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.

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.

Fragrance addition improves visitation by honeybees and fruit quality in kiwifruit (Actinidia deliciosa).

BACKGROUND: Kiwifruit is an important horticultural crop all over the world and its development is important in Argentina. This dioecious crop has a short blooming period with nectarless flowers, and its fruit production depends on cross-pollination. Here, we tested whether kiwifruit quality increases by using honeybees exposed to female flowers treated with an artificial fragrance. The three experimental treatments were: A, sprinkled female flowers with 1:1 sugar syrup + Lavandula hybrida extract solution (a new attractant substance especially developed for this study named Lavandin Grosso); B, sprinkled female flowers with 1:1 water + sugar syrup (female flowers with additional sugar syrup reward); C (control; female flowers exposed to honeybees). RESULTS: The results showed a higher number of visits of honeybees to the female flowers sprinkled with the attractant substance, Lavandin Grosso, as well as higher fruit quality (weight, number of seeds, regularity in fruit size). CONCLUSION: Our study demonstrates the potential of fragrance-treated flowers to improve yield production in kiwifruit. © 2021 Society of Chemical Industry.

Chronic bee paralysis virus (CBPV) in South American non-Apis bees.

Chronic bee paralysis virus (CBPV) is a positive single-stranded RNA virus that exhibits a worldwide distribution. Although the effects of this virus on honeybees' health are well known, its presence in other bee species has not been fully studied. In this work, CBPV was detected in several native bees from Argentina, including Bombus pauloensis, Halictillus amplilobus, Peponapis fervens, and members of the genus Xylocopa. Here, we report for the first time the presence of CBPV in native bees from South America.

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.

Abscisic acid enhances cold tolerance in honeybee larvae.

The natural composition of nutrients present in food is a key factor determining the immune function and stress responses in the honeybee (Apis mellifera). We previously demonstrated that a supplement of abscisic acid (ABA), a natural component of nectar, pollen, and honey, increases honeybee colony survival overwinter. Here we further explored the role of ABA in in vitro-reared larvae exposed to low temperatures. Four-day-old larvae (L4) exposed to 25°C for 3 days showed lower survival rates and delayed development compared to individuals growing at a standard temperature (34°C). Cold-stressed larvae maintained higher levels of ABA for longer than do larvae reared at 34°C, suggesting a biological significance for ABA. Larvae fed with an ABA-supplemented diet completely prevent the low survival rate due to cold stress and accelerate adult emergence. ABA modulates the expression of genes involved in metabolic adjustments and stress responses: Hexamerin 70b, Insulin Receptor Substrate, Vitellogenin, and Heat Shock Proteins 70. AmLANCL2, the honeybee ABA receptor, is also regulated by cold stress and ABA. These results support a role for ABA increasing the tolerance of honeybee larvae to low temperatures through priming effects.

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.

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.

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.

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.

Body size variability of Varroa destructor and its role in acaricide tolerance.

Phenotypic plasticity has been defined as the ability of a genotype to produce different phenotypes when exposed to distinct environments throughout its ontogeny. Morphological variability of individuals is an example of this plasticity. Taking into account that several studies have reported a wide morphological variability in Varroa destructor populations, we evaluated if the body size plasticity of the parasite constituted a key factor able to modulate mites survival when they were exposed to a drug bioassays. Drug bioassays against mites were conducted using three different Syzygium aromaticum essential oil concentrations (0.5, 1, and 5 µl/capsule) and controls. After 4 h of exposition, mite mortality was registered. The width (WS) and length (LS) of the dorsal shield were measured in dead mites. General lineal models were carried to determine if V. destructor survival to acaricides was related to the explanatory variables. Data modelling confirmed that WS and LS variables, together with time interaction, were significantly related to V. destructor survival when the parasites were exposed to acaricides. The models proposed demonstrated that for the smaller S. aromaticum essential oil concentration, the larger the parasite body, the greater the probability that it remains alive at the end of the bioassay. Such relationship was inverse for the other two concentrations tested. Possible causes explaining the body size variability in V. destructor individuals were discussed.

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.

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.

Hydrolats from Humulus lupulus and Their Potential Activity as an Organic Control for Varroa destructor.

Varroa destructor is a parasitic mite, which is considered a severe pest for honey bees causing serious losses to beekeeping. Residual hydrolats from steam extraction of hop essential oils, generally considered as a waste product, were tested for their potential use as acaricides on V. destructor. Four hop varieties, namely Cascade, Spalt, Victoria, and Mapuche, showed an interesting performance as feasible products to be used in the beekeeping industry. Some volatile oxidized terpenoids were found in the hydrolats, mainly ß-caryophyllene oxide, ß-linalool, and isogeraniol. These compounds, together with the presence of polyphenols, flavonoids, and saponins, were probably responsible for the promissory LC50 values obtained for mites after hydrolat exposition. Victoria hydrolat was the most toxic for mites (LC50: 16.1 µL/mL), followed by Mapuche (LC50 value equal to 30.1 µL/mL), Spalt (LC50 value equal to 114.3 µL/mL), and finally Cascade (LC50: 117.9 µL/mL). Likewise, Spalt had the highest larval survival, followed by Victoria and Mapuche. Cascade was the variety with the highest larval mortality. In addition, none of the extracts showed mortality higher than 20% in adult bees. The Victoria hydrolat presented the best results, which makes it a good compound with the prospect of an acaricide treatment against V. destructor.

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.

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.

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.

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.

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.