Apidologie Original article * INRA, DIB and Springer-Verlag France, 2016 DOI: 10.1007/s13592-016-0474-2 Pathogens, parasites, and parasitoids associated with bumble bees (Bombus spp.) from Uruguay 1 2 3 2 Santiago PLISCHUK , Sheena SALVARREY , Natalia ARBULO , Estela SANTOS , 4 4 5 5 Jeffrey H. SKEVINGTON , Scott KELSO , Pablo D. REVAINERA , Matías D. MAGGI , 2 1,6 Ciro INVERNIZZI , Carlos E. LANGE 1Centro de Estudios Parasitológicos y de Vectores (CCT La Plata CONICET—UNLP), Boulevard 120 # 1460, 1900, La Plata, Argentina 2Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay 3Centro Universitario Regional del Este, Universidad de la República, Rocha, Uruguay 4Canadian National Collection of Insects, Arachnids and Nematodes, Agriculture and Agri-Food Canada, Ottawa, Canada 5Centro de Investigación en Abejas Sociales (UNMdP), CONICET, Mar del Plata, Argentina 6Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICPBA), La Plata, Argentina Received6July2016– Revised 24 August 2016 – Accepted 21 September 2016 Abstract – As elsewhere in the world, bumble bees play a vital role as pollinators in Uruguay, but knowledge on their health status is still limited. Between September 2012 and May 2013, 403 adult individuals of the two species of Bombus known for the country (Bombus atratus , Bombus bellicosus ) were collected in six localities. We found that 177 (119 B. atratus ,58B. bellicosus ) were harboring one or two types of pathogens, parasites, or parasitoids. Identification of these natural enemies carried out by morphological or molecular procedures revealed the presence of two species of Microsporidia [Nosema ceranae (prevalence: 18.2 % in B. atratus ;44.9%inB. bellicosus ), Tubulinosema pampeana (prevalence: 13 % in B. atratus )], two species of Nematoda [Sphaerularia bombi (prevalence: 40.4 % in B. atratus ;40%inB. bellicosus ) and an unidentified Mermithidae (prevalence: 0.8 % in B. bellicosus )], and one species of Diptera parasitoid (prevalence: 3.2 % in B. atratus ; 4.2 % in B. bellicosus ). Except N. ceranae , none of the other species have been previously reported in Uruguay. Conopidae / Microsporidia / Nematoda / pollinator 1. INTRODUCTION Bombus atratus Franklin1 and B. bellicosus Smith (Santos et al. 2013). Both are relatively Of the 250 species of Bombus bumble bees common in southern South America and abundant currently recognized worldwide (Cameron et al. in the Pampas region. Since much of this region 2007),twoareknowntooccurinUruguay, has become a heavily used agro-ecosystem, these insects provide essential pollination services not only for relict natural vegetation but also for a Electronic supplementary material The online version of variety of crops (Arbulo et al. 2011). Despite their this article (doi:10.1007/s13592-016-0474-2) contains central relevance in such an important process and supplementary material, which is available to authorized users. 1 The widely adopted name B. atratus is used in this Corresponding author: S. Plischuk, contribution although according to some authors (Moure [email protected] and Melo 2012), the valid one would be Bombus Manuscript Editor: James Nieh pauloensis . Plischuk S. et al. although pathogens and parasites have been sig- quantified using an Improved Neubauer hemocytometer naled as a relevant mortality factor (Kissinger by counting of spores (Undeen and Vávra 1997). et al. 2011 and references therein), the current Due to morphological similarity between species of health status of bumble bees in Uruguay is still Nosema , the microsporidia genus known to be associ- poorly known being the microsporidium Nosema ated with bumble bees, molecular identification was ceranae Fries et al. the only pathogen reported in carried out by Multiplex PCR using specific primers the country associated with these insects (Arbulo for16SSSUrRNAofNosema ceranae (218MITOC), et al. 2015). With the aim to increase the knowl- Nosema apis Zander (321APIS), and Nosema bombi edge about the diversity of pathogens, parasites, Fantham and Porter (BOMBICAR) (see Plischuk et al. and parasitoids naturally harbored by Bombus 2009). Briefly, genetic material was extracted from species known to inhabit this area, we provide infected tissues before homogenization using a QIAamp novel information on the presence, prevalence, Viral RNA Mini Kit (Qiagen) following the manufac- intensity of infections, and co-infection rates of turer’s instructions but modifying the final volume of the parasitic natural enemy complex2 associated 60 μL of elution with AVE buffer to a final volume of to B. atratus and B. bellicosus . 