Prevalence of species in a feral bee population: a 20-year survey Juliana Rangel, Kristen Baum, William L. Rubink, Robert N. Coulson, J. Spencer Johnston, Brenna E. Traver

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Juliana Rangel, Kristen Baum, William L. Rubink, Robert N. Coulson, J. Spencer Johnston, et al.. Prevalence of Nosema species in a feral population: a 20-year survey. Apidologie, Springer Verlag, 2016, 47 (4), pp.561-571. ￿10.1007/s13592-015-0401-y￿. ￿hal-01532328￿

HAL Id: hal-01532328 https://hal.archives-ouvertes.fr/hal-01532328 Submitted on 2 Jun 2017

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2016) 47:561–571 Original article * INRA, DIB and Springer-Verlag France, 2015 DOI: 10.1007/s13592-015-0401-y

Prevalence of Nosema species in a feral honey bee population: a 20-year survey

1 2 3 4 Juliana RANGEL , Kristen BAUM , William L. RUBINK , Robert N. COULSON , 1 5 J. Spencer JOHNSTON , Brenna E. TRAVER

1Department of Entomology, Texas A&M University, 2475 TAMU, College Station, TX 77843-2475, USA 2Department of Integrative Biology, Oklahoma State University, 501 Life Sciences West, Stillwater, OK 74078, USA 3P.O. Box 2686, Edinburg, TX 78540, USA 4Knowledge Engineering Laboratory, Department of Entomology, Texas A&M University, College Station, TX 77843-2475, USA 5Department of Biology, Penn State Schuylkill, 200 University Drive, Schuylkill Haven, PA 17972, USA

Received 26 January 2015 – Revised 14 July 2015 – Accepted 20 October 2015

Abstract – Nosema spp. are microsporidian pathogens of honey bees that cause nosemosis, a disease implicated in colony losses worldwide. Few studies have measured Nosema spp. levels in feral honey bees. We evaluated the presence and infection intensity of and in a feral Africanized honey bee population in south Texas from 1991 to 2001 and in 2013. Overall, less than 6 % of samples had Nosema spp. spores. N. apis was only found in samples from 1991 to 1995. Conversely, N. ceranae was found every year examined, ranging from 16.7 % infection in 1991 to 85.7 % in 2013. There were no effects of temperature or rainfall on infection with either species over time. This suggests that feral honey bees are relatively free of Nosema spp. compared to managed colonies. More studies on the incidence of Nosema spp. in feral honey bee populations are needed.

Apis mellifera / Africanized feral honey bees / Nosema apis / Nosema ceranae /qPCR

1. INTRODUCTION Vegetative stages reproduce within the cells to form spores that are released upon lysis of the Nosemosis, a disease of honey bees that infects cell, which then are freed to infect other midgut epithelial cells of the midgut (Bailey 1981; epithelial cells (Bailey and Ball 1991). Two spe- Matheson 1993), can be caused by the presence cies of Nosema can infect the honey bee Apis of species in the Nosema mellifera : Nosema apis and Nosema ceranae . (). Nosemosis is transmitted horizon- N. apis affects the western European honey tally between adults through the oral–fecal route, bee (Apis mellifera L.) and is found worldwide where uninfected adults become infected by con- (Matheson 1996). N. ceranae was first discovered tact with food or feces contaminated with Nosema in the Asian honey bee in 1996 (Fries spp. spores (Fries 1993, 1996). Following inges- 1996), was later identified in managed Apis tion, spores germinate within the midgut by mellifera colonies in Spain and Taiwan (Higes ejecting a polar filament that injects the Nosema et al. 2006; Huang et al. 2007), and is now wide- spp. sporoplasm into epithelial cells of the midgut. spread in Europe (Higes et al. 2006, 2009a; Chauzat 2007; Huang et al. 2007, 2008; Klee et al. 2007; Paxton et al. 2007; Topolska and S. Corresponding author: J. Rangel, Kasprzak 2007), North America (Chen et al. [email protected] 2008;Williamsetal.2008;TraverandFell Manuscript editor: Yves Le Conte 2011a, b), Central America (Calderón 2008; 562 J. Rangel et al.

