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.

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.

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 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.

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.

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.

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.

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.

Quantifying the impact of an invasive Hornet on Bombus terrestris Colonies.

The invasive hornet Vespa velutina nigrithorax is considered a proliferating threat to pollinators in Europe and Asia. While the impact of this species on managed honey bees is well-documented, effects upon other pollinator populations remain poorly understood. Nonetheless, dietary analyses indicate that the hornets consume a diversity of prey, fuelling concerns for at-risk taxa. Here, we quantify the impact of V. velutina upon standardised commercially-reared colonies of the European bumblebee, Bombus terrestris terrestris. Using a landscape-scale experimental design, we deploy colonies across a gradient of local V. velutina densities, utilising automated tracking to non-invasively observe bee and hornet behaviour, and quantify subsequent effects upon colony outcomes. Our results demonstrate that hornets frequently hunt at B. terrestris colonies, being preferentially attracted to those with high foraging traffic, and engaging in repeated-yet entirely unsuccessful-predation attempts at nest entrances. Notably however, we show that B. terrestris colony weights are negatively associated with local V. velutina densities, indicating potential indirect effects upon colony growth. Taken together, these findings provide the first empirical insight into impacts on bumblebees at the colony level, and inform future mitigation efforts for wild and managed pollinators.

The prevalence of Acarapis woodi in Spanish honey bee (Apis mellifera) colonies.

Acarapis woodi is an internal obligate parasite of the respiratory system of honey bees which provokes significant economic losses in many geographical areas. The main aim of this study was assess the A. woodi role in the "higher honey bee colony losses phenomenon" in Spain. Therefore, a new polymerase chain reaction (PCR) was developed to amplify the mitochondrial cytochrome oxidase I gene (COI) and so the actual prevalence of A. woodi in Spanish honey bee colonies in 2006 and 2007 was determined as part of a wider survey. The results revealed a greater prevalence than expected in most of the geographical areas studied where has been generally underestimated One problem encountered in this study was to distinguish between A. woodi and other species (Acarapis dorsalis and Acarapis externus) at the molecular level. Furthermore, the patterns of genetic divergence across sequences raised serious doubts about the current classification of these organisms.

Effectiveness of electric harps in reducing Vespa velutina predation pressure and consequences for honey bee colony development.

BACKGROUND: Vespa velutina has become a species of concern in invaded regions of Europe and Asia, due to its impacts on biodiversity, apiculture and society. This hornet, a ferocious hunter of pollinating insects, poses a serious threat to biodiversity and pollination services. Despite ongoing efforts, its extermination in continental Europe is hampered by a lack of effective control methods, thus effective mitigation measures are primary concerns. The aims of this work were: (i) to study the effects of V. velutina predating on honey bee colonies, and (ii) to assess the effectiveness of electric harps in reducing hunting pressure and predation. We assessed the predation pressure and compared honey bee colony performance, body weight of workers, and winter survivorship for protected versus unprotected colonies in 36 experimental hives across three apiaries. RESULTS: Electric harps protected honey bees by reducing predation pressure and therefore mitigating foraging paralysis. Consequently, foraging activity, pollen income, brood production and worker body weight were higher in protected colonies which in turn showed greater winter survivorship than those that were unprotected, especially at sites with intermediate to high levels of predation. CONCLUSION: The predation of V. velutina affects foraging activity, breeding, body weight and colony survivorship of Apis mellifera. Electric harps contribute significantly to mitigate the impact of this invasive hornet on apiaries; however, they should be deployed in tandem with additional measures to preserve honey bee colony stocks, such as facilitating access to food sources for colonies during the periods of highest predation pressure. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of pathogens in the invasive hornet Vespa velutina and in native Hymenoptera (Apidae, Vespidae) from SW-Europe.

