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Deformed wing virus (DWV) is a honey bee virus, whose emergence from relative obscurity is driven by the recent host-switch, adaptation, and global dispersal of the ectoparasitic mite Varroa destructor (a highly efficient vector of DWV) to reproduction on honey bees (Apis mellifera). Our study examines how varroa affects the continuing evolution of DWV, using the Azores archipelago, where varroa is present on only three out of the eight Islands, as a natural experimental system for comparing different evolutionary conditions and trajectories. We combined qPCR of 494 honey bee colonies sampled across the archipelago with amplicon deep sequencing to reveal how the DWV genetic landscape is altered by varroa. Two of the varroa-free Islands were also free of DWV, while a further two Islands were intriguingly dominated by the rare DWV-C major variant. The other four Islands, including the three varroa-infested Islands, were dominated by the common DWV-A and DWV-B variants. The varroa-infested Islands had, as expected, an elevated DWV prevalence relative to the uninfested Islands, but not elevated DWV loads, due the relatively high prevalence and loads of DWV-C on the varroa-free Islands. This establishes the Azores as a stable refuge for DWV-C and provides the most convincing evidence to date that at least some major strains of DWV may be capable of not just surviving, but actually thriving in honey bees in the absence of varroa-mediated transmission. We did not detect any change in DWV genetic diversity associated with island varroa status but did find a positive association of DWV diversity with virus load, irrespective of island varroa status.
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The worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands naïve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.
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Varroidae , Animales , Abejas/virología , Abejas/parasitología , Varroidae/virología , Azores , Virus de Insectos/genética , Virus de Insectos/aislamiento & purificación , Virus de Insectos/clasificación , Virus ARN/genética , Virus ARN/aislamiento & purificación , Virus ARN/clasificaciónRESUMEN
The gut microbiota of honey bees has received increasing interest in the past decades due to its crucial role in their health, and can be disrupted by pathogen infection. Nosema ceranae is an intracellular parasite that affects the epithelial cells of the midgut, altering gut homeostasis and representing a major threat to honey bees. Previous studies indicated that younger worker bees are more susceptible to experimental infection by this parasite, although the impact of infection and of age on the gut bacterial communities remains unclear. To address this, honey bees were experimentally infected with a consistent number of N. ceranae spores at various ages post-emergence (p.e.) and the gut bacteria 7 days post-infection (p.i.) were analysed using real-time quantitative PCR, with the results compared to non-infected controls. Infected bees had a significantly higher proportion and load of Gilliamella apicola. In respect to the age of infection, the bees infected just after emergence had elevated loads of G. apicola, Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Bartonella apis, and Bombella apis. Moreover, the G. apicola load was higher in bees infected at nearly all ages, whereas older non-infected bees had higher loads of Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Ba. apis, and Bo apis. These findings suggest that N. ceranae infection and, in particular, the age of bees at infection modulate the gut bacterial community, with G. apicola being the most severely affected species.
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Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.
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Parásitos , Trypanosomatina , Humanos , Abejas , Animales , Ecosistema , Trypanosomatina/parasitología , Evolución BiológicaRESUMEN
Evidence suggests that acaricide residues, such as tau-fluvalinate and coumaphos, are very prevalent in honey bee colonies worldwide. However, the endpoints and effects of chronic oral exposure to these compounds remain poorly understood. In this study, we calculated LC50 and LDD50 endpoints for coumaphos and tau-fluvalinate, and then evaluated in vivo and in vitro effects on honey bees using different biomarkers. The LDD50 values for coumaphos were 0.539, and for tau-fluvalinate, they were 12.742 in the spring trial and 8.844 in the autumn trial. Chronic exposure to tau-fluvalinate and coumaphos resulted in significant changes in key biomarkers, indicating potential neurotoxicity, xenobiotic biotransformation, and oxidative stress. The Integrated Biomarker Response was stronger for coumaphos than for tau-fluvalinate, supporting their relative lethality. This study highlights the chronic toxicity of these acaricides and presents the first LDD50 values for tau-fluvalinate and coumaphos in honey bees, providing insights into the risks faced by colonies.
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Acaricidas , Piretrinas , Abejas , Animales , Cumafos/toxicidad , Acaricidas/toxicidad , Piretrinas/toxicidad , Nitrilos/toxicidadRESUMEN
Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker's lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.
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Crithidia , Trypanosomatina , Abejas , Animales , Crithidia/parasitología , España , Trypanosomatina/genética , Trypanosomatina/parasitologíaRESUMEN
Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.