Rangel et al. 2013), South America (Invernizzi honey bees (Fries 2003). Feral and managed hon- et al. 2009; Medici et al. 2012; Mendoza et al. ey bee colonies in Central and South America, as 2014), Africa (Fries 2003; Higes et al. 2009b), and well as some regions in the southern United States Australia (Giersch et al. 2009). While honey bee are considered “Africanized,” or hybrids formed colonies infected with N. apis exhibit fecal streak- from a mixture of genes derived from European ing on the hives and comb due to severe and African ancestors (Taylor 1988;Taylorand (Hassanein 1951;Fries1993), those infected with Spivak 1984;Rubinketal.1996;Pinto2004). N. ceranae do not show obvious external symp- Interestingly, infection of workers by Nosema in toms. Despite the key symptomatic differences Latin America is observed mostly in Africanized caused by different Nosema species, infection honey bees. However, that observation may reflect with one or both species causes significant de- incomplete sampling, as most studies of nosemosis clines in a colony’s overall worker population in Africanized honey bees in that region have been (Wang and Mofller 1970; Higes et al. 2008)and done in managed colonies (Calderón 2008; honey production (Hassanein 1951; Fries et al. Invernizzi et al. 2009;Rangeletal.2013; 1984; Rinderer and Sylvester 1978; Anderson Mendoza et al. 2014). and Giacon 1992;Maloneetal.1995). There have only been three studies reporting The decrease in the worker population (colony Nosema levels in unmanaged honey bees living as size) is mainly caused by worker mortality associated feral colonies in diverse habitats, and those three with digestive disorders and shortened lifespan studies only reported N. apis infection. Manning (Hassanein 1951). Antúnez et al. (2009)showed et al. (2007) reported high levels of N. apis infection evidence that N. ceranae infections result in immu- in feral honey bee colonies around southwestern nosuppression in workers, hypothesizing that Australia from 2002 to 2003. While all the colonies N. ceranae infection can make a colony more sus- surveyed were infected with N. apis , the presence ceptible to other pathogens, particularly viruses and severity of infection were significantly affected (Bailey et al. 1983;Dainatetal.2012). Several studies by the location of the colony and the weather during have also reported detrimental synergistic interactions sample collection. In the USA, only two studies between N. ceranae and other stressors such as have reported the prevalence of Nosema spp. in pesticides, on honey bee health (Alaux et al. 2010; feral Africanized honey bees. Gilliam and Taber Vidau et al. 2011;Wuetal.2012; Higes et al. 2013). (1991) first reported the presence of N. apis in feral Colonies may be infected by one or both strains honey bees from Arizona, where they found a very of Nosema . A recent study found direct evidence low level of infection, with 9 of 21 individuals suggesting that N. ceranae and N. apis undergo sampled being infected with only one to five severe interspecific competition inside the gut of N. apis spores per infected bee. More recently, honey bees and that when co-infecting bees with Szalanski (2014) explored the prevalence of both microsporidia, N. ceranae infection exhibit- Nosema spp. in feral Africanized honey bees in ed stronger inhibition of N. apis than vice versa Arkansas, New Mexico, Mississippi, New (Natsopoulou et al. 2014). Because the spores of Mexico, Oklahoma, Texas, and Utah. Using PCR- both species look very similar using traditional restriction fragment length polymorphism (PCR- miscroscopy, the cause of nosemosis, whether by RFLP), the authors found that only 8.3 % of the N. apis or N. ceranae , or both, is most reliably samples analyzed tested positive for N. apis , with distinguished using polymerase chain reaction infections being more common in Texas than in any (PCR) (Martin-Hernandez et al. 2007; Roudel other state surveyed. et al. 2013) and quantitative polymerase chain All of the aforementioned surveys were reaction (qPCR) techniques (Higes et al. 2006; done only for N. apis and were conducted on Klee et al. 2007; Paxton et al. 2007; Traver and samples collected in a single year. Therefore, Fell 2011a, b;Rangeletal.2013). data are lacking on the presence of N. apis and N. apis will infect the African honey bee, Apis N. ceranae in feral honey bee populations in mellifera scutellata , causing diagnostic symptoms the USA over time. In this study, we surveyed that are similar to those found in infected European the same population of feral honey bees in Prevalence of Nosema in feral honey bees 563 south Texas from 1991 to 2001 and again in 2.2. Genomic DNA extraction 2013 and used molecular techniques to deter- and quantitative real-time PCR mine the presence and levels of infection in these colonies by N. apis , N. ceranae , or both, Genomic DNA was extracted from individual abdo- over the last two decades. mens of ten workers from each cavity sampled per year as described previously (Traver et al. 2009; Traver and Fell 2011a). Briefly, a Bender buffer lysis followed by a 2. METHODS proteinase K digestion and phenol/chloroform extrac- tion was performed. After the first separation step, the 2.1. Study site and sample collection organic layer was saved for spore analysis. DNA was precipitated with isopropanol and resuspended over- The feral honey bees sampled for this study were night in DEPC-treated water kept at room temperature. collected at the Welder Wildlife Refuge (WWR), locat- This procedure resulted in the extraction of genomic ed in San Patricio County, TX (28° 07′ 18″, −97° 26′ DNA from vegetative cells but not from mature spores. 