Vairimorpha (Nosema) ceranae can promote Serratia development in honeybee gut: an underrated threat for bees?

The genus Serratia harbors opportunistic pathogenic species, among which Serratia marcescens is pathogenic for honeybees although little studied. Recently, virulent strains of S. marcescens colonizing the Varroa destructor mite's mouth were found vectored into the honeybee body, leading to septicemia and death. Serratia also occurs as an opportunistic pathogen in the honeybee's gut with a low absolute abundance. The Serratia population seems controlled by the host immune system, but its presence may represent a hidden threat, ready to arise when honeybees are weakened by biotic and abiotic stressors. To shed light on the Serratia pathogen, this research aims at studying Serratia's development dynamics in the honeybee body and its interactions with the co-occurring fungal pathogen Vairimorpha ceranae. Firstly, the degree of pathogenicity and the ability to permeate the gut epithelial barrier of three Serratia strains, isolated from honeybees and belonging to different species (S. marcescens, Serratia liquefaciens, and Serratia nematodiphila), were assessed by artificial inoculation of newborn honeybees with different Serratia doses (104, 106, and 108 cells/mL). The absolute abundance of Serratia in the gut and in the hemocoel was assessed in qPCR with primers targeting the luxS gene. Moreover, the absolute abundance of Serratia was assessed in the gut of honeybees infected with V. ceranae at different development stages and supplied with beneficial microorganisms and fumagillin. Our results showed that all tested Serratia strains could pass through the gut epithelial barrier and proliferate in the hemocoel, with S. marcescens being the most pathogenic. Moreover, under cage conditions, Serratia better proliferates when a V. ceranae infection is co-occurring, with a positive and significant correlation. Finally, fumagillin and some of the tested beneficial microorganisms could control both Serratia and Vairimorpha development. Our findings suggest a correlation between the two pathogens under laboratory conditions, a co-occurring infection that should be taken into consideration by researches when testing antimicrobial compounds active against V. ceranae, and the related honeybees survival rate. Moreover, our findings suggest a positive control of Serratia by the environmental microorganism Apilactobacillus kunkeei in a in vivo model, confirming the potential of this specie as beneficial bacteria for honeybees.

Detrimental effects of amitraz exposure in honey bees (Apis mellifera) infected with Nosema ceranae.

In recent years, there has been growing concern on the potential weakening of honey bees and their increased susceptibility to pathogens due to chronic exposure to xenobiotics. The present work aimed to study the effects on bees undergoing an infection by Nosema ceranae and being exposed to a frequently used in-hive acaricide, amitraz. To achieve this, newly emerged bees were individually infected with N. ceranae spores and/or received a sublethal concentration of amitraz in their diets under laboratory conditions. Mortality, food intake, total volume excrement, body appearance, and parasite development were registered. Bees exposed to both stressors jointly had higher mortality rates compared to bees exposed separately, with no difference in the parasite development. An increase in sugar syrup consumption was observed for all treated bees while infected bees fed with amitraz also showed a diminishment in pollen intake. These results coupled with an increase in the total number of excretion events, alterations in behavior and body surface on individuals that received amitraz could evidence the detrimental action of this molecule. To corroborate these findings under semi-field conditions, worker bees were artificially infected, marked, and released into colonies. Then, they were exposed to a commercial amitraz-based product by contact. The recovered bees showed no differences in the parasite development due to amitraz exposure. This study provides evidence to which extent a honey bee infected with N. ceranae could potentially be weakened by chronic exposure to amitraz treatment.

The gut microbiome of solitary bees is mainly affected by pathogen assemblage and partially by land use.

Pollinators, including solitary bees, are drastically declining worldwide. Among the factors contributing to this decline, bee pathogens and different land uses are of relevance. The link between the gut microbiome composition and host health has been recently studied for social pollinators (e.g. honeybees), whereas the information related to solitary bees is sparse. This work aimed at the characterization of the gut microbiome of the solitary bees Xylocopa augusti, Eucera fervens and Lasioglossum and attempted to correlate the gut microbial composition with the presence and load of different pathogens and land uses. Solitary bees were sampled in different sites (i.e. a farm, a natural reserve, and an urban plant nursery) showing different land uses. DNA was extracted from the gut, 16S rRNA gene amplified and sequenced. Eight pathogens, known for spillover from managed bees to wild ones, were quantified with qPCR. The results showed that the core microbiome profile of the three solitary bees significantly varied in the different species. Pseudomonas was found as the major core taxa in all solitary bees analyzed, whereas Lactobacillus, Spiroplasma and Sodalis were the second most abundant taxa in X. augusti, E. fervens and Lasioglossum, respectively. The main pathogens detected with qPCR were Nosema ceranae, Nosema bombi and Crithidia bombi, although differently abundant in the different bee species and sampling sites. Most microbial taxa did not show any correlation with the land use, apart from Snodgrassella and Nocardioides, showing higher abundances on less anthropized sites. Conversely, the pathogens species and load strongly affected the gut microbial composition, with Bifidobacterium, Apibacter, Serratia, Snodgrassella and Sodalis abundance that positively or negatively correlated with the detected pathogens load. Therefore, pathogens presence and load appear to be the main factor shaping the gut microbiome of solitary bees in Argentina.

