. Overall, winter losses of honey bee colonies in Latin America (20.6%) position this region between Europe (12.5%) and the United States (40.4%). These results highlight the magnitude of bee colony losses occurring in the region and suggest difficulties in maintaining overall colony health and economic survival for beekeepers and meliponiculturists.

Bacterial diversity using metagenomics of 16s rDNA in water kefir, an innovative source of probiotics for bee nutrition.

Water kefir is a sparkling, slightly acidic fermented beverage made from sugar, water, and water kefir grains, which are a mixture of yeast and bacteria. These grains produce a variety of fermentation compounds such as lactic acid, acetaldehyde, acetoin, ethanol and carbon dioxide. In this study, a high-throughput sequencing technique was used to characterize the bacterial composition of the original water kefir from which potential probiotics were obtained. We studied the bacterial diversity of both water kefir grains and beverages. DNA was extracted from three replicate samples of both grains and beverages using the Powerlyzer Microbial Kit. The hypervariable V1-V2 region of the bacterial 16S ribosomal RNA gene was amplified to prepare six DNA libraries. Between 1.4M and 2.4M base-pairs were sequenced for the library. In total, 28721971 raw reads were obtained from all the samples. Estimated species richness was higher in kefir beverage samples compared to grain samples. Moreover, a higher level of microbial alpha diversity was observed in the beverage samples. Particularly, the predominant bacteria in beverages were Anaerocolumna and Ralstonia, while in grains Liquorilactobacillus dominated, with lower levels of Leuconostoc and Oenococcus. Although the bacterial diversity in kefir grains was low because only three genera were the most represented, all of them are LAB bacteria with the potential to serve as probiotics in the artificial feeding of bees.

Probiotics and in-hive fermentation as a source of beneficial microbes to support the gut microbial health of honey bees.

Managed populations of honey bees (Apis mellifera Linnaeus; Hymenoptera: Apidae) are regularly exposed to infectious diseases. Good hive management including the occasional application of antibiotics can help mitigate infectious outbreaks, but new beekeeping tools and techniques that bolster immunity and help control disease transmission are welcome. In this review, we focus on the applications of beneficial microbes for disease management as well as to support hive health and sustainability within the apicultural industry. We draw attention to the latest advances in probiotic approaches as well as the integration of fermented foods (such as water kefir) with disease-fighting properties that might ultimately be delivered to hives as an alternative or partial antidote to antibiotics. There is substantial evidence from in vitro laboratory studies that suggest beneficial microbes could be an effective method for improving disease resistance in honey bees. However, colony level evidence is lacking and there is urgent need for further validation via controlled field trials experimentally designed to test defined microbial compositions against specific diseases of interest.

Microbiological and Chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition / Caracterización microbiológica y química del kéfir de agua: una fuente innovadora de potenciales probióticos para la nutrición de las abejas

Abstract We evaluated the microbial composition of water kefir grains and beverage overthe course of one year to determine whether the number and type of microorganisms changedover the time. Bacteria and yeast colonies with different morphologies were isolated fromwater kefir and their antimicrobial activity was evaluated against Paenibacillus larvae andAscosphaera apis. A chemical characterization of kefir was also carried out. Our results con-firmed that bacteria and yeasts were more numerous in kefir grains compared with those in thebeverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

Resumen Evaluamos la composición microbiana del kéfir de agua durante un ano para determinar si la cantidad y el tipo de microorganismos cambiaban con el tiempo. Se aislaron colonias de bacterias y de levaduras con diferentes morfologías, y su actividad antimicrobiana se evaluó frente a Paenibacillus larvaey Ascosphaeraapis. También se realizó una caracterización química del kéfir. Nuestros resultados confirmaron que las bacterias y las levaduras eran más numerosas en los gránulos de kéfir en comparación con la parte líquida. Los recuentos de microorganismos disminuyeron, aunque una cantidad igualmente importante se encontró en el kéfir después de un año. Se aislaron del kéfir once cepas que inhibieron los mencionados patógenos de abejas. Los resultados genotípicos demostraron que estos aislamientos eran Lentilactobacillus hilgardii, Lentilactobacillus buchneri y Saccharomyces cerevisiae. Por lo tanto, el kéfir de agua podría ser una fuente innovadora de potenciales cepas probióticas para contribuir a la nutrición y sanidad de las abejas.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition.