34″). The WWR lies between the Gulf Prairies and DNA purity and concentration were assessed using a Marshes and the South Texas Plains ecoregions, which Nanodrop 2000 Spectrophotometer (Thermo Scientific, constitute a transitional zone composed mostly of mes- Wilmington, DE, USA). Subsamples from five random quite, chaparral brushland, open grassland, and abun- DNA samples were pooled together to reduce the sam- dant live oak tress (Drawe et al. 1978; Blankenship ple size for analysis. Pools of five DNA samples were 2000). Typically, this area experiences a year-round collected twice for each sample, as there was individual humid climate, with hot summers and cool winters DNA from ten bees from each colony sampled. (Blankenship 2000;Baum2003). A qPCR was subsequently performed following the Baum (2003; 2005) surveyed a 6.25-km2 area of the protocols described by Traver and Fell (2011a). Briefly, WWR every year between 1991 and 2001 and found a Nosema species–specific TaqMan Gene Expression as- total of 109 trees housing feral honey bee colonies, with says (Applied Biosystems, Foster City, CA, USA) were most of the colonies living in standing oak trees (85 %) used to quantify infection levels and to identify the while the remaining colonies lived in elm or fallen oaks. Nosema species present in each colony sampled. During the survey, honey bee workers were collected Absolute quantitation, using standard curves with from all active colonies every year for mitochondrial known N. ceranae and N. apis DNA copy numbers, and genomic DNA analysis. This was done to investi- was used to estimate the levels of infection of each gate the patterns of hybridization between bees of Nosema species across all colonies sampled per year. European and African descent occurring in this popula- All qPCRs were performed using an Applied tion of feral honey bees since the late 1990s (Baum Biosystems StepOne™ Real-Time PCR System. A 20- 2003; 2005; Pinto 2004; Pinto 2005). In 2013, μL reaction for duplex qPCR was performed according 5.60 km2 of the original WWR study area was re- to the TaqMan Gene Expression Master Mix protocol surveyed, and 89 of the original 109 trees that had been (Applied Biosystems, Foster City, CA, USA) using identified earlier were re-visited and sampled for honey 50 ng of total pooled genomic DNA per reaction. bees if a colony was present. Standard controls as well as the experimental samples In the current study, honey bee workers were select- were run together and the average cycle threshold value ed from the 2013 population and from a subsample of (C T) was used to calculate the average copy number of the colonies sampled in previous years. The colonies each species of Nosema present in each DNA sample used for Nosema spp. analysis were those with honey (Traver and Fell 2011a). bees that inhabited the same tree cavities over multiple years, and those with at least 60 workers that could be 2.3. Nosema spore counts examined for spore counts. In total, we analyzed worker samples from 18 tree cavities inhabited by honey bees Spores were counted as previously described, a in seven different years: 1991, 1992, 1994, 1995, 1998, method that carefully ensures that only Nosema spores, 2001, and 2013. No samples were collected between not those of other microorganisms, are tallied (Cantwell 2001 and 2013. 1970; Mulholland et al. 2012). Briefly, ten workers were 564 J. Rangel et al. examined per colony by dissecting out their abdomens. honey bees at the WWR exhibited low spore Each abdomen pellet was crushed and microscopically counts. Of the 464 individual worker abdomens examined to estimate the average number of Nosema examined for spore counts, only 28 samples had spp. spores found per worker from each colony. Spores spores present, representing only 5.9 % of the total were counted on a Bright-Line Hemacytometer number of bees analyzed (Table I). In the bees (Hausser Scientific, Horsham, PA) to estimate the num- with spores present, the mean number of spores ber of spores per individual worker abdomen as de- per bee ranged significantly across years scribed previously (Cantwell 1970; Mulholland et al. (χ2=24.57; d.f. = 9; P =<0.01), with the lowest 2012). The average number of spores per colony, for number of spores observed in 1995 (357±357 each year, was calculated by averaging the number of spores per bee) and the highest number observed spores per bee from each cavity sampled. in 2001 (13,405±10,161; Table I).

