Long-term prevalence of the protists Crithidia bombi and Apicystis bombi and
detection of the microsporidium Nosema bombi in invasive bumble bees
Santiago PLISCHUK
1,*
, Karina ANTÚNEZ
2
, Marina HARAMBOURE
1
,
Graciela M. MINARDI
1
, and Carlos E. LANGE
1,3
1
Centro de Estudios Parasitológicos y de Vectores (CONICET – UNLP). La
Plata, Argentina.
2
Instituto de Investigaciones Biológicas “Clemente Estable”. Montevideo,
Uruguay.
3
Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
(CICPBA), Argentina.
*Corresponding author: S. Plischuk. CEPAVE, Boulevard 120 # 1460
(B1902CHX) La Plata, Argentina. Tel./Fax: (0054) 0221 423 2140.
E-mail: santiago@cepave.edu.ar
Running title: Pathogens in invasive bumble bees
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‘Accepted Article’, doi: 10.1111/1758-2229.12520
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Summary
An initial survey in 2009 carried out at a site in northwestern Patagonia region,
Argentina, revealed for the first time in South America the presence of the flagellate
Crithidia bombi and the neogregarine Apicystis bombi, two pathogens associated with
the Palaearctic invasive bumble bee Bombus terrestris. In order to determine the long-
term persistence and dynamics of this microparasite complex, four additional
collections at the same site (San Carlos de Bariloche) were conducted along the
following seven years. Both protists were detected in all collections: prevalence was 2%
- 21.6% for C. bombi and 1.2% - 14% for A. bombi. In addition, the microsporidium
Nosema bombi was recorded for the first time in the country in the last two collections,
at prevalences of 12.4% and 2.4% and unusually high infection intensities (Average =
6.56 x 10
7
spores per individual). Due to the exceptional dispersal ability of the exotic
B. terrestris, these three multihost pathogens should be considered as potential threats to
South American native bumble bees.
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Introduction
An initial study in 2009 on the entomopathogenic protists associated with the
Palaearctic invasive bumble bee Bombus terrestris (Hymenoptera: Apidae) in Argentina
had revealed the presence of two species, the enteric flagellate Crithidia bombi
(Euglenozoa: Kinetoplastea) and the neogregarine of the fat body Apicystis bombi
(Apicomplexa: Conoidasida), both never reported to occur in the eight native bumble
bee species known for the country (Plischuk and Lange, 2009). Diagnosis of the
pathogens at that time was based on morphological characters, host-pathogen
relationships, and host species involved, but later work by molecular means confirmed
the identity of both protists (Plischuk et al., 2011; Schmid-Hempel et al., 2014). In
order to further the knowledge on the occurrence and diversity of pathogens associated
to B. terrestris in Argentina, we performed four additional collections at the same site
where the first findings were registered. This communication presents the obtained data
and reports on the first record of the microsporidium Nosema bombi in Argentina.
Figure 1 placement
Fig. 1. Sampling site of Bombus terrestris in Argentina (♦). Black circle
indicates the main site of release of B. terrestris (Quillota, Chile). Empty circle
indicates site of release of Bombus ruderatus (Temuco, Chile). Shaded area shows the
known distribution of B. terrestris in Argentina.
Sampling of adult specimens of B. terrestris was performed at a site located 8
km West of San Carlos de Bariloche, Río Negro province (41°07'33"S; 71°23'55"W).
With the exception of the first collection (Season 1) which was conducted from October
2009 to April 2010 (n = 107), all other three were carried out in January as season 2
(2011; n = 269), season 3 (2015; n = 162), and season 4 (2016; n = 166). Bumble bees
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were captured one by one with cylindrical acetate sheet vials (20 cm long, 5 cm
diameter) with removable ends while foraging on flowers of autochthonous
Alstroemeria aurea Graham (Alstroemeriaceae) and exotics Lavandula angustifolia
Mill. (Lamiaceae) and Hypericum calycinum L. (Hypericaceae). After collection,
bumble bees were frozen (-32° C) until their identification according to Fernández and
Sharkey (2006) and Abrahamovich et al. (2007), and later processing.
Figure 2 placement
Fig. 2. Nosema bombi in Bombus terrestris from San Carlos de Bariloche,
Argentina. A: Spores. B: Parts of Malpighian tubules of B. terrestris heavily filled with
spores packed together tightly (upper) and more loosely (lower). C: Immature oocyst of
the neogregarine Apicystis bombi among spores of N. bombi from the only mixed
infection found. [Bars: A-C: 5 µm; B: 25µm; Phase-Contrast Microscopy].
Processing was performed under a Nikon SMZ745T stereoscopic microscope
(x10, x40), dissecting each bumble bee and extracting small pieces of different tissues
and organs in order to prepare fresh smears with one-quarter-strength Ringer’s solution
(Poinar and Thomas, 1984). Detection of pathogens was done using phase-contrast
microscopy (x400, x1000) (Lange and Lord, 2012; Solter et al., 2012). After detections,
each infected individual was individually homogenized in 2 ml of double distilled water
and infection intensity was quantified using an Improved Neubauer hemocytometer as
described by Undeen and Vávra (1997).
To perform molecular analyses of microsporidia, 200 µl of the suspensions
prepared for spore quantification were used for DNA extraction with the PureLink®
Genomic DNA Mini Kit (Invitrogen), according to manufacturer´s instructions. DNA
samples were analyzed by multiplex PCR for simultaneous detection of Nosema apis
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and Nosema ceranae using primers 321APIS and 218MITOC, as described by Martín-
Hernández et al. (2007). Samples were also subjected to PCR to detect the presence of
Nosema bombi, using primers BOMBICAR according to Plischuk et al. (2009).
Amplified DNA was electrophoresed on a 0.8% agarose gel, stained with GelRed
(Biotium) and visualized using a UV-Transiluminator. Positive samples were purified
and sequenced at Macrogen Inc. (Seoul, South Korea). Obtained sequences were
compared with data available in the GenBank (Altschul et al., 1997).
Results and Discussion
Two deliberate introductions of bumble bees for pollination services were
carried out in Chile late in the twentieth century. The first one was in 1982-83 when
close to 400 queens of Bombus ruderatus were imported from New Zealand and
released East of Temuco (Arretz and MacFarlane, 1986; Schmid-Hempel et al., 2014)
(Fig. 1). The second introduction, although exact information is not available, seems to
have occurred in 1997-98 when an unknown number of individuals of B. terrestris were
brought from Israel and possibly Belgium in several localities in central Chile,
particularly Quillota (Schmid-Hempel et al., 2014) (Fig. 1). In 1996 and 2006 both B.
ruderatus and B. terrestris were respectively detected in Argentine Patagonia on the
eastern slope of the Andes (Roig-Alsina and Aizen, 1996; Torretta et al., 2006). It has
been suggested that one or more invasion episodes have occurred (and likely still occur)
across the lowest mountain passes during periods of mild weather (Torretta et al., 2006;
Plischuk and Lange, 2009). Populations of B. terrestris have established not only along
western Patagonia region but also reached the Atlantic coast (Fig. 1), showing ubiquity
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