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

Genetic diversification of an invasive honey bee ectoparasite across sympatric and allopatric host populations.

Invasive parasites are major threats to biodiversity. The honey bee ectoparasite, Varroa destructor, has shifted host and spread almost globally several decades ago. This pest is generally considered to be the main global threat to Western honey bees, Apis mellifera, although the damages it causes are not equivalent in all its new host's populations. Due to the high virulence of this parasite and the viruses it vectors, beekeepers generally rely on acaricide treatments to keep their colonies alive. However, some populations of A. mellifera can survive without anthropogenic mite control, through the expression of diverse resistance and tolerance traits. Such surviving colonies are currently found throughout the globe, with the biggest populations being found in Sub-Saharan Africa and Latin America. Recently, genetic differences between mite populations infesting surviving and treated A. mellifera colonies in Europe were found, suggesting that adaptations of honey bees drive mite evolution. Yet, the prevalence of such co-evolutionary adaptations in other invasive populations of V. destructor remain unknown. Using the previous data from Europe and novel genetic data from V. destructor populations in South America and Africa, we here investigated whether mites display signs of adaptations to different host populations of diverse origins and undergoing differing management. Our results show that, contrary to the differences previously documented in Europe, mites infesting treated and untreated honey bee populations in Africa and South America are genetically similar. However, strong levels of genetic differentiation were found when comparing mites across continents, suggesting ongoing allopatric speciation despite a recent spread from genetically homogenous lineages. This study provides novel insights into the co-evolution of V. destructor and A. mellifera, and confirms that these species are ideal to investigate coevolution in newly established host-parasite systems.

Equine rhinosporidiosis in Buenos Aires, Argentina

Abstract Rhinosporidiosis is caused by Rhinosporidium seeberi, a parasitic organism of the family Rhinosporideacea family, class Micomycetozoa. The disease is endemic in India; however, some cases were reported in Europe, Africa, North America, and South America. The aim of the present study is to report three cases of rhinosporidiosis in wild horses in different cities of Buenos Aires province, Argentina. We confirm the presence of R. seeberi in the analyzed samples using histopathological and PCR sequencing techniques.

Resumen La rinosporidiosis es una enfermedad causada por Rhinosporidium seeberi, un organismo parasitario clasificado en la familia Rhinosporideacea, clase Micomycetozoa. Es una enfermedad endémica de la India, pero se notificaron algunos casos en Europa, África, América del Norte y América del Sur. El objetivo del presente estudio fue describir tres casos de rinosporidiosis en caballos de vida libre en diferentes ciudades de la provincia de Buenos Ares, Argentina. Confirmamos la presencia de R. seeberi en las muestras analizadas utilizando técnicas histopatológicas, PCR y secuenciación.

Equine rhinosporidiosis in Buenos Aires, Argentina.

Rhinosporidiosis is caused by Rhinosporidium seeberi, a parasitic organism of the family Rhinosporideacea family, class Micomycetozoa. The disease is endemic in India; however, some cases were reported in Europe, Africa, North America, and South America. The aim of the present study is to report three cases of rhinosporidiosis in wild horses in different cities of Buenos Aires province, Argentina. We confirm the presence of R. seeberi in the analyzed samples using histopathological and PCR sequencing techniques.

Current status of resistance to ivermectin in Rhipicephalus sanguineus sensu stricto infesting dogs in three provinces in Argentina.