2.4. Temperature and precipitation 3.2. Nosema spp. infection intensities across across years years

Because most of the bees were collected in the N. apis was found only in colonies sampled month of July across years, we evaluated if mean tem- from 1991 to 1995 (Figure 1 and Table I). The perature or rainfall patterns in July at the WWR had an percentage of colonies infected with N. apis was effect on the prevalence of either Nosema species. To highest in 1991 (33.3 %) and 1995 (14.3 %). The answer this question, we queried the monthly climato- mean N. apis DNA copy number increased from logical summary from the Welder Wildlife Foundation nearly 12,000 DNA copies in 1991 to nearly weather station (elevation, 49 ft.; latitude, 28.114° N; 340,000 DNA copies in 1994, with only 24 copies longitude, 97.418° W) operated by the National observed in 1995 (Figure 2 and Table II). Colonies Oceanic and Atmospheric Administration of the US were co-infected with N. apis and N. ceranae Department of Commerce from 1991 to 2001, and again from 1991 to 1995 at varying levels. We did not for 2013. The data include mean temperature (°C) and find N. apis in samples collected from 1998 to total precipitation (mm) recorded at the station in July. 2013, however (Figure 1 and Table II). Although the feral honey bees at the WWR had 2.5. Statistical analysis low spore counts, N. ceranae was found in at least two of the colonies sampled every year. The per- AKruskal–Wallis test was performed to determine centage of colonies infected with N. ceranae var- whether the mean number of Nosema spp. spores per ied between years, ranging from 16.7 % in 1991, bee varied significantly across years. We used a least 100 % in 1994, and 85.7 % in 2013 (Figure 1 and squares analysis of variance test with the software JMP Table I). The mean N. ceranae DNA copy num- 11 Pro (SAS Institute Inc., Cary, NC) to test whether ber per bee increased by 7 orders of magnitude temperature and rainfall had a significant effect on the from about 155 DNA copies in 1991 to over 88+ average copy numbers of N. ceranae or N. apis found million DNA copies in 2013 (Figure 2 and across years. All descriptive statistics are reported as the Table II). mean±1 standard error of the mean (SEM). We set the level of statistical significance at α=0.05 for all tests. 3.3. Average temperature and rainfall patterns 3. RESULTS The mean temperature recorded in July at 3.1. Nosema spp. spore counts the WWR meteorological station ranged from 32.0 to 34.8 °C. The temperature increased We scored worker samples from 18 tree cavi- relatively steadily from 1991 to 1998, dropped ties inhabited by honey bees in 1991, 1992, 1994, by almost 3 °C in 1999, and returned to tem- 1995, 1998, 2001, and 2013 using spore counts peratures above 34.7 °C in 2001 and 2013 and qPCR (Table I). Overall, colonies from feral (Figure 2). There were no obvious patterns in Prevalence of Nosema in feral honey bees 565

Table I. Mean spore count (±SEM) from feral honey bee colony samples that tested positive for Nosema by spore analysis.

