USDA-ARS, Pollinating Insect Research Unit, 1410 N 800 E, Logan, UT 84341, USA.
Department of Entomology, University of Arkansas, 319 AGRI, Fayetteville, AR 72701, USA.
J Invertebr Pathol. 2018 Mar;153:147-155. doi: 10.1016/j.jip.2018.03.009. Epub 2018 Mar 14.
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.
球囊巴尔通体（Crithidia bombi）和扩展巴尔通体（Crithidia expoeki）（动基体目）是熊蜂（Bombus spp.）的常见寄生虫。球囊巴尔通体于 20 世纪 80 年代被描述，而扩展巴尔通体则是最近利用分子工具发现的。这两个物种在其熊蜂宿主中分布广泛，但自 2010 年首次描述扩展巴尔通体以来，针对感染物种的熊蜂研究很少。由于它们复杂生命周期的每个阶段都存在变异性，因此形态鉴定物种非常困难，尽管通过 DNA 测序可以很容易地区分它们。然而，DNA 测序可能很昂贵，尤其是在需要诊断许多样本的情况下。为了在大规模调查中可靠且经济地区分巴尔通体物种，我们开发了一种多重 PCR 协议，该协议使用物种特异性引物和通用动基体原虫引物组进行检测，以发现意外的亲缘关系。我们将该方法应用于来自北美的 356 只阳性熊蜂，这是对该地区每种寄生虫分布和宿主范围的首次观察。球囊巴尔通体比扩展巴尔通体更为常见（90.2%），而扩展巴尔通体阳性样本大多数为球囊巴尔通体的混合感染（13.8%）。这种两步检测方法还表明，有 2.2%的样本对既不是球囊巴尔通体也不是扩展巴尔通体的动基体原虫呈阳性。测序显示，有两个个体对蜜蜂巴尔通体（C. mellificae）呈阳性，一个对Lotmaria passim 呈阳性，三个对两种未分类的动基体原虫呈阳性。这种两步法可有效诊断已知感染熊蜂的巴尔通体物种，并发现未知的潜在共生体。
