The recent revision of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) was flawed and misleads the bee scientific community.

The genus Vairimorpha was proposed for several species of Nosema in 1976 (Pilley, 1976), almost 70 years after Nosema apis Zander (Zander, 1909). Tokarev and colleagues proposed the redefinition of 17 microsporidian species in four genera, Nosema, Vairimorpha, Rugispora, and Oligosporidium, based on phylogenetic trees of two genetic markers (SSU rRNA and RPB1) (Tokarev et al., 2020). Several issues should invalidate this new classification, leading to the synonymization of Vairimorpha within Nosema.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

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.

Trypanosomatids in honey bee colonies in Spain: A new specific qPCR method for specific quantification of Lotmaria passim, Crithidia mellificae and Crithidia bombi.

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.

Bee Trypanosomatids: First Steps in the Analysis of the Genetic Variation and Population Structure of Lotmaria passim, Crithidia bombi and Crithidia mellificae.

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.

Detection of Microsporidia in Pollinator Communities of a Mediterranean Biodiversity Hotspot for Wild Bees.

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.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Nosema apis and Nosema ceranae Tissue Tropism in Worker Honey Bees (Apis mellifera).

The microsporidia Nosema apis and Nosema ceranae are major honey bee pathogens that possess different characteristics in terms of the signs they produce, as well as disease development and transmission. Although the ventricular epithelium is generally considered the target tissue, indirect observations led to speculation that N. ceranae may also target other structures, possibly explaining at least some of the differences between these 2 species. To investigate the tropism of Nosema for honey bee tissues, we performed controlled laboratory infections by orally administering doses of 50 000 or 100 000 fresh mature spores of either species. The fat body was isolated from the infected bees, as well as organs from the digestive (esophagus, ventriculus, ileum, rectum), excretory (Malpighian tubules), circulatory (aorta, heart), respiratory (thoracic tracheas), exocrine (hypopharyngeal, mandibular and labial, cephalic, thoracic salivary glands), and sensory/nervous (brain, eyes and associated nerve structures, thoracic nerve ganglia) systems. Tissues were examined by light and electron microscopy at 7, 10, and 15 days postinfection. Both Nosema species were found to infect epithelial cells and clusters of regenerative cells in the ventriculus, and while the ileum and rectum contained spores of the microsporidia in the lumen, these structures did not show overt lesions. No stages of the parasites or cellular lesions were detected in the other organs tested, confirming the high tropism of both species for the ventricular epithelium cells. Thus, these direct histopathological observations indicate that neither of these 2 Nosema species exhibit tropism for honey bee organs other than the ventriculus.

Assessing the resistance to acaricides in Varroa destructor from several Spanish locations.

Varroosis is the disease caused by the ectoparasitic mite Varroa destructor, one of the most destructive diseases of honeybees. In Spain, there is great concern because there are many therapeutic failures after acaricide treatments intended to control varroosis outbreaks. In some of these cases it is not clear whether such failures are due to the evolution of resistance. Therefore, it is of high interest the development of methodologies to test the level of resistance in mite populations. In this work, a simple bioassay methodology was used to test whether some reports on low efficacy in different regions of Spain were in fact related to reduced Varroa sensitivity to the most used acaricides. This bioassay proved to be very effective in evaluating the presence of mites that survive after being exposed to acaricides. In the samples tested, the mortality caused by coumaphos ranged from 2 to 89%; for tau-fluvalinate, it ranged from 5 to 96%. On the other hand, amitraz caused 100% mortality in all cases. These results suggest the presence of Varroa resistant to coumaphos and fluvalinate in most of the apiaries sampled, even in those where these active ingredients were not used in the last years. The bioassay technique presented here, either alone or in combination with other molecular tools, could be useful in detecting mite populations with different sensitivity to acaricides, which is of vital interest in selecting the best management and/or acaricide strategy to control the parasite in apiaries.

Nosema ceranae in Apis mellifera: a 12 years postdetection perspective.

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat.

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.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Genotype diversity in the honey bee parasite Nosema ceranae: multi-strain isolates, cryptic sex or both?

BACKGROUND: There is great controversy as to whether Microsporidia undergo a sexual cycle. In the paradigmatic case of Nosema ceranae, although there is no morphological evidence of sex, some meiosis-specific genes are present in its reduced genome and there is also high intraspecific variability, with incongruent phylogenies having been systematically obtained. The possibility of sexual recombination is important from an epidemiological standpoint, particularly as N. ceranae is considered to be a major factor in the current disquieting epidemic of widespread bee colony losses. This parasite apparently originated in oriental honey bees, spreading out of Asia and Australia to infect honey bees worldwide. This study had three main objectives: i) to obtain genetic markers that are not part of known multi-copy arrays for strain determination; ii) to shed light on the intraspecific variability and recombination of N. ceranae; and iii) to assess the variability in N. ceranae populations. The answers to these questions are critical to understand the capacity of adaptation of microsporidia. RESULTS: Biallelic polymorphisms were detected at a number of specific points in the five coding loci analyzed from European and Australian isolates of N. ceranae. Heterozygous genotypes were abundant and cloning experiments demonstrate that they reflect the existence of multiple alternative sequences in each isolate. The comparisons of different clones and genotypes clearly indicate that new haplotypes are generated by homologous recombination. CONCLUSIONS: The N. ceranae isolates from honey bees correspond to genotypically distinct populations, revealing that individual honey bees may not be infected by a particular clone but rather, a pool of different strains. Homologous recombination implies the existence of a cryptic sex cycle yet to be described in N. ceranae. There are no diagnostic alleles associated with Australian or European origins, nor are there differences between the two hosts, A. cerana and A. mellifera, supporting the absence of biological barriers for N. ceranae transmission. Diversity is high among microsporidia of both these origins, and the maintenance of a high heterozygosis in the recently invaded European populations, could hypothetically underlie the stronger virulence of N. ceranae observed in A. mellifera.