Invasive species contribute to deteriorate the health of ecosystems due to their direct effects on native fauna and the local parasite-host dynamics. We studied the potential impact of the invasive hornet Vespa velutina on the European parasite-host system by comparing the patterns of diversity and abundance of pathogens (i.e. Microsporidia: Nosematidae; Euglenozoa: Trypanosomatidae and Apicomplexa: Lipotrophidae) in European V. velutina specimens with those in the native European hornet Vespa crabro, as well as other common Hymenoptera (genera Vespula, Polistes and Bombus). We show that (i) V. velutina harbours most common hymenopteran enteropathogens as well as several new parasitic taxa. (ii) Parasite diversity in V. velutina is most similar to that of V. crabro. (iii) No unambiguous evidence of pathogen release by V. velutina was detected. This evidence together with the extraordinary population densities that V. velutina reaches in Europe (around of 100,000 individuals per km2 per year), mean that this invasive species could severely alter the native pathogen-host dynamics either by actively contributing to the dispersal of the parasites and/or by directly interacting with them, which could have unexpected long-term harmful consequences on the native entomofauna.

Residual Tau-Fluvalinate in Honey Bee Colonies Is Coupled with Evidence for Selection for Varroa destructor Resistance to Pyrethroids.

Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy-Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.

Longitudinal analysis on parasite diversity in honeybee colonies: new taxa, high frequency of mixed infections and seasonal patterns of variation.

To evaluate the influence that parasites have on the losses of Apis mellifera it is essential to monitor their presence in the colonies over time. Here we analysed the occurrence of nosematids, trypanosomatids and neogregarines in five homogeneous colonies for up to 21 months until they collapsed. The study, which combined the use of several molecular markers with the application of a massive parallel sequencing technology, provided valuable insights into the epidemiology of these parasites: (I) it enabled the detection of parasite species rarely reported in honeybees (Nosema thomsoni, Crithidia bombi, Crithidia acanthocephali) and the identification of two novel taxa; (II) it revealed the existence of a high rate of co-infections (80% of the samples harboured more than one parasite species); (III) it uncovered an identical pattern of seasonal variation for nosematids and trypanosomatids, that was different from that of neogregarines; (IV) it showed that there were no significant differences in the fraction of positive samples, nor in the levels of species diversity, between interior and exterior bees; and (V) it unveiled that the variation in the number of parasite species was not directly linked with the failure of the colonies.

Experimental evidence of harmful effects of Crithidia mellificae and Lotmaria passim on honey bees.

The trypanosomatids Crithidia mellificae and Lotmaria passim are very prevalent in honey bee colonies and potentially contribute to colony losses that currently represent a serious threat to honey bees. However, potential pathogenicity of these trypanosomatids remains unclear and since studies of infection are scarce, there is little information about the virulence of their different morphotypes. Hence, we first cultured C. mellificae and L. passim (ATCC reference strains) in six different culture media to analyse their growth rates and to obtain potentially infective morphotypes. Both C. mellificae and L. passim grew in five of the media tested, with the exception of M199. These trypanosomatids multiplied fastest in BHI medium, in which they reached a stationary phase after around 96 h of growth. Honey bees inoculated with either Crithidia or Lotmaria died faster than control bees and their mortality was highest when they were inoculated with 96 h cultured L. passim. Histological and Electron Microscopy analyses revealed flagellated morphotypes of Crithidia and Lotmaria in the lumen of the ileum, and adherent non-flagellated L. passim morphotypes covering the epithelium, although no lesions were evident. These data indicate that parasitic forms of these trypanosomatids obtained from the early stationary growth phase infect honey bees. Therefore, efficient infection can be achieved to study their intra-host development and to assess the potential pathogenicity of these trypanosomatids.

The toxic unit approach as a risk indicator in honey bees surveillance programmes: A case of study in Apis mellifera iberiensis.

The influence of genetic diversity and exposure to xenobiotics on the prevalence of pathogens was studied within the context of a voluntary epidemiological study in Spanish apiaries of Apis mellifera iberiensis, carried out during the spring season of years 2014 and 2015. As such, the evolutionary lineages of the honey bee colonies were identified, a multiresidue analysis of xenobiotics was carried out in beebread and worker bee samples, and the Toxic Unit (TUm) was estimated for each sampled apiary. The relationship between lineages and the most prevalent pathogens (Nosema ceranae, Varroa destructor, trypanosomatids, Black Queen Cell Virus; and Deformed Wing Virus) was analysed with contingency tables, and the possible relationships between TUm and the prevalence of these pathogens were studied by using a factor analysis. The statistical analysis supported the associations between V. destructor and Deformed Wing Virus (DWV), and between N. ceranae and Black Queen Cell Virus (BQCV), but the association between these pathogens and trypanosomatids was not observed. TUm values varied between 5.5 × 10-6 and 3.65 × 10-1. When TUm < 3.35 × 10-4, it was mainly determined by coumaphos, tau-fluvalinate and/or chlorfenvinphos. At higher values, other insecticides also contributed to TUm, although a clear predominance was not seen up to TUm ≥ 1.83 × 10-2, when it was mainly defined by acrinathrin, spinosad and/or imidacloprid. The possible cumulative effect from the joint action of xenobiotics was >10% in the 63% of the cases. The prevalence of pathogens did not appear to be influenced by the distribution of evolutionary lineages and, while the prevalence of V. destructor was not found to be determined by TUm, there was a trend towards an increasing prevalence of N. ceranae when TUm ≥ 23 10-4. This study is an example of using TUm approach beyond the field of the ecotoxicology.