Effects of Synthetic Acaricides and Nosema ceranae (Microsporidia: Nosematidae) on Molecules Associated with Chemical Communication and Recognition in Honey Bees.

Acaricides and the gut parasite Nosema ceranae are commonly present in most productive hives. Those stressors could be affecting key semiochemicals, which act as homeostasis regulators in Apis mellifera colonies, such as cuticular hydrocarbons (CHC) involved in social recognition and ethyl oleate (EO) which plays a role as primer pheromone in honey bees. Here we test the effect of amitraz, coumaphos, tau-fluvalinate and flumethrin, commonly applied to treat varroosis, on honey bee survival time, rate of food consumption, CHC profiles and EO production on N. ceranae-infected and non-infected honey bees. Different sublethal concentrations of amitraz, coumaphos, tau-fluvalinate and flumethrin were administered chronically in a syrup-based diet. After treatment, purified hole-body extracts were analyzed by gas chromatography coupled to mass spectrometry. While N. ceranae infection was also shown to decrease EO production affecting survival rates, acaricides showed no significant effect on this pheromone. As for the CHC, we found no changes in relation to the health status or consumption of acaricides. This absence of alteration in EO or CHC as response to acaricides ingestion or in combination with N. ceranae, suggests that worker honey bees exposed to those highly ubiquitous drugs are hardly differentiated by nest-mates. Having determined a synergic effect on mortality in worker bees exposed to coumaphos and Nosema infection but also, alterations in EO production as a response to N. ceranae infection it is an interesting clue to deeper understand the effects of parasite-host-pesticide interaction on colony functioning.

Sub-lethal effects of the consumption of Eupatorium buniifolium essential oil in honeybees.

When developing new products to be used in honeybee colonies, further than acute toxicity, it is imperative to perform an assessment of risks, including various sublethal effects. The long-term sublethal effects of xenobiotics on honeybees, more specifically of acaricides used in honeybee hives, have been scarcely studied, particularly so in the case of essential oils and their components. In this work, chronic effects of the ingestion of Eupatorium buniifolium (Asteraceae) essential oil were studied on nurse honeybees using laboratory assays. Survival, food consumption, and the effect on the composition of cuticular hydrocarbons (CHC) were assessed. CHC were chosen due to their key role as pheromones involved in honeybee social recognition. While food consumption and survival were not affected by the consumption of the essential oil, CHC amounts and profiles showed dose-dependent changes. All groups of CHC (linear and branched alkanes, alkenes and alkadienes) were altered when honeybees were fed with the highest essential oil dose tested (6000 ppm). The compounds that significantly varied include n-docosane, n-tricosane, n-tetracosane, n-triacontane, n-tritriacontane, 9-tricosene, 7-pentacosene, 9-pentacosene, 9-heptacosene, tritriacontene, pentacosadiene, hentriacontadiene, tritriacontadiene and all methyl alkanes. All of them but pentacosadiene were up-regulated. On the other hand, CHC profiles were similar in healthy and Nosema-infected honeybees when diets included the essential oil at 300 and 3000 ppm. Our results show that the ingestion of an essential oil can impact CHC and that the effect is dose-dependent. Changes in CHC could affect the signaling process mediated by these pheromonal compounds. To our knowledge this is the first report of changes in honeybee cuticular hydrocarbons as a result of essential oil ingestion.

Impacts of dietary supplementation with p-coumaric acid and indole-3-acetic acid on survival and biochemical response of honey bees treated with tau-fluvalinate.