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Apicultura , Trypanosomatina , Animales , Abejas , Trypanosomatina/genética , Trypanosomatina/parasitología , Crithidia/genética , Crithidia/parasitología , Simbiosis , AzoresRESUMEN
Pollination services to increase crop production are becoming more and more important, as we are facing both climate change and a growing world population. Both are predicted to impact food security worldwide. High-density, commercial beekeeping has become a key link in the food supply chain, and diseases have become a central issue in hive losses around the world. American Foulbrood (AFB) disease is a highly contagious bacterial brood disease in honey bees (Apis mellifera), leading to hive losses worldwide. The causative agent is the Gram+ bacterium Paenibacillus larvae, which is able to infect honey bee larvae during the first 3 days of their lives. It can be found in hives around the world with viable spores for decades. Antibiotics are largely ineffective in treating the disease as they are only efficient against the vegetative state. Once a hive shows the clinical manifestation of the disease, the only effective way to eradicate it and prevent the spread of the disease is by burning the hive, the equipment, and the colony. Because of its virulent nature and detrimental effects on honey bee colonies, AFB is classified as a notifiable disease worldwide. Effective, safe, and sustainable methods are needed to ensure the wellbeing of honey bee colonies. Even though insects lack antibodies, which are the main requisites for trans-generational immune priming (TGIP), they can prime their offspring against persisting pathogens. Here, we demonstrate an increased survival of infected honey bee larvae after their queen was vaccinated, compared to offspring of control queens (placebo vaccinated). These results indicate that TGIP in insects can be used to majorly enhance colony health, protect commercial pollinators from deadly diseases, and reduce high financial and material losses to beekeepers. Classification: biological sciences, applied biological sciences.
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Avian trichomonosis is a parasitic disease caused mainly by Trichomonas gallinae and other Trichomonas species. It can be asymptomatic, or it can produce a necrotic lesion in the upper digestive tract and spread to other organs, causing the death of the infected birds. In this study, we aimed to evaluate an adapted real-time PCR method for the diagnosis of different genotypes and species of avian oropharyngeal trichomonads. Fifty-six samples from the oropharynx of Bonelli's eagles (Aquila fasciata) obtained between 2018 and 2019 were analyzed using the real-time PCR and the end-point PCR, both targeting trichomonads ITS, and the results were compared by a coefficient of agreement. All positive samples were sequenced. The analysis showed a higher percentage of detection of real-time PCR ITS compared with end-point PCR ITS (64.3 vs 55.4%), and good agreement value (Kappa = 0.816). Melting temperature value for resulting amplicons of real-time PCR for avian trichomonads was 83.45 ± 0.72 °C. Genotypes A, D, and III were found among the sequences. Moreover, Trichomonas gypaetinii, a common species in scavenger birds, is reported for the first time in Bonelli's eagles.
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Enfermedades de las Aves , Águilas , Tricomoniasis , Trichomonas , Animales , Trichomonas/genética , Águilas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Enfermedades de las Aves/diagnóstico , Enfermedades de las Aves/parasitología , Tricomoniasis/diagnóstico , Tricomoniasis/veterinaria , Tricomoniasis/parasitologíaRESUMEN
Nosema ceranae is a highly prevalent intracellular parasite of honey bees' midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control.
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In the last years, an increase has been observed in the adulteration of bee pollen. Consequently, different tools are required to authenticate the origin of this product, such as a study of the profile and composition of a specific family of compounds. The present study investigates the potential of betaines and related compounds as markers of the apiary of origin and harvest period of 71 bee pollen samples. These were collected from four apiaries (Pistacho, Tío Natalio, Monte and Fuentelahiguera), located in the same geographical area (Guadalajara, Spain) and sampled during three consecutive harvest periods in the same year (April-May, June, July-August). They were analyzed by means of a previously developed methodology, which involved solvent extraction, hydrophilic interaction liquid chromatography coupled to mass spectrometry, and a statistical analysis of the data (canonical discriminant analysis). Variable amounts of betaines and related compounds were found in the samples, with four of these being identified in all of them (betonicine, betaine, trigonelline and choline); betonicine was the predominant compound in a concentration range of 264 to 52384 mg/kg. It was possible to statistically assign over 50 % of the samples to the corresponding apiary of origin, the best results being obtained for the Tío Natalio apiary (75 %); this classification was even better in the case of the harvest period, as more than 75 % of the samples were correctly assigned, and in two periods (April-May and June) a 90 % rate was obtained.
