|Strange, James - Jamie|
|Szalanski, Allen - University Of Arkansas|
Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2018
Publication Date: 3/15/2018
Citation: Tripodi, A.D., Strange, J.P., Szalanski, A.L. 2018. Novel multiplex PCR reveals multiple trypanosomatid species infecting North American bumble bees (Hymenoptera: Apidae: Bombus). Journal of Invertebrate Pathology. 153:147-155.
Interpretive Summary: Trypanosomatids, including those in the genus Crithidia, are parasites that can cause disease in animals. In humans, trypanosomatids vectored by insects cause sleeping sickness, Chagas disease, and leishmaniasis. Although bumble bees do not vector human disease, they are susceptible to infection by two species of Crithidia, C. bombi and C. expoeki. Because trypanosomatids are quite small and vary in shape, distinguishing these two species can be difficult. Species identification usually requires DNA sequencing, which is relatively expensive. We developed a new molecular method to distinguish the two species of bumble-bee infecting trypanosomatids using a visualization-based diagnosis that is cheaper than DNA sequencing. This method can additionally indicate when bumble bees are harboring trypanosomatids that are not either of the expected species, allowing scientists to conduct exploratory discovery work. We applied this method to determine which trypanosomatid species were infecting 298 bumble bee specimens, and found that C. bombi was most common, with 89% of infected bumble bees testing positive for this species. Most specimens that had C. expoeki were co-infected with C. bombi, although 8% of bee samples only carried C. expoeki. Additionally, we found that 2% of the bumble bee samples had a trypanosomatid that was neither C. bombi nor C. expoeki. Three of these bumble bees tested positive for trypanosomatids that are parasites of honey bees, C. mellificae and Lotmaria passim, and two tested positive for an unidentified trypanosomatid. This new tool will help scientists identify agents of disease in bumble bees and learn more about how trypanosomatids cause disease in bumble bees.
Technical Abstract: Crithidia bombi and Crithidia expoeki (Trypanosomatidae) are common parasites of bumble bees (Bombus spp.). Crithidia bombi was described in the 1980s, and C. expoeki was recently discovered using molecular tools. Both species have cosmopolitan distributions among their bumble bee hosts, but there have been few studies that have identified the species causing Crithidia infections in bumble bees since the original description of C. expoeki in 2010. Morphological identification of species is difficult due to variability within each stage of their complex lifecycles, although they can be easily differentiated through DNA sequencing. However, DNA sequencing can be expensive, particularly if there are many samples to diagnose. In order to reliably and inexpensively distinguish Crithidia species for a large-scale survey, we developed a multiplex PCR protocol using species-specific primers and included a universal trypanosomatid primer set to detect unexpected relatives. We applied this method to 298 trypanosomatid-positive bumble bees from North America as a first-look at the distribution and host range of each parasite in the region. Crithidia bombi was more common (89%) than C. expoeki (22%), with most C. expoeki-positive samples existing as co-infections with C. bombi (14%). This two-step detection method also revealed that 2% of samples were positive for trypanosmatids that were neither C. bombi nor C. expoeki. Sequencing revealed that two individuals were positive for C. mellificae, one for Lotmaria passim, and two for another unclassified trypanosomatid. This method is effective in diagnosing known bumble bee infecting Crithidia species, and allowing for the discovery of unknown potential symbionts.