Pollinator populations are in decline worldwide. Multiple factors have been cited as potential causes to these declines. In honey bees, a combination of stressors is known to cause colony losses. Adequate nutrition is a key factor for honey bee growth and colony development. Several studies show that the nutritional quality of the diet is directly proportional to the ability of the bee to face challenges or stressors. We explored the effect of p-coumaric (600 µM) and indole-3-acetic acid (2, 20 or 200 µM) supplementation on the survival and activity of key detoxification enzymes of honey bees exposed to tau-fluvalinate. The dietary supplementation with p-coumaric and indole-3-acetic acids (20 µM) enhanced the survival of bees exposed to tau-fluvalinate (approximately 20%). We also showed that dietary p-coumaric acid increased the levels of cytochrome P450 and glutathione reductase activity in bees treated with tau-fluvalinate, as well as in the untreated controls, while glutathione-S-transferase activity was lower in treated bees than in untreated. In bees fed with indole-3-acetic acid, cytochrome P450 showed increased levels, however, glutathione-S-transferase showed the lowest activity. Moreover, the results showed that supplementation with p-coumaric and indole-3-acetic acids did not alter acetyl cholinesterase activity, nor did treatment with tau-fluvalinate. Altogether, the enzymatic changes related to the detoxification mechanisms observed in bees that were fed with p-coumaric and indole-3-acetic acids could be responsible for the increased survival of bees treated with tau-fluvalinate compared to those that received a control diet. The results presented in this study, together with previous studies, provide evidence of the importance of dietary phytochemicals in the response of honey bees to pesticide exposure. Moreover, these results are the first report of the beneficial effect of the phytohormone indole-3-acetic acid on the survival of honey bees treated with tau-fluvalinate.

Laurus nobilis L. Extracts against Paenibacillus larvae: Antimicrobial activity, antioxidant capacity, hygienic behavior and colony strength.

The aim of this work was to compare the antimicrobial activity against Paenibacillus larvae and the antioxidant capacity of two Laurus nobilis L. extracts obtained by different extraction methods. The hydroalcoholic extract was moreover added as supplementary diet to bees in field conditions to test behavioural effects and colony strength. Both laurel extracts were subjected to different phytochemical analysis to identify their bioactive compounds. Antimicrobial activity was analyzed by the minimal inhibitory concentration (MIC) determination by means the agar dilution method. The hydroalcoholic extract (HE) was able to inhibit the bacterial growth of all P. larvae strains, with 580 µg/mL mean value. This better antibacterial activity in relation to the essential oil (EO) could be explained by the presence of some phenolic compounds, such as flavonoids, evidenced by characteristic bands resulting from the Fourier Transform Infrared Spectroscopy (FTIR) analysis. Antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging ability and ferric reducing antioxidant power (FRAP) assays. The HE showed the highest antioxidant activity as measured by DPPH, with IC50 values of 257 ±â€¯12 µg/mL. The FRAP assay method showed that the HE was 3-fold more effective reducing agent than the EO. When the bee colonies were supplied with laurel HE in sugar paste an improvement in their general condition was noticed, although neither the hygienic behavior nor the proportions of the breeding cells varied statistically due to the treatment. In conclusion, the inhibition power against P. larvae attributable to the phenolic compounds, the antioxidant capacity of the HE, and the non-lethal effects on adult honey bees on field trials suggest the HE of laurel as a promising substance for control American foulbrood disease.

Nosema ceranae in South American Native Stingless Bees and Social Wasp.

Besides the incipient research effort, the role of parasites as drivers of the reduction affecting pollinator populations is mostly unknown. Given the worldwide extension of the beekeeping practice and the diversity of pathogens affecting Apis mellifera populations, honey bee colonies are a certain source of parasite dispersion to other species. Here, we communicate the detection of the microsporidium Nosema ceranae, a relatively new parasite of honey bees, in stingless bees (Meliponini) and the social wasp Polybia scutellaris (Vespidae) samples from Argentina and Brazil by means of duplex PCR. Beyond the geographic location of the nests, N. ceranae was detected in seven from the eight Meliponini species analyzed, while Nosema apis, another common parasite of A. mellifera, was absent in all samples tested. Further research is necessary to determine if the presence of the parasite is also associated with established infection in host tissues. The obtained information enriches the current knowledge about pathologies that can infect or, at least, be vectored by native wild pollinators from South America.

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.

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.

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.

Susceptibility of Varroa destructor (Acari: Varroidae) to synthetic acaricides in Uruguay: Varroa mites' potential to develop acaricide resistance.

The purpose of this study was to estimate the acaricide susceptibility of Varroa destructor populations from Uruguay, which had never been exposed to synthetic acaricides. It was also to determine whether acaricide resistance to coumaphos occurred in apiaries in which acaricide rotation had been applied. Bioassays with acaricides against mite populations that had never been exposed to synthetic acaricides were performed, also against mite populations in which control failures with coumaphos had been reported. Additionally, coumaphos' effectiveness in honeybee colonies was experimentally tested. The lethal concentration that kills 50% of the exposed animals (LC(50)) for susceptible mite populations amounted to 0.15 µg/Petri dish for coumaphos and to less than 0.3 µg/Petri dish for the other acaricides. Coumaphos LC(50) was above 40 µg/Petri dish for resistant mites. The effectiveness of coumaphos in honeybee colonies parasitized by V. destructor ranged from 17.6% to 93.9%. LC(50) for mite populations susceptible to the most commonly applied miticides was determined, and the first case of coumaphos resistance recorded in Uruguay was established.