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Compuestos de Amonio , Betaína , Animales , Abejas , Betaína/análisis , Cromatografía Liquida , Análisis Discriminante , Polen/químicaRESUMEN
Nosema ceranae is a highly prevalent pathogen of Apis mellifera, which is distributed worldwide. However, there may still exist isolated areas that remain free of N. ceranae. Herein, we used molecular tools to survey the Azores to detect N. ceranae and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that N. ceranae was not only present but also the dominant species in the Azores. In 2014/2015, N. apis was rare and N. ceranae prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, N. ceranae prevalence increased significantly (p < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that N. ceranae colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the N. ceranae invasion, and it is remarkable that Santa Maria is also free of Varroa destructor, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.
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There is mounting evidence that acaricides are among the most prevalent medicinal compounds in honey bee hive matrices worldwide. According to OCDE guideline No. 245 chronic lethal concentration of tau-fluvalinate (at concentrations ranging from 77.5 to 523.18 ppm), coumaphos (59.8 ppm) and dimethoate (0.7 ppm) were determined. The activity of the biomarkers acetylcholinesterase (AChE), carboxylesterase (CbE), glutathione S-transferase (GST), catalase (CAT) and malondialdehyde (MDA) was analysed and as they are implicated in neurotoxicity, biotransformation and antioxidant defences, these values were combined into an integrated biomarker response (IBR). There was enhanced AChE, CAT and GST activity in honey bees exposed to tau-fluvalinate, while dimethoate inhibited AChE activity. Both dimethoate and coumaphos inhibited CbE activity but they enhanced CAT activity and MDA formation. Our results highlight how these biomarkers may serve to reveal honey bee exposure to commonly used acaricides.
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Acaricidas , Piretrinas , Acaricidas/toxicidad , Acetilcolinesterasa , Animales , Biomarcadores , Cumafos/toxicidad , Dimetoato/toxicidad , Piretrinas/toxicidadRESUMEN
Nosema ceranae is an intracellular parasite that infects honeybees' gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or ß-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees' age had a significant impact in shaping the bacteriome structure and composition of the bees' gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees' microbiome.
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Microbioma Gastrointestinal , Nosema , Animales , Abejas/genética , Nosema/genética , ARN Ribosómico 16S/genética , Estaciones del AñoRESUMEN
Crithidia acanthocephali is a trypanosomatid species that was initially described in the digestive tract of Hemiptera. However, this parasite was recently detected in honey bee colonies in Spain, raising the question as to whether bees can act as true hosts for this species. To address this issue, worker bees were experimentally infected with choanomastigotes from the early stationary growth phase and after 12 days, their hindgut was extracted for analysis by light microscopy and TEM. Although no cellular lesions were observed in the honey bee's tissue, trypanosomatids had differentiated and adopted a haptomonad morphology, transforming their flagella into an attachment pad. This structure allows the protozoa to remain attached to the gut walls via hemidesmosomes-such as junctions. The impact of this species on honey bee health, as well as the pathogenic mechanisms involved, remains unknown. Nevertheless, these results suggest that insect trypanosomatids may have a broader range of hosts than initially thought.
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Bee pollen is currently one of the most commonly consumed food supplements, as it is considered to be a good source of bioactive substances and energy. It contains various health-promoting compounds, such as proteins, amino acids, lipids, as well as glucosinolates. In the present study, the glucosinolate content was determined, by means of ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass detector, in 72 bee pollen samples from four different apiaries in Guadalajara (Spain), harvested in three different periods. In addition, 11 commercial multifloral samples from different Spanish regions were also analyzed. The aim was to verify the suitability of these compounds as biomarkers of their geographical origin, and to test their potential for distinguishing the harvesting period. By means of a canonical discriminant analysis, it was possible to differentiate the apiary of origin of most of the samples, and these could also be clearly differentiated from the commercial ones, simply as a result of the glucosinolate content. In addition, it was also demonstrated for the first time that bee pollen samples were capable of being differentiated according to the time of harvesting and their glucosinolate content.