Intensive use of macrocyclic lactones for parasite control exerts strong selective pressure for arthropods such as ticks to become resistant to them. Rhipicephalus sanguineus sensu stricto is a tick and disease vector of significant public health and veterinary importance worldwide. We assessed the toxicological response to the macrocyclic lactone ivermectin (IVM) in R. sanguineus s.s. infesting dogs in Argentina. Samples of nine tick populations were obtained by inspecting dogs at veterinary clinics, hospitals, or rural areas in the provinces of San Luis, Rio Negro, and Buenos Aires. Pet owners were interviewed to gather data on the history of dog treatment with ectoparasiticides. The larval immersion test was used to assess the toxicological response of R. sanguineus s.s. to IVM. Dose-response mortality regressions, lethal concentrations (LC), and slope were calculated by probit analysis. The lowest LC concentrations were used to designate the reference susceptible population because a laboratory reference strain of R. sanguineus s.s. does not exist in Argentina. Compared with the most susceptible tick population in this study, six populations (66.66%) were classified as resistant to IVM. A clear interpopulation variation in the level of IVM resistance was present (resistance ratios at LC50% ranged from 1.0 to 18.33 and at LC99% ranged from 1.0 to 8.96). In San Luis Province, all tick populations were classified as resistant. The highest level of IVM resistance (resistance ratio at LC50%:18.83 and LC99%:8.96) was found in a population of R. sanguineus s.s. from a rural area in the province of Buenos Aires. It is concluded that populations of R. sanguineus s.s. from dogs in three provinces of Argentina were resistant to IVM. Clear interpopulation variation in the level of IVM resistance was present.

Intra-Colonial Viral Infections in Western Honey Bees (Apis Mellifera).

RNA viruses play a significant role in the current high losses of pollinators. Although many studies have focused on the epidemiology of western honey bee (Apis mellifera) viruses at the colony level, the dynamics of virus infection within colonies remains poorly explored. In this study, the two main variants of the ubiquitous honey bee virus DWV as well as three major honey bee viruses (SBV, ABPV and BQCV) were analyzed from Varroa-destructor-parasitized pupae. More precisely, RT-qPCR was used to quantify and compare virus genome copies across honey bee pupae at the individual and subfamily levels (i.e., patrilines, sharing the same mother queen but with different drones as fathers). Additionally, virus genome copies were compared in cells parasitized by reproducing and non-reproducing mite foundresses to assess the role of this vector. Only DWV was detected in the samples, and the two variants of this virus significantly differed when comparing the sampling period, colonies and patrilines. Moreover, DWV-A and DWV-B exhibited different infection patterns, reflecting contrasting dynamics. Altogether, these results provide new insight into honey bee diseases and stress the need for more studies about the mechanisms of intra-colonial disease variation in social insects.

Viruses that affect Argentinian honey bees (Apis mellifera).

Beekeeping is a widespread activity in Argentina, mainly producing honey that has gained both national and international recognition. There are more than 3,000,000 hives in the country, mainly concentrated in Buenos Aires Province (approximately 1,000,000 hives). In recent decades, worrying rates of hive loss have been observed in many countries around the world. In Latin America, the estimated loss of hives is between 13% (Peru and Ecuador) and 53% (Chile). Argentina had annual losses of 34% for the period of October 1, 2016 to October 1, 2017. The causes of these losses are not clear but probably involve multiple stressors that can act simultaneously. One of the main causes of loss of bee colonies worldwide is infestation by the ectoparasitic mite Varroa destructor in combination with viral infections. To date, 10 viruses have been detected that affect honey bees (Apis mellifera) in Argentina. Of these, deformed wing virus, sacbrood virus, acute bee paralysis virus, chronic bee paralysis virus, and Israeli acute bee paralysis can be transmitted by mites. Deformed wing virus and the AIK complex are the viruses most often associated with loss of hives worldwide. Considering that bee viruses have been detected in Argentina in several hymenopteran and non-hymenopteran insects, these hosts could act as important natural reservoirs for viruses and play an important role in their dispersal in the environment. Further studies to investigate the different mechanisms by which viruses spread in the environment will enable us to develop various strategies for the control of infected colonies and the spread of viruses in the habitat where they are found.

