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.

Decoupling the effects of nutrition, age, and behavioral caste on honey bee physiology, immunity, and colony health.

Nutritional stress, especially a dearth of pollen, has been linked to honey bee colony losses. Colony-level experiments are critical for understanding the mechanisms by which nutritional stress affects individual honey bee physiology and pushes honey bee colonies to collapse. In this study, we investigated the impact of pollen restriction on key markers of honey bee physiology, main elements of the immune system, and predominant honey bee viruses. To achieve this objective, we uncoupled the effects of behavior, age, and nutritional conditions using a new colony establishment technique designed to control size, demography, and genetic background. Our results showed that the expression of storage proteins, including vitellogenin (vg) and royal jelly major protein 1 (mrjp1), were significantly associated with nursing, pollen ingestion, and older age. On the other hand, genes involved in hormonal regulation including insulin-like peptides (ilp1 and ilp2) and methyl farnesoate epoxidase (mfe), exhibited higher expression levels in young foragers from colonies not experiencing pollen restriction. In contrast, pollen restriction induced higher levels of insulin-like peptides in old nurses. On the other hand, we found a strong effect of behavior on the expression of all immune genes, with higher expression levels in foragers. In contrast, the effects of nutrition and age were significant only the expression of the regulatory gene dorsal. We also found multiple interactions of the experimental variables on viral titers, including higher Deformed wing virus (DWV) titers associated with foraging and age-related decline. In addition, nutrition significantly affected DWV titers in young nurses, with higher titers induced by pollen ingestion. In contrast, higher levels of Black queen cell virus (BQCV) were associated with pollen restriction. Finally, correlation, PCA, and NMDS analyses proved that behavior had had the strongest effect on gene expression and viral titers, followed by age and nutrition. These analyses also support multiple interactions among genes and virus analyzed, including negative correlations between the expression of genes encoding storage proteins associated with pollen ingestion and nursing (vg and mrjp1) with the expression of immune genes and DWV titers. Our results provide new insights into the proximal mechanisms by which nutritional stress is associated with changes in honey bee physiology, immunity, and viral titers.

Insights into the effects of sublethal doses of pesticides glufosinate-ammonium and sulfoxaflor on honey bee health.

Insect pollinators are threatened worldwide, being the exposure to multiple pesticides one of the most important stressor. The herbicide Glyphosate and the insecticide Imidacloprid are among the most used pesticides worldwide, although different studies evidenced their detrimental effects on non-target organisms. The emergence of glyphosate-resistant weeds and the recent ban of imidacloprid in Europe due to safety concerns, has prompted their replacement by new molecules, such as glufosinate-ammonium (GA) and sulfoxaflor (S). GA is a broad-spectrum and non-selective herbicide that inhibits a key enzyme in the metabolism of nitrogen, causing accumulation of lethal levels of ammonia; while sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and generates excitatory responses including tremors, paralysis and mortality. Although those molecules are being increasingly used for crop protection, little is known about their effects on non-target organisms. In this study we assessed the impact of chronic and acute exposure to sublethal doses of GA and S on honey bee gut microbiota, immunity and survival. We found GA significantly reduced the number of gut bacteria, and decreased the expression of glucose oxidase, a marker of social immunity. On the other hand, S significantly increased the number of gut bacteria altering the microbiota composition, decreased the expression of lysozyme and increased the expression of hymenoptaecin. These alterations in gut microbiota and immunocompetence may lead to an increased susceptibility to pathogens. Finally, both pesticides shortened honey bee survival and increased the risk of death. Those results evidence the negative impact of GA and S on honey bees, even at single exposition to a low dose, and provide useful information to the understanding of pollinators decline.

Seasonal Dynamics of the Honey Bee Gut Microbiota in Colonies Under Subtropical Climate : Seasonal Dynamics of Honey Bee Gut Microbiota.