Year of Total no. of Total no. of workers % workers with Mean spore SEM spore collection workers that tested positive nosema spores count per bee count per bee analyzed for nosema spores

1991 60 9 15.0 5.0×103 1.7×103 1992 70 2 2.9 1.4×103 1.1×103 1994 70 3 4.3 1.8×103 1.1×103 1995 70 1 1.4 357.1 357.1 1998 68 2 2.9 1.1×103 817.1 2001 67 7 10.4 1.7×104 13.8×10469 2013 69 4 5.8 1.3×104 10.2×104 Total 474 28

The samples were collected at the Welder Wildlife Refuge, San Patricio County, TX, from 1991 to 2001 and again for 2013

rainfall across the study years, with the total 4. DISCUSSION precipitation in July ranging from a low of 0 mm in 1993 to a high of about 140 mm in For over 20 years, the feral honey bee popula- 1999, which also was the year with the lowest tion at the WWR in south Texas has served as a mean temperature in July (Figure 2). Overall, valuable model for studies of biological invasion, there were no clear effects of temperature or species hybridization, and survival under adverse rainfall on the average DNA copy number of environmental conditions. We provide the first N. ceranae (F-ratio=3.14; P =0.15) or N. apis report of the combined prevalence and infection (F-ratio=0.89; P =0.48) found in the colonies intensities of the microsporidia N. apis and sampled over time. N. ceranae in a population of feral honey bees in the USA. Overall, we found that the Nosema levels in this population are relatively low, with 6.E+09 Nosema ceranae Nosema apis N. apis being present only in samples collected 2.E+08 from 1991 to 1995 and subsequently disappearing 1.E+07 by 1998. Conversely, the levels of N. ceranae

4.E+05 escalated by 7 orders of magnitude from 1991 to 2013, suggesting that the decrease in prevalence 2.E+04 of N. apis might have been in part due to com- 6.E+02 petitive displacement by N. ceranae as has been

mean DNA copy number / sample 3.E+01 10 hypothesized in other regions for managed colo-

Log 1.E+00 nies (Paxton et al. 2007). Furthermore, average 1990 1992 1994 1996 1998 2000 2013 rainfall or temperature did not have a clear impact Year of sample collection on the levels of either Nosema species prevalence Figure 1. Nosema ceranae (white bars )andN. apis of infection levels over time. (gray bars ) mean copy numbers (±SEM) for feral hon- ey bee colonies inhabiting tree cavities at the Welder N. apis was once the only Nosema species to Wildlife Refuge, San Patricio County, TX, from 1991 to infect Apis mellifera (Bailey and Ball 1991), with 2001 and again for 2013. Two sets of five worker infection shown to reduce worker longevity by up abdomens were used for a total of ten worker abdomens to 50 % and cause queen supersedure within 2– examined from each colony sampled. Species identifi- 6 weeks (Moeller 1956; Bailey 1981). Related cation was done using qPCR analysis. studies suggested that queen supersedure events 566 J. Rangel et al.

Mean temperature Precipitation 40 150

35

C) 125 o 30 100 25

20 75

15 50

10 Total precipitation (mm)

Mean temperature in July ( 25 5

0 0 1989 1992 1995 1998 2001 2004 2007 2010 2013 Year of data collection Figure 2. Average temperature and total precipitation in July recorded at the Welder Wildlife Foundation meteo- rological station of the National Oceanographic and Atmospheric Administration (elevation 49 ft., latitude 28.114° N, longitude 97.418° W). The values are provided for 1991 to 2001 and again for 2013. The gap between 2001 and 2013 represents years in which data were not queried, as no honey bees were sampled those years.

occurred because of a 17–45 % size reduction in More recently, N. ceranae has been identi- the secretory globules of the hypopharyngeal fied in managed honey bee colonies, first in glands of workers inoculated with N. apis spores Europe and Asia (Higes et al. 2006; Huang (Wang and Moeller 1969). Furthermore, artificial et al. 2007) and now on almost every continent inoculation of workers with N. apis spores nega- where apiculture is practiced, including North tively affected honey yield (Fries et al. 1994), America (Chen et al. 2008; Williams et al. 2008; suggesting that N. apis infection was directly Traver and Fell 2011 a, b). The infectivity of associated with decreased colony productivity. N. ceranae seems more severe than that of

Table II. Infection levels of Nosema ceranae and N. apis found in feral honey bee colonies sampled at the Welder Wildlife Refuge, San Patricio County, TX, from 1991 to 2001, and again in 2013.

