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

Bartolomé, Carolina; Buendía, María; Benito, M.; Rúa, Pilar De la; Gallego, Carlos; Martín-Hernández, Raquel; Higes, Mariano; Maside, Xulio

doi:10.1016/j.jip.2018.03.015

Cited by 23 publications

(21 citation statements)

References 46 publications

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“…Field samples have detected C. bombi in the Bombus subgenus Psithyrus spp. in Switzerland (Schmid-Hempel and Tognazzo, 2010) and A. mellifera in Spain (Bartolomé et al ., 2018). However, experimental inoculations of A. mellifera with C. bombi indicated that although parasites remained viable in the gut and feces for up to 7 day post-inoculation, quantities were below the levels used for inoculation, indicating a lack of successful infection (Ruiz-González and Brown, 2006).…”

Section: Methodsmentioning

confidence: 99%

“…Experimental inoculations have indicated infectivity in Osmia cornuta and Osmia bicornis (Schwarz et al ., 2015; Strobl et al ., 2019). Molecular analyses of field samples have also detected the parasite in B. terrestris (Bartolomé et al ., 2018). Previously not distinguished from C. mellificae , L. passim is the recently described parasite species now believed to be the dominant trypanosomatid in honey bees worldwide (Ravoet et al ., 2015; Schwarz et al ., 2015) and correlated with collapse of colonies (Cornman et al ., 2012).…”

Section: Methodsmentioning

confidence: 99%

“…Previously not distinguished from C. mellificae , L. passim is the recently described parasite species now believed to be the dominant trypanosomatid in honey bees worldwide (Ravoet et al ., 2015; Schwarz et al ., 2015) and correlated with collapse of colonies (Cornman et al ., 2012). Thus far, infectivity has only been shown experimentally in A. mellifera (Schwarz et al ., 2015; Liu et al ., 2020), but the parasite was detectable by polymerase chain reaction (PCR) in field samples of B. terrestris (Bartolomé et al ., 2018).…”

Section: Methodsmentioning

confidence: 99%

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Cross-infectivity of honey and bumble bee-associated parasites across three bee families

et al. 2020

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Abstract Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts – including four managed species – from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cross-infectivity of honey and bumble bee-associated parasites across three bee families

et al. 2020

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“…due to the occurrence of mutations in the priming sites or to the sequence composition of the amplicon) can go easily undetected. These drawbacks can be partially alleviated by adding a cloning step prior to sequencing (Maharramov et al ., 2013; Gómez‐Moracho et al ., 2014; Ravoet et al ., 2015; Cameron et al ., 2016), which is expensive and time consuming, or by performing specific multiplex PCR assays (Martín‐Hernández et al ., 2007; Xu et al ., 2017; Bartolomé et al ., 2018; Tripodi et al ., 2018), which allow the detection of a limited number of targets per reaction.…”

Section: Introductionmentioning

confidence: 99%

“…Although these methods have permitted the finding of several trypanosomatid (Bartolomé et al ., 2018; Tripodi et al ., 2018) and nosematid species (Li et al ., 2012) in Bombus specimens, their technical limitations urge the use of more powerful tools to better assess the extent of parasite diversity. Nowadays, the most comprehensive and cost‐effective way to investigate this matter at a large scale is to apply high‐throughput sequencing methods, which have much greater depth and resolution than Sanger sequencing (Kulski, 2016) and enable the simultaneous identification of multiple pathogens across large numbers of samples without having to clone the amplicons.…”

Section: Introductionmentioning

confidence: 99%

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

Bartolomé

Jabal-Uriel

Buendía-Abad

et al. 2020

Environmental Microbiology

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Summary 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.

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

et al. 2021

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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.

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

Cited by 23 publications

References 46 publications

Cross-infectivity of honey and bumble bee-associated parasites across three bee families

Cross-infectivity of honey and bumble bee-associated parasites across three bee families

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

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

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