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To study the influence of thiamethoxam exposure on colony strength and pathogen prevalence, an apiary (5 colonies) was placed in front of a plot sown with winter oilseed rape (wOSR), just before the flowering phase. Before sowing, the seeds were treated with an equivalent application of 18 g thiamethoxam/ha. For comparison, a second apiary (5 colonies) was located in front of a separate 750 m plot sown with untreated wOSR. Dead foragers at the entrance of hives were assessed every 2-3 days throughout the exposure period, while the colony strength (number of combs covered with adult honey bees and brood) and pathogens were monitored each month until the following spring. Foraging on the wOSR crop was confirmed by melissopalynology determination of the corbicular pollen collected periodically, while the chemical analysis showed that exposure to thiamethoxam was mainly through nectar. There was an increase in the accumulation of dead bees in the apiary exposed to thiamethoxam relating with the control, which was coped with an increment of bee brood surface and adult bee population. However, we did not find statistically significant differences between apiaries (α = 0.05) in terms of the evolution of pathogens. We discuss these results under hormesis perspective.
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Insect pollination is crucial for the maintenance of natural and managed ecosystems but the functioning of this ecosystem service is threatened by a worldwide decline of pollinators. Key factors in this situation include the spread and interspecific transmission of pathogens worldwide through the movement of managed pollinators. Research on this field has been mainly conducted in some particular species, while studies assessing the interspecific transmission of pathogens at a community level are scarce. However, this information is pivotal to design strategies to protect pollinators. Herein, we analysed the prevalence of two common microsporidia pathogens of managed honey bees (Nosema ceranae and N. apis) in bee communities of semiarid Mediterranean areas from the Southeast of the Iberian Peninsula. Our results confirm the ability of N. ceranae to disperse across wild bee communities in semiarid Mediterranean ecosystems since it was detected in 36 Apoidea species (39% of the sampling; for the first time in nine genera). The prevalence of the pathogen did not show any phylogenetic signal which suggests a superfamily host range of the pathogen or that wild bees may be acting only as vectors of N. ceranae. In addition, N. apis was detected in an Eucera species, which is the second time it has been detected by molecular techniques in a host other than the honey bee. Our study represents the primary assessment of the prevalence of microsporidia at community level in Mediterranean areas and provides outstanding results on the ability of Nosema pathogens to spread across the landscape.
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Mariposas Nocturnas , Nosema , Animales , Abejas , Biodiversidad , Ecosistema , Nosema/genética , Filogenia , PolinizaciónRESUMEN
The remodelling of flagella into attachment structures is a common and important event in the trypanosomatid life cycle. Lotmaria passim and Crithidia mellificae can parasitize Apis mellifera, and as a result they might have a significant impact on honeybee health. However, there are details of their life cycle and the mechanisms underlying their pathogenicity in this host that remain unclear. Here we show that both L. passim promastigotes and C. mellificae choanomastigotes differentiate into haptomonad stages covering the ileum and rectum of honeybees. These haptomonad cells remain attached to the host surface via zonular hemidesmosome-like structures, as revealed by transmission electron microscopy. This work describes for the first known time the haptomonad morphotype of these species and their hemidesmosome-like attachments in A. mellifera, a key trait used by other trypanosomatid species to proliferate in the insect host hindgut.
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Crithidia , Trypanosomatina , Animales , AbejasRESUMEN
Trypanosomatids are among the most prevalent parasites in bees but, despite the fact that their impact on the colonies can be quite important and that their infectivity may potentially depend on their genotypes, little is known about the population diversity of these pathogens. Here we cloned and sequenced three non-repetitive single copy loci (DNA topoisomerase II, glyceraldehyde-3-phosphate dehydrogenase and RNA polymerase II large subunit, RPB1) to produce new genetic data from Crithidia bombi, C. mellificae and Lotmaria passim isolated from honeybees and bumblebees. These were analysed by applying population genetic tools in order to quantify and compare their variability within and between species, and to obtain information on their demography and population structure. The general pattern for the three species was that (1) they were subject to the action of purifying selection on nonsynonymous variants, (2) the levels of within species diversity were similar irrespective of the host, (3) there was evidence of recombination among haplotypes and (4) they showed no haplotype structuring according to the host. C. bombi exhibited the lowest levels of synonymous variation (πS= 0.06 ± 0.04 %) - and a mutation frequency distribution compatible with a population expansion after a bottleneck - that contrasted with the extensive polymorphism displayed by C. mellificae (πS= 2.24 ± 1.00 %), which likely has a more ancient origin. L. passim showed intermediate values (πS= 0.40 ± 0.28 %) and an excess of variants a low frequencies probably linked to the spread of this species to new geographical areas.