title: Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim.
abstract: 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.
authors.affiliation: Laboratory of Molecular Entomology and Bee Pathology, Ghent University, Ghent, Belgium. Electronic address: jorgen.ravoet@gmail.com.
authors.forename: Jorgen
authors.initials: J
authors.lastname: Ravoet
authors.affiliation: USDA-ARS Bee Research Laboratory, Beltsville Agricultural Research Center - East, Beltsville, United States.
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Schwarz
authors.affiliation: Laboratory of Molecular Entomology and Bee Pathology, Ghent University, Ghent, Belgium.
authors.forename: Tine
authors.initials: T
authors.lastname: Descamps
authors.affiliation: Institute of Bee Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
authors.forename: Orlando
authors.initials: O
authors.lastname: Yañez
authors.affiliation: Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
authors.forename: Cansu Ozge
authors.initials: CO
authors.lastname: Tozkar
authors.affiliation: Bee Pathology Laboratory, Centro Apícola Regional, Marchamalo, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Martin-Hernandez
authors.affiliation: Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, Santiago de Compostela, Spain; Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Bartolomé
authors.affiliation: Laboratory of Molecular Entomology and Bee Pathology, Ghent University, Ghent, Belgium.
authors.forename: Lina
authors.initials: L
authors.lastname: De Smet
authors.affiliation: Bee Pathology Laboratory, Centro Apícola Regional, Marchamalo, Spain.
authors.forename: Mariano
authors.initials: M
authors.lastname: Higes
authors.affiliation: Laboratory of Socioecology and Social Evolution, K.U. Leuven, Leuven, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Wenseleers
authors.affiliation: Institute of Integrative Biology, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland.
authors.forename: Regula
authors.initials: R
authors.lastname: Schmid-Hempel
authors.affiliation: Institute of Bee Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Neumann
authors.affiliation: Department of Biological Sciences, Xi'an Jiaotong-Liverpool University, Jiangsu, China.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kadowaki
authors.affiliation: USDA-ARS Bee Research Laboratory, Beltsville Agricultural Research Center - East, Beltsville, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Evans
authors.affiliation: Laboratory of Molecular Entomology and Bee Pathology, Ghent University, Ghent, Belgium.
authors.forename: Dirk C
authors.initials: DC
authors.lastname: de Graaf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000469:parasitology*; D003421:Crithidia / Q000469:parasitology*; D003937:Diagnosis, Differential; D017125:Genes, Protozoan; D005838:Genotype; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014351:Trypanosomatina / Q000469:parasitology*
keywords: Apis mellifera; Crithidia mellificae; Honey bee; ITS1; Lotmaria passim; gp63