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina.

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.

Host range of Triatoma virus does not extend to Aedes aegypti and Apis mellifera.

Vector control is the most effective method to prevent transmission of Chagas disease. Control is mostly made through chemical insecticides although they have negative impact on wild pollinators, such as bees. Reducing pesticide use through biological alternatives could minimize the damage to these beneficial insects. Triatoma virus (TrV) is a pathogen able to kill triatomines and thus a valid candidate to be used as biological control agent. In this study we evaluate the capacity of TrV to infect an important beneficial insect (Apis mellifera) as well as a plague insect (Aedes aegypti). Results indicate that TrV does not infect the bees or mosquitoes tested in this study. The possible specificity of TrV for kissing bugs reinforces the possible use of TrV as a biological control agent for triatomines.

kMucormicosys due to Saksenaea vasiformis in a dog.

A 2-year-old female Border collie was examined for dermatitis with a partial alopecic zone around her left front member. Six months later the lesion became swollen, alopecic with ulcerated areas. Microscopy analysis of samples showed numerous non-septate, branching, thin-walled and irregular shaped hyphal elements. Fungal cultures and molecular studies identified Saksenaea vasiformis. Treatments with griseofulvin, itraconazole and surgical debridement were used, however, fourteen months later the dog was euthanatized because of the unfavorable clinical outcome.

Ascosphaera apis, the entomopathogenic fungus affecting larvae of native bees (Xylocopa augusti): first report in South America / Infección por Ascosphaera apis en larvas de abejas carpinteras (Xylocopa augusti): primer reporte en América del Sur

Background. Nowadays several invertebrate pollinators of crops and wild plants are in decline as result of multiple and, sometimes, unknown factors; among them, the modern agricultural practices, pests and diseases are postulated as the most important factors. Bees of the genus Xylocopa are considered effective pollinators of passion fruit crops in tropical regions, as well as important pollinators in wild plants, but these bees are attacked by several pathogens that affect different stages in their life cycle. The fungal species of the genus Ascosphaera are commonly associated with social and solitary bee larvae causing chalkbrood disease. Aims. The aim of the present study was to demonstrate the presence of Ascosphaera apis affecting larvae of Xylocopa augusti in South America. Methods. For this purpose, A. apis was isolated from affected larvae in YGPSA medium. Final identification was run out by three techniques: (1) Microscopic examination of the hyphae and sizes of the fruiting bodies; (2) Mating test, and specific sexual compatibility test, and (3) PCR detection, using specific primers. Results. This study demonstrates for the first time the presence of A. apis affecting larvae of X. augusti in South America. Conclusions. The evidence of A. apis affecting the larvae of X. augusti, and the fact that the sharing of pathogens between different bee species has been underestimated, suggests the need for further epidemiological studies in order to determine not only the prevalence of this pathogen among wild pollinators, but also its relationship to the sudden collapse of honey bee colonies in this region (AU)

Antecedentes. En la actualidad, las poblaciones de una gran cantidad de insectos polinizadores están disminuyendo debido a múltiples factores, no siempre conocidos, como las prácticas agrícolas modernas, las plagas y las nuevas enfermedades. Las abejas del género Xylocopa se consideran unos importantes polinizadores de los cultivos de maracuyá en el trópico, así como de gran cantidad de plantas silvestres. Estos polinizadores silvestres se ven afectados por diversos agentes patógenos en diferentes etapas de su ciclo de vida. En particular, varias especies del hongo entomopatógeno Ascosphaera están comúnmente asociadas a las larvas de abejas sociales y solitarias, en las que causan una infección conocida como cría yesificada. Objetivos. El objetivo del presente estudio fue demostrar la presencia de Ascosphaera apis en larvas de Xylocopa augusti en Sudamérica. Métodos. Se aisló A. apis de las larvas afectadas en medio YGPSA. La identificación final fue realizada utilizando tres técnicas: 1) examen microscópico del tamaño de las hifas y los cuerpos de fructificación; 2) test de apareamiento y test específico de compatibilidad sexual, y 3) detección mediante PCR, utilizando iniciadores específicos. Resultados. En este estudio se ha registrado por primera vez en Sudamérica la presencia de A. apis en larvas enfermas de la abeja nativa X. augusti. Conclusiones. La presencia de A. apis en larvas enfermas de X. augusti, sumada al hecho de que se ha subestimado la presencia de los mismos patógenos en diferentes especies de abejas, evidencia la necesidad de realizar más estudios epidemiológicos para determinar no solo la prevalencia de este patógeno entre polinizadores silvestres, sino también su relación con el repentino colapso de las colonias de abejas de la miel en esta región (AU)