N. ceranae N. apis

Year of N Mean DNA SEM % colonies Mean DNA SEM % colonies collection copy number infected copy number infected with per bee with per bee N. apis N. ceranae 1991 12 155.1 121.7 16.7 11,615.6 8839.7 33.3 1992 14 2131.0 1230.4 35.7 1905.0 1905.0 7.1 1994 14 14,344.8 3555.9 100 338,286.7 201,187.3 35.7 1995 14 108.9 65.3 21.4 24.1 16.4 14.3 1998 14 577.1 222.7 50.0 0 0 0 2001 14 4.28×107 4.28×107 42.9 0 0 0 2013 14 8.87×107 4.73×107 85.7 0 0 0

N represents the number of colonies sampled for Nosema per year using qPCR analysis for species identification. Individual worker abdomens (n =10 for each sample) were examined for DNA copy number for each Nosema spp. from each colony every year of sample collection. The mean DNA copy number was obtained for each worker group sampled SEM standard error of the mean Prevalence of Nosema in feral honey bees 567

N. apis (Bailey et al. 1983;Dainatetal.2012; Overall, pathogens and diseases affecting honey Antúnez et al. 2009), perhaps because some bees are primarily a problem of commercial bee- parasite variants may induce higher virulence keeping (Sherman et al. 1998). prac- in certain populations (Fries 2010, Higes et a. tices, like moving combs between colonies and 2013, Roudel et al. 2013). This suggests that , manipulating several apiaries with the N. ceranae may be outcompeting N. apis in same tools and equipment, or concentrating colo- many regions where infection of both species nies into apiaries, among others, undoubtedly pro- has been analyzed using molecular techniques mote pathogen and parasite infectivity and trans- (Traver and Fell 2011a, b;Rangeletal.2013). mission. However, this does not imply that unman- Our current knowledge on the co-infection of aged or feral honey bee colonies are not prone to honey bees by both species of Nosema is based diseases or that they do not serve as reservoirs for on studies using only managed colonies kept in diseases. Under the right environmental conditions, apiaries. Those that have looked at the levels of the warm temperature inside a feral honey bee nest Nosema in feral honey bee populations have only can drive and pathogen transmission. reported data on N. apis infection. For example, However, previous studies have suggested that feral Manning et al. (2007) looked at the levels of colonies exhibit effective nest hygiene mechanisms N. apis in feral colonies, settled swarms, and that counteract these problems, including the secre- managed colonies in southwestern Australia in tion of antimicrobial and antifungal chemicals into the early 2000s. They found that 77 % of the feral honey, the use of tree resins (i.e., propolis) to coat colonies were infected with N. apis , with the crevices in the nest structure, defecation outside the infection ranging from 0 to 25 million spores/ nest, and the removal of dead and sick individuals bee. Interestingly, the severity of N. apis infection (Visscher 1980; Seeley 1985;Drumand in feral colonies was significantly lower than in Rothenbuhler 1985). Given that the level of managed colonies, suggesting that feral colonies Nosema infections in feral honey bees appears to in that area of Australia have strains of N. apis be low in all the studies conducted so far, it is likely that are less virulent, have better strategies for that these bee populations are using effective mech- combating N. apis , or a combination of both. anisms to control the levels of N. ceranae or In the USA, only two studies have explored N. apis in colonies. The use of propolis as an infection of Nosema in unmanaged colonies, both antimicrobial product used by honey bees to “self reporting infection by N. apis alone. Similar to medicate” when confronted with certain infectious our findings, Gilliam and Taber (1991) reported agents such as the fungal pathogen Ascophaera very low N. apis spore counts in a feral honey bee apis (Simone-Finstrom and Spivak 2012) could population in Arizona, with only 42 % of sampled potentially be a well-suited strategy used by feral colonies testing positive for N. apis spores. More honey bees to combat Nosema infections. recently, Szalanski (2014) explored the preva- However, this remains to be investigated. lence of N. apis in feral Africanized honey bees The overall low levels of Nosema infection in in five different states, including Texas. They the WWR population of feral honey bees might in found that less than 10 % of the