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

Abstract

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.

Workflow of Lotmaria passim isolation: Experimental infection with a low-passage strain causes higher honeybee mortality rates than the PRA-403 reference strain

“…However, there is no consensus as to the precise influence that these protozoans might have on bee health, at either the individual or the colony level. The majority of studies to date have focused on different protocols to detect trypanosomatid prevalence and diversity ( Arismendi et al, 2016 , Bartolomé et al, 2018 , Bartolomé et al, 2020 , Vejnovic et al, 2018 , Xu et al, 2018 ), yet few have investigated the mortality rates they produce following experimental infection by different isolates and species ( Gómez-Moracho et al, 2020 ; Higes et al, 2016 ; Liu et al, 2020 ).…”

“…In a novel characterization, bee-infecting trypanosomatid parasites were reclassified into two different species that each belongs to different genera: C. mellificae and Lotmaria passim ( Schwarz et al, 2015 ). Subsequently, other species have been detected in honeybees, such as Crithidia bombi ( Bartolomé et al, 2018 ) and Crithidia acanthocephali ( Bartolomé et al, 2020 ), expanding the host species and the network of known trypanosomatid species that could infect or co-infect the same host. However, of all these species, L. passim seems to display the highest prevalence in apiaries worldwide, with values ranging 15–70% of honeybee colonies ( Castelli et al, 2018 ; Cepero et al, 2014 ; Ravoet et al, 2013 ; Runckel et al, 2011 ; Stevanovic et al, 2016 ).…”

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

“…Trypanosomatids appeared as a combination of three species – Lotmaria passim Evans and Schwarz, 2014, Crithidia mellificae Langridge and McGhee, 1967 and C. bombi – (Table 1), which is in good agreement with previous results from honeybee and bumblebee studies (Bartolomé et al ., 2018; Bartolomé et al ., 2020) and stresses the importance of applying next generation technologies to get a better overview of the parasite diversity in individual samples. This is further supported by the first detection of Crithidia acanthocephali Hanson and McGhee, 1961 in bumblebees, a protozoan that was recently found to be rather frequent in Apis mellifera Linnaeus, 1758 colonies following the same Ion PGM protocol (Bartolomé et al ., 2020). A revision of taxonomic ambiguities by DNA barcoding (Boucinha et al ., 2020) proved that the sequences of this trypanosomatid were identical to those of Crithidia flexonema Wallace, Clark, Dyer and Collins 1960, so from now on both names are used for this taxon.…”

“…With respect to neogregarines, the PCR‐Sanger sequencing protocol detected the presence of Apicystis bombi Liu, Macfarlane and Pengelly, 1974 in only two of the samples (B13.48 and B13.83), whereas the use of a different set of primers combined with Ion PGM sequencing permitted the detection of this species in all the specimens investigated (Table 1), as well as the identification of a new neogregarine taxon recently found in honeybees (GenBank accession number MN031271; Bartolomé et al ., 2020).…”

“…The increasing evidence of trypanosomatid co‐occurrence in pollinators (Ravoet et al ., 2015; Bartolomé et al ., 2018; Tripodi et al ., 2018; Bartolomé et al ., 2020) prompted us to evaluate the performance of one of these massive parallel sequencing technologies (Ion PGM TM System; Thermo Fisher Scientific) in 12 trypanosomatid‐positive bumblebees detected in the study by Jabal‐Uriel et al . (2017).…”