Ascosphaera apis, the entomopathogenic fungus affecting larvae of native bees (Xylocopa augusti): First report in South America.

BACKGROUND: Nowadays several invertebrate pollinators of crops and wild plants are in decline as result of multiple and, sometimes, unknown factors; among them, the modern agricultural practices, pests and diseases are postulated as the most important factors. Bees of the genus Xylocopa are considered effective pollinators of passion fruit crops in tropical regions, as well as important pollinators in wild plants, but these bees are attacked by several pathogens that affect different stages in their life cycle. The fungal species of the genus Ascosphaera are commonly associated with social and solitary bee larvae causing chalkbrood disease. AIMS: The aim of the present study was to demonstrate the presence of Ascosphaera apis affecting larvae of Xylocopa augusti in South America. METHODS: For this purpose, A. apis was isolated from affected larvae in YGPSA medium. Final identification was run out by three techniques: (1) Microscopic examination of the hyphae and sizes of the fruiting bodies; (2) Mating test, and specific sexual compatibility test, and (3) PCR detection, using specific primers. RESULTS: This study demonstrates for the first time the presence of A. apis affecting larvae of X. augusti in South America. CONCLUSIONS: The evidence of A. apis affecting the larvae of X. augusti, and the fact that the sharing of pathogens between different bee species has been underestimated, suggests the need for further epidemiological studies in order to determine not only the prevalence of this pathogen among wild pollinators, but also its relationship to the sudden collapse of honey bee colonies in this region.

Evaluación del método epsilométrico Etest para la determinación de la sensibilidad a tetraciclina en Paenibacillus larvae, agente causal de la loque americana de las abejas / [Evaluation of the Epsilometer (Etest) method for the detection of tetracycline susceptibility in Paenibacillus larvae, the causal agent of American foulbrood disease of honeybees].

American foulbrood (AFB) is a bacterial disease caused by the spore-forming, grampositive bacterium Paenibacillus larvae, which affects honeybee broods worldwide. The aim of this work was to compare the Epsilometer test (Etest) to the agar dilution method for testing a collection of 22 P. larvae strains to tetracycline by using MYPGP and Iso- Sensitest agars. Results showed that a categorical agreement of 100

was found when using Iso-Sensitest, while a categorical agreement of 86.36

was found (with 3 minor errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confirmed with future studies involving a larger number of isolates.

Simultaneous detection of bee viruses by multiplex PCR.

Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies.

[Evaluation of the Epsilometer (Etest) method for the detection of tetracycline susceptibility in Paenibacillus larvae, the causal agent of American foulbrood disease of honeybees]. / Evaluación del método epsilométrico Etest para la determinación de la sensibilidad a tetraciclina en Paenibacillus larvae, agente causal de la loque americana de las abejas.

American foulbrood (AFB) is a bacterial disease caused by the spore-forming, grampositive bacterium Paenibacillus larvae, which affects honeybee broods worldwide. The aim of this work was to compare the Epsilometer test (Etest) to the agar dilution method for testing a collection of 22 P. larvae strains to tetracycline by using MYPGP and Iso- Sensitest agars. Results showed that a categorical agreement of 100% was found when using Iso-Sensitest, while a categorical agreement of 86.36% was found (with 3 minor errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confirmed with future studies involving a larger number of isolates.