Honey bees (Apis mellifera) provide invaluable benefits for food production and maintenance of biodiversity of natural environments through pollination. They are widely spread across the world, being adapted to different climatic conditions. To survive the winter in cold temperate regions, honey bees developed different strategies including storage of honey and pollen, confinement of individuals during the winter, and an annual cycle of colony growth and reproduction. Under these conditions, winter honey bees experience physiological changes, including changes in immunity and the composition of honey bee gut microbiota. However, under tropical or subtropical climates, the life cycle can experience alterations, i.e., queens lay eggs during almost all the year and new honey bees emerge constantly. In the present study, we characterized nurses' honey bee gut microbiota in colonies under subtropical region through a year, combining qPCR, PCR-DGGE, and 16S rDNA high-throughput sequencing. We also identified environmental variables involved in those changes. Our results showed that under the mentioned conditions, the number of bacteria is stable throughout the year. Diversity of gut microbiota is higher in spring and lower in summer and winter. Gradual changes in compositions occur between seasons: Lactobacillus spp. predominate in spring while Gilliamella apicola and Snodgrasella alvi predominate in summer and winter. Environmental variables (mainly precipitations) affected the composition of the honey bee gut microbiota. Our findings provide new insights into the dynamics of honey bee gut microbiota and may be useful to understand the adaptation of bees to different environmental conditions.

Impact of Chronic Exposure to Sublethal Doses of Glyphosate on Honey Bee Immunity, Gut Microbiota and Infection by Pathogens.

Glyphosate is the most used pesticide around the world. Although different studies have evidenced its negative effect on honey bees, including detrimental impacts on behavior, cognitive, sensory and developmental abilities, its use continues to grow. Recent studies have shown that it also alters the composition of the honey bee gut microbiota. In this study we explored the impact of chronic exposure to sublethal doses of glyphosate on the honey bee gut microbiota and its effects on the immune response, infection by Nosema ceranae and Deformed wing virus (DWV) and honey bee survival. Glyphosate combined with N. ceranae infection altered the structure and composition of the honey bee gut microbiota, for example by decreasing the relative abundance of the core members Snodgrassella alvi and Lactobacillus apis. Glyphosate increased the expression of some immune genes, possibly representing a physiological response to mitigate its negative effects. However, this response was not sufficient to maintain honey bee health, as glyphosate promoted the replication of DWV and decreased the expression of vitellogenin, which were accompanied by a reduced life span. Infection by N. ceranae also alters honey bee immunity although no synergistic effect with glyphosate was observed. These results corroborate previous findings suggesting deleterious effects of widespread use of glyphosate on honey bee health, and they contribute to elucidate the physiological mechanisms underlying a global decline of pollination services.

Unraveling Honey Bee-Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations.

The ectoparasite Varroa destructor is the greatest biotic threat of honey bees Apis mellifera in vast regions of the world. Recently, the study of natural mite-resistant populations has gained much interest to understand the action of natural selection on the mechanisms that limit the mite population. In this study, the components of the A. mellifera-V. destructor relationship were thoroughly examined and compared in resistant and susceptible honey bee populations from two regions of Uruguay. Mite-resistant honey bees have greater behavioral resistance (hygienic and grooming behaviors) than susceptible honey bees. At the end of the summer, resistant honey bees had fewer mites and a lower deformed wing virus (DWV) viral load than susceptible honey bees. DWV variant A was the only detected variant in honey bees and mites. Molecular analysis by Short Tandem Repeat showed that resistant honey bees were Africanized (A. m. scutellata hybrids), whereas susceptible honey bees were closer to European subspecies. Furthermore, significant genetic differentiation was also found between the mite populations. The obtained results show that the natural resistance of honey bees to V. destructor in Uruguay depends on several factors and that the genetic variants of both organisms can play a relevant role.

Detection of Lotmaria passim in Africanized and European honey bees from Uruguay, Argentina and Chile.

Trypanosomatids affecting honey bees, Crithidia mellificae and Lotmaria passim, have been poorly studied in South America. We therefore analyzed their presence in Africanized and European honeybees from Uruguay, Argentina and Chile collected between 1990 and 2011 and assessed their association with other bee parasites and pathogens. Crithidia mellificae was not detected while L. passim was wide-spread. This report shows that L. passim has been present in this region at least since 2007 and it infects both Africanized and European honey bees. L. passim infected colonies showed high V. destructor parasitization levels, suggesting an association between them.