S-element insertions are associated with the evolution of the Hsp70 genes in Drosophila melanogaster.

The "selfish DNA" theory postulates that transposable elements (TEs) are intragenomic parasites, and that natural selection against deleterious effects associated with their presence is the main force preventing their genomic spread in natural populations. In agreement with this model, TEs in Drosophila melanogaster populations are usually found at low frequencies in most genomic locations. Only a few cases of fixation of TE insertions have been reported, usually in regions of low recombination, where selection is expected to be less effective. Here, we report a population genetics study on the apparent fixation of an S-element in a highly recombining region in two natural populations of D. melanogaster. Three similar fragments of an S-element are inserted into the 5' regions of three members of a heat shock gene family, Hsp70 (Hsp70Aa and Hsp70Ab in polytene chromosome band 87A, and Hsp70Bb in 87C). A PCR-based analysis suggests that the insertions are fixed or at high frequencies in the entire species. A population survey of the levels of nucleotide sequence variation at the insertion site in 87C in two natural populations of D. melanogaster provided evidence for reduced levels of variation in the region, normal levels of recombination, and selection, reflected in a significant departure from neutrality of the variant frequency spectrum. This was particularly strong for the S-element inverted repeats (IRs) and suggests that these are of functional significance for the host.

Evolution of amino-acid sequences and codon usage on the Drosophila miranda neo-sex chromosomes.

We have studied patterns of DNA sequence variation and evolution for 22 genes located on the neo-X and neo-Y chromosomes of Drosophila miranda. As found previously, nucleotide site diversity is greatly reduced on the neo-Y chromosome, with a severely distorted frequency spectrum. There is also an accelerated rate of amino-acid sequence evolution on the neo-Y chromosome. Comparisons of nonsynonymous and silent variation and divergence suggest that amino-acid sequences on the neo-X chromosome are subject to purifying selection, whereas this is much weaker on the neo-Y. The same applies to synonymous variants affecting codon usage. There is also an indication of a recent relaxation of selection on synonymous mutations for genes on other chromosomes. Genes that are weakly expressed on the neo-Y chromosome appear to have a faster rate of accumulation of both nonsynonymous and unpreferred synonymous mutations than genes with high levels of expression, although the rate of accumulation when both types of mutation are pooled is higher for the neo-Y chromosome than for the neo-X chromosome even for highly expressed genes.

Rates and patterns of chromosomal evolution in Drosophila pseudoobscura and D. miranda.

Comparisons of gene orders between species permit estimation of the rate of chromosomal evolution since their divergence from a common ancestor. We have compared gene orders on three chromosomes of Drosophila pseudoobscura with its close relative, D. miranda, and the distant outgroup species, D. melanogaster, by using the public genome sequences of D. pseudoobscura and D. melanogaster and approximately 50 in situ hybridizations of gene probes in D. miranda. We find no evidence for extensive transfer of genes among chromosomes in D. miranda. The rates of chromosomal rearrangements between D. miranda and D. pseudoobscura are far higher than those found before in Drosophila and approach those for nematodes, the fastest rates among higher eukaryotes. In addition, we find that the D. pseudoobscura chromosome with the highest level of inversion polymorphism (Muller's element C) does not show an unusually fast rate of evolution with respect to chromosome structure, suggesting that this classic case of inversion polymorphism reflects selection rather than mutational processes. On the basis of our results, we propose possible ancestral arrangements for the D. pseudoobscura C chromosome, which are different from those in the current literature. We also describe a new method for correcting for rearrangements that are not detected with a limited set of markers.