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About 20% of bumble bee species are in decline in North America, and the microsporidian pathogen, Nosema bombi , has been correlated with these declines. We conducted a comprehensive survey of N. bombi infections in the bumble bee communities throughout the flight season along an elevation gradient in Northern Arizona. Focusing on two species, Bombus (Pyrobombus) huntii and Bombus (Pyrobombus) centralis , we used a combination of PCR and microscopy to distinguish between sporulating and non/low, sporulating N. bombi infections. Surprisingly high levels of PCR-positive infections with no detectable spore loads were found in B. huntii (31–63%) and B. centralis (56.5–66.5%), while the prevalence of sporulating infections was low (3.0–11.8% and 0–12.9% respectively). We determined the prevalence of sporulating N. bombi infection in six other co-occurring, but rarer, bumble bee species (0–62.5%,), but did not test them using PCR. The prevalence of sporulating N. bombi infections in B. (Bombias) nevadensis was significantly higher than in either B. huntii or B. centralis (29%). The declining bumble bee, Bombus sensu strico occidentalis , had the highest prevalence of sporulating N. bombi infections (62.5%), but we purposely captured very few B. occidentalis because of its declining status. PCR was a more sensitive measure of N. bombi prevalence and revealed that wild bumble bees have a much higher prevalence of N. bombi than has previously been recognized. Microscopy and PCR together provide complementary, not redundant, information that deepens our understanding of the dynamic interactions between N. bombi and their bumble bee hosts. [ABSTRACT FROM AUTHOR]

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 bumble bee studies that have identified infections to species 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 with 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 with a universal trypanosomatid primer set to detect unexpected relatives. We applied this method to 356 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 (90.2%) than C. expoeki (21.3%), with most C. expoeki -positive samples existing as co-infections with C. bombi (13.8%). This two-step detection method also revealed that 2.2% 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 three for two unclassified trypanosomatids. This two-step method is effective in diagnosing known bumble bee infecting Crithidia species, and allowing for the discovery of unknown potential symbionts. [ABSTRACT FROM AUTHOR]

Abstract: Several bumble bee (Bombus) species in North America have undergone range reductions and rapid declines in relative abundance. Pathogens have been suggested as causal factors, however, baseline data on pathogen distributions in a large number of bumble bee species have not been available to test this hypothesis. In a nationwide survey of the US, nearly 10,000 specimens of 36 bumble bee species collected at 284 sites were evaluated for the presence and prevalence of two known Bombus pathogens, the microsporidium Nosema bombi and trypanosomes in the genus Crithidia. Prevalence of Crithidia was ⩽10% for all host species examined but was recorded from 21% of surveyed sites. Crithidia was isolated from 15 of the 36 Bombus species screened, and were most commonly recovered from Bombus bifarius, Bombus bimaculatus, Bombus impatiens and Bombus mixtus. Nosema bombi was isolated from 22 of the 36 US Bombus species collected. Only one species with more than 50 sampled bees, Bombus appositus, was free of the pathogen; whereas, prevalence was highest in Bombus occidentalis and Bombus pensylvanicus, two species that are reportedly undergoing population declines in North America. A variant of a tetranucleotide repeat in the internal transcribed spacer (ITS) of the N. bombi rRNA gene, thus far not reported from European isolates, was isolated from ten US Bombus hosts, appearing in varying ratios in different host species. Given the genetic similarity of the rRNA gene of N. bombi sampled in Europe and North America to date, the presence of a unique isolate in US bumble could reveal one or more native North American strains and indicate that N. bombi is enzootic across the Holarctic Region, exhibiting some genetic isolation. [Copyright &y& Elsevier]