samples tested part be due to the time of year when samples were positive for N. apis , with infections being more collected. In our study, Nosema levels were mea- common in Texas than in any other state. These sured from samples collected in July, which is one results coincide with the first documented records of the hottest months of the year in Texas. of Nosema infections in feral honey bees by Incidentally, this region of the USA suffered one Bailey (1958, p. 93), who stated that “no firm of the most devastating droughts recorded in re- general conclusion about disease in wild colonies cent history in 2011 (Combs 2012), which likely can be drawn from the evidence given above, but influenced the colony-level dynamics of Nosema its incidence usually seems to be low relative to in feral honey bees at the WWR for our 2013 that in beekeepers’ colonies.” Likewise, Ratnieks sampling period. More studies are needed to de- and Nowakowski (1989) reported no incidence of termine the seasonal changes in Nosema levels in any disease in feral honey bee swarms. this population during the entire year, particularly 568 J. Rangel et al. in the fall, and if the likelihood of Nosema infec- ACKNOWLEDGMENTS tion is correlated with weather, as was found previously in other regions (Doull and Cellier We thank Chris Garza, Kyle Harrison, Tuula 1961, Cantwell and Shimanuki 1970,Manning Kantola, Rong Ma, Cassandra Sanabria, Elizabeth et al. 2007). Such studies have shown that Walsh, and Michael Wong, for their help in locating Nosema levels are lowest during the summer and collecting honey bee samples from tree cavities at months, while they go up in spring or autumn the WWR in 2013. We are also grateful to Dr. Maria Tchakerian and Tuula Kantola for their help in creating (Doull and Cellier 1961, Manning et al. 2007). maps with GPS coordinates to locate tree cavities at the The endogenous life cycle of N. ceranae has WWR. We thank WWR director Dr. Terry Blankenship been shown to be better adapted to withstand abrupt and assistant director Dr. Selma Glasscock for their temperature changes, supporting the observed epi- generosity in providing lodging and logistical support demiology of this microsporidian (Martín- to our field team. We thank Grace Mulholland for her Hernández et al. 2009). A recent study tested the technical assistance during the molecular analysis of survival capability of N. ceranae in different ex- samples. This project was funded in part by a startup treme temperature and desiccation settings (Fenoy fund to J. Rangel by Texas AgriLife Research Hatch et al. 2009). The authors found that compared to Project TEX09557, and by a National Science Founda- control spores that were kept at 4 °C, about 90 % of tion REU-EXITE grant to C. Sanabria, E. Walsh, and N .ceranae spores heated at 60 °C for 6 h remained M. Wong (Grant no. 1062178). Funding for B. Traver was provided by the Agriculture and Food Research viable. Furthermore, Cantwell and Shimanuki Initiative Competitive Grant no. 2010-67012-19924 (1970) heated combs at 48.9 °C for 24 h and noted from the US Department of Agriculture National Insti- areductioninNosema spore counts from tute of Food and Agriculture. 2.4 million spores/bee in unheated combs, to 1.2 million spores/bee in heated combs. Interestingly, heated combs subsequently exhibited Présence et niveau d 'infestation des espèces de Nosema an increase in honey production. Although not dans une population d 'abeilles sauvages : une étude sur clearly stated in the methods, the authors most likely 20 ans measured N. apis levels, as they do not mention the molecular tools needed to assess N. ceranae levels. Apis mellifera / abeilles africanisées sauvages / Nosema apis Nosema ceranae Combined, results from both studies suggest that / / PCR quantitative / Texas N. ceranae can tolerate the highest temperatures Prävalenz von Nosemaartenineinerwildlebenden encountered in the summer at the WWR, which Honigbienenpopulation : Eine 20 - jährige might be one of the reasons why it may have Langzeituntersuchung outcompeted N. apis in this system, especially when faced with increasing temperatures. Apis mellifera / Afrikanisierte Bienen / Nosema apis / In conclusion, our study is the first to report the Nosema ceranae / qPCR levels of both N. apis and N. ceranae in a feral honey bee population in the USA over a span of 20 years. 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