Nosemosis control in European honey bees, Apis mellifera, by silencing the gene encoding Nosema ceranae polar tube protein 3.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities for combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera, and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 (ptp3) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae-infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3-dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared with untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future, and warrant further investigation.

Epormenis cestri secretions in Sebastiania schottiana trees cause mass death of honey bee Apis mellifera larvae in Uruguay.

For more than 60 years, sporadic cases of massive summer honey bee larvae mortality in colonies located near freshwater systems with abundant riparian vegetation have been reported in Uruguay. This odd phenomenon, known as "River disease" by beekeepers, can lead to colony death by depopulation. The aim of this study was to detect the causes of larvae death. Different experiments and analyses were performed using affected apiaries located between two important water courses. 1 day old larvae were the most susceptible and substances that killed the larvae were present in the nectar but not in the pollen. A palynological analysis of nectar samples showed that bees collect this resource from commonly pollinated floral species in the country. However, abundant fungi spores and conidia were found, which indicates that the bees also collected honeydews. In the riparian vegetation, bees were observed collecting the secretions of the planthopper Epormenis cestri on Sebastiania schottiana trees. It was found that the mortality period of larvae overlaps with the presence of E. cestri. Larvae maintained in the laboratory were fed (i) nectar from healthy colonies, (ii) nectar from affected colonies, and (iii) secretions of E. cestri. The mortality of the larvae that received nectar from colonies affected with River disease and secretions of E. cestri was higher than the mortality of those receiving nectar from healthy colonies. This represents the first report of planthopper honeydew causing mass larval mortality in honey bees.

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.

Seasonal Variation of Honeybee Pathogens and its Association with Pollen Diversity in Uruguay.

Honeybees are susceptible to a wide range of pathogens, which have been related to the occurrence of colony loss episodes reported mainly in north hemisphere countries. Their ability to resist those infections is compromised if they are malnourished or exposed to pesticides. The aim of the present study was to carry out an epidemiological study in Uruguay, South America, in order to evaluate the dynamics and interaction of honeybee pathogens and evaluate their association with the presence of external stress factors such as restricted pollen diversity and presence of agrochemicals. We monitored 40 colonies in two apiaries over 24 months, regularly quantifying colony strength, parasite and pathogen status, and pollen diversity. Chlorinated pesticides, phosphorus, pyrethroid, fipronil, or sulfas were not found in stored pollen in any colony or season. Varroa destructor was widespread in March (end of summer-beginning of autumn), decreasing after acaricide treatments. Viruses ABPV, DWV, and SBV presented a similar trend, while IAPV and KBV were not detected. Nosema ceranae was detected along the year while Nosema apis was detected only in one sample. Fifteen percent of the colonies died, being associated to high V. destructor mite load in March and high N. ceranae spore loads in September. Although similar results have been reported in north hemisphere countries, this is the first study of these characteristics in Uruguay, highlighting the regional importance. On the other side, colonies with pollen of diverse botanical origins showed reduced viral infection levels, suggesting that an adequate nutrition is important for the development of healthy colonies.

American Foulbrood in Uruguay: twelve years from its first report.

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a deleterious disease that affects honeybees. In Uruguay it was first reported in 1999. In 2001 the bacterium was spread all over the country, and its prevalence in honey was estimated in 51%. Two P. larvae genotypes were found; ERIC I - BOX A, worldwide distributed and ERIC I - BOX C, exclusively detected in Argentina until then. In the present manuscript we analyzed the evolution of AFB outbreaks from 1999 to 2009, presented a new nation-wide survey carried out during 2011 when a prevalence of 2% was found and discuss national strategies for prevention of the disease. Since Uruguay is a small country where almost all beekeepers are registered, Uruguayan experience can be useful to be applied in other countries.