Evidence for weak genetic recombination at the PTP2 locus of Nosema ceranae.

The microsporidian Nosema ceranae is an emergent pathogen that threatens the health of honeybees and other pollinators all over the world. Its recent rapid spread across a wide variety of host species and environments demonstrated an enhanced ability of adaptation, which seems to contradict the lack of evidence for genetic recombination and the absence of a sexual stage in its life cycle. Here we retrieved fresh data of the patterns of genetic variation at the PTP2 locus in naturally infected Apis mellifera colonies, by means of single genome amplification. This technique, designed to prevent the formation of chimeric haplotypes during polymerase chain reaction (PCR), provides more reliable estimates of the diversity levels and haplotype structure than standard PCR-cloning methods. Our results are consistent with low but significant rates of recombination in the history of the haplotypes detected: estimates of the population recombination rate are of the order of 30 and support recent evidence for unexpectedly high levels of variation of the parasites within honeybee colonies. These observations suggest the existence of a diploid stage at some point in the life cycle of this parasite and are relevant for our understanding of the dynamics of its expanding population.

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.

Stable genetic diversity despite parasite and pathogen spread in honey bee colonies.

In the last decades, the rapid spread of diseases, such as varroosis and nosemosis, associated with massive honey bee colonies mortality around the world has significantly decreased the number and size of honey bee populations and possibly their genetic diversity. Here, we compare the genetic diversity of Iberian honey bee colonies in two samplings performed in 2006 and 2010 in relation to the presence of the pathogenic agents Nosema apis, Nosema ceranae, and Varroa destructor in order to determine whether parasite and pathogen spread in honey bee colonies reflects changes in genetic diversity. We found that the genetic diversity remained similar, while the incidence of N. ceranae increased and the incidence of N. apis and V. destructor decreased slightly. These results indicate that the genetic diversity was not affected by the presence of these pathogenic agents in the analyzed period. However, the two groups of colonies with and without Nosema/Varroa detected showed significant genetic differentiation (G test). A detailed analysis of the allelic segregation of microsatellite loci in Nosema/Varroa-negative colonies and parasitized ones revealed two outlier loci related to genes involved in immune response.

Development and validation of a liquid chromatography with mass spectrometry method to determine resveratrol and piceid isomers in beeswax.

This paper represents the first report of a liquid chromatography coupled to electrospray ionization mass spectrometry method for simultaneously analyzing resveratrol and piceid isomers (cis and trans) in beeswax. An efficient extraction procedure has been proposed (average analyte recoveries were between 89 and 95%); this involved a solid-liquid extraction using a mixture of ethanol and water (80:20, v/v) and a concentration step in a rotary evaporator. The separation of all the compounds was achieved using a C18 column and a mobile phase composed of ammonium formate 0.03 M in water and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification ranged from 1.0 to 1.7 and 3.5 to 5.5 µg/kg, respectively. Finally, the proposed method was applied to analyze beeswax samples collected from experimental and organic apiaries.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim.

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Permanent prevalence of Nosema ceranae in honey bees (Apis mellifera) in Hungary.

Nosema ceranae is present in honey bee (Apis mellifera L.) colonies worldwide. Studies on the comparative virulence of N. ceranae and N. apis showed significant differences in individual mortality, and the prevalence of N. ceranae seems to be predominant in both the continental and the Mediterranean climate regions. This study attempted to monitor the geographical and seasonal distribution of these two Nosema species in Hungary, using a simple laboratory method. The distribution of N. ceranae and N. apis infection rates along all seasons was homogeneous (P = 0.57). In co-infected samples, the intensity of N. ceranae infection was always significantly higher than that of N. apis infection (P < 0.001). The infection rate of infected bees in exterior samples was higher than in interior samples in each season; however, the differences were not statistically significant. The species N. ceranae had been present in Hungary already in 2004. Statistical analysis of data shows that the infection level is best represented by sampling exterior bees to establish the proportion of infected bees rather than by determining the mean spore count.

Ribosomal gene polymorphism in small genomes: analysis of different 16S rRNA sequences expressed in the honeybee parasite Nosema ceranae (Microsporidia).

To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian.

High levels of genetic diversity in Nosema ceranae within Apis mellifera colonies.

Nosema ceranae is a widespread honeybee parasite, considered to be one of the pathogens involved in the colony losses phenomenon. To date, little is known about its intraspecific genetic variability. The few studies on N. ceranae variation have focused on the subunits of ribosomal DNA, which are not ideal for this purpose and have limited resolution. Here we characterized three single copy loci (Actin, Hsp70 and RPB1) in three N. ceranae isolates from Hungary and Hawaii. Our results provide evidence of unexpectedly high levels of intraspecific polymorphism, the coexistence of a wide variety of haplotypes within each bee colony, and the occurrence of genetic recombination in RPB1. Most haplotypes are not shared across isolates and derive from a few frequent haplotypes by a reduced number of singletons (mutations that appear usually just once in the sample), which suggest that they have a fairly recent origin. Overall, our data indicate that this pathogen has experienced a recent population expansion. The presence of multiple haplotypes within individual isolates could be explained by the existence of different strains of N. ceranae infecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host-parasite interactions.