46 μL. Multiplex reactions were performed in a volume of 25 μL using 1.5 μLofMgCl2 (50 mM), 2.5 μLof PCR 10× buffer, 0.2 μL of Taq polymerase (50 U/μL), 2. MATERIALS AND METHODS 1 μL of dNTPs (10 mM), 0.25 of each primer (40 μM), and 2.5 μL of template DNA. Final volume was A total of 403 adult bumble bees (285 B. atratus , achieved with distilled water. Samples were denatured 118 B. bellicosus ) were individually collected at six at 94 °C for 2 min followed by 10 cycles of 94 °C for localities belonging to five departments3 in southern 15 s, 61.8 °C for 30 s, and 72 °C for 45 s. After that, 20 Uruguay [INIA-La Estanzuela (Dept. Colonia), Monte- cycles were added but increasing 5 s to the last step of video (Dept. Montevideo), Sarandí Grande (Dept. Flor- each one, followed by a final extension step of 72 °C for ida), Villa Soriano (Dept. Soriano), Sauce (Dept. 7 min. Positive controls were provided by Dr. Mariano Canelones), Canelón Chico (Dept. Canelones); Higes, Centro Agrario de Marchamalo, Spain. Amplifi- Table I, Figure 1] between September 2012 and April cation products were analyzed by 2 % agarose gels in 2013. They were captured while foraging using plastic TBE 0.5× buffer (Tris 10.8 g, boric acid 5.5 g, EDTA jars directly from flowers and conserved until dissection 0.5 M pH 8, 4 mL per 100 mL) and treated with GelRed either in 70 % ethanol or frozen (−32 °C). Individuals nucleic acid stain (Biotium, Hayward, USA). were dissected using a Nikon SMZ745T stereoscopic Tubulinosema pampeana Plischuk et al., a recently microscope (×10, ×40) (Lange 1996). Initially, bumble described microsporidium from B. atratus, can be read- bees were checked for parasites and parasitoids of the ily identified and differentiated with compound micros- metasomal cavity such as nematodes and juvenile dip- copy from Nosema species by its spore size and ap- teran stages. Afterwards, tracheae were scrutinized for pearance as well as affected tissue and typical gross mites. Finally, small portions of different tissues and pathology (Plischuk et al. 2015). organs were extracted in order to prepare fresh smears Observation of Nematoda characters was done using with one-quarter-strength Ringer’s solution (Poinar and simple stereoscopic microscopy (×10, ×40) as well as Thomas 1984) for detection of microsporidia and pro- compound (bright field) microscopy (×400, ×1000) tists (Lange and Lord 2012;Solteretal.2012). Obser- (Plischuk and Lange 2012). Identification was carried vations were done using phase-contrast microscopy out following appropriate keys (Pouvreau 1962;Poinar (Nikon E200; ×400, ×1000). Microsporidia spore sus- 1977;Siddiqi2000). pensions were obtained by homogenization of the Parasitoid larvae hard structures were observed whole infected insect in 2 mL of distilled water and and photographed using both simple (×10, ×40) and compound (bright field) microscopy (×400, ×1000). Soft larval tissues are completely digested 2 Into Bparasitic natural enemy complex^ we include path- ogens, parasites, and parasitoids as defined by Onstad et al. when exposed to a Proteinase K-based total DNA (2006). extraction protocol. The middle of the larval body 3 Main national geographic subdivisions. was excised for DNA extraction, allowing the Parasitic natural enemies of bumble bees Table I. Sample localities and prevalence (%) of pathogens (Microsporidia), parasites (Nematoda), and parasitoids (Diptera) of Bombus atratus and Bombus bellicosus from Uruguay during season 2012–2013 (n =403) Locality Species Caste Microsporidia Nematoda Diptera (department) (n ) Nosema Tubulinosema Sphaerularia Mermithidae Conopidae ceranae pampeana bombi INIA–La Estanzuela B. atratus W(75) 45 –––9 ′ (Colonia) 34° 20 S; M(2) –– – – – 57° 41′ W B. bellicosus W(50) 76 ––28 M(13) 15 –––– Montevideo B. atratus Q(93) 22 37 41 –– (Montevideo) 34° B. bellicosus Q(5) –– 40 –– 50′ S; 56° 13′ W Sarandí Grande B. atratus W(27) 22 –––8 ′ (Florida) 33° 43 S; M(19) 11 –––5 56° 20′ W B. bellicosus W(22) 45 –––5 M(10) –– – – – Villa Soriano (Soriano) B. atratus W(15) 7 –––– ′ ′ 33° 24 S; 58° 19 W B. bellicosus W(2) –– – – – Sauce (Canelones) B. atratus Q(1) –– – – – ′ ′ 34° 40 S; 56° 02 W W(40) 18 2 ––– M(2) –– – – – B. bellicosus W(15) 7 –––– M(1) –– – – – Canelón Chico B. atratus W(11) – 18 ––– (Canelones) 34° 39′ S; 56° 11′ W W workers, M males, Q queens cephalopharyngeal skeleton and posterior spiracles following manufacturer’s recommendations prior to to be retained as vouchers for each of the specimens sequencing. Sequencing reactions were carried out studied. Total DNA was extracted using a DNeasy® using an ABI BigDye Terminator v3.1 Cycle Se- Tissue kit (Qiagen Inc., Santa Clara, CA, USA) quencing kit (PE Applied Biosystems, Foster City, following the manufacturer’s protocol. DNA ampli- CA) in a volume of 10 μL containing 5 μL purified fications were carried out in 25-μL reactions with PCR template, 1.5 μL 5× sequencing buffer, 2 μL 15.7 μLddH2O, 2.5 μL 10× PCR buffer, 0.65 μL ddH2O, 0.5 μL10μM primer, and 1 μL ABI Big 25 mM MgCl2,1.0μL of each 10 μMprimer,2.0μL Dye reagent and were purified using the ABI etha- 10 mM dNTPs, 0.15 μLExTaq Hot Start DNA nol/ EDTA/ sodium acetate precipitation protocol. polymerase (Takara Bio Inc., Otsu, Shiga, Japan), They were analyzed on an ABI 3130xl Genetic An- and 2 μL genomic DNA template. A 658-base-pair alyzer (PE Applied Biosystems) and identified by fragment of the 5′ end of the COI gene was amplified comparison to known sequences in GenBank using using the primer pair LCO1490 (5′-GGTC BLAST.