Journal of Invertebrate Pathology 101 (2009) 150–153
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Short Communication
Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay Ciro Invernizzi a,*, Carolina Abud a, Ivanna H. Tomasco a, Jorge Harriet b, Gustavo Ramallo c, Juan Campá b, Helena Katz b, Gabriela Gardiol b, Yamandú Mendoza c a
Facultad de Ciencias, Iguá 4225, CP 11400, Montevideo, Uruguay Dirección de Laboratorios Veterinarios, Ruta 8 Km. 17.500, CP 12100, Montevideo, Uruguay c Instituto de Investigación Agropecuaria, Ruta 11 Km, CP 70000, Colonia, Uruguay b
a r t i c l e
i n f o
Article history: Received 14 November 2008 Accepted 31 March 2009 Available online 7 April 2009 Keywords: Honeybee Apis mellifera Nosemosis Microsporidia Nosema apis Nosema ceranae PCR–RFLP Uruguay
a b s t r a c t The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR–RFLP diagnosis, 29 samples of infected honeybees obtained in 2007–2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant. Ó 2009 Elsevier Inc. All rights reserved.
For several decades, the only known causal agent of Nosemosis in European honeybees (Apis mellifera) was the unicellular microsporidium Nosema apis (Zander, 1909). This pathogen reproduces obligately in the epithelial cells of the ventriculus, severely affecting digestive functions, which leads to denutrition, physiological aging and reduction in bee longevity. As a result, the affected colonies diminish honey production (Fries, 1997; Hornitzky, 2008). In 2006, Higes et al. reported a new microsporidium, Nosema ceranae, as the main etiological agent of Nosemosis in Spain. Shortly after, the presence of N. ceranae was confirmed in Europe, America and Asia (Fries et al., 2006; Chauzat et al., 2007; Cox-Foster et al., 2007; Klee et al., 2007; Huang et al., 2007; Paxton et al., 2007; Chen et al., 2008; Sarlo et al., 2008; Williams et al., 2008). N. ceranae was detected in 1994 in its original host, the Asian honeybee Apis cerana, by Fries et al. (1996). The dispersion of N. ceranae to diverse regions of the world has been occurring for over 10 years: the species has been in Europe (Finland) since 1998 (Paxton et al., 2007) and in the USA since 1995 (Chen et al., 2008), at least. The appearance of this new microsporidium has caught the attention of beekeepers and researchers because it could be the cause of the high colony loss in Europe (Higes et al., 2005; Chauzat et al., 2006) and in the USA (a phenomenon known as Colony Collapse Disorder, CCD) (Oldroyd, 2007). In this context, the virulence * Corresponding author. Tel.: +598 2 525 8618; fax: +598 2 525 8617. E-mail address:
[email protected] (C. Invernizzi). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.03.006
of N. ceranae was experimentally demonstrated in laboratory tests (Higes et al., 2007; Paxton et al., 2007) as well as in natural field conditions (Higes et al., 2008). In Spain, from 1999 to 2005, Martín-Hernández et al. (2007) found two characteristics of Nosemosis, (1) a marked increase in prevalence and (2) a loss in the seasonality, that are likely associated to N. ceranae. The prevalence and distribution of this species in South America is far from being understood. The presence of N. ceranae has been verified in bees from São Paulo, Brasil (Klee et al., 2007) and the southeast of Provincia de Buenos Aires, Argentina (Sarlo et al., 2008). In Uruguay, historically, Nosemosis has been recognized by the presence of Nosema spores through light microscopy, assuming N. apis to be the causal agent. The goal of this study was to determine which species of Nosema have been present in Uruguay in a recent past. A total of 29 samples of bees were analyzed: 26 from 2007 to 2008, two from 2004 and one deposited in Apiculture Laboratory of the Administration of Veterinary Laboratories (DILAVE) with no exact date of collection but dating back to before 1990. Samples were obtained from colonies with Nosemosis, determined by observation of spores at the light microscope, from diverse regions of Uruguay (Fig. 1). The sample taken before 1990 is of unknown geographic origin within Uruguay. Samples were deposited in the tissues collection of Laboratorio de Evolución in Facultad de Ciencias. Bee abdomens were homogenized in a mortar and suspended in ethanol 95°. DNA extraction was done from 30 ll of the pellet
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Fig. 1. Geographic origin of samples of bees infected with Nosema ceranae. The color of the circles show the year the sample was obtained and the number inside them indicates the number of samples used. Grey: 2007 and 2008. Black: 2004. An additional sample taken before 1990 and of unknown geographic origin was used.
obtained after 1 min of centrifugation at 14,000 rpm, using standard protocols. The diagnosis of Nosema species was done by PCR/RFLP, as suggested by Klee et al. (2007) with minor modifications. PCR products were separated by polyacrylamide gel electrophoresis and visualized with nitrate silver staining. Additional bands were obtained in some PCR runs but never interfered with the diagnosis. Negative and positive controls were used in all PCR and enzyme digestion procedures. Samples infected with N. ceranae, N. apis and co-infected with both species (provided by Dr. M. Higes from Centro Apícola Regional de Marchamalo, Spain) were used as positive controls. To verify the species diagnosis obtained through the previous procedure, three PCR products were sequenced (Macrogen Inc.) and compared to sequences reported in Genbank, as well as those reported by Klee et al. (2007; Genbank accession numbers U26534 and DQ078785). Diagnosis through enzyme digestion was confirmed with the sequences obtained, which corresponded to N. ceranae. All the samples analyzed were positive for N. ceranae, clearly showing its current presence in different regions of Uruguay (Fig. 1), and indicating that the pathogen has been in the country for at least 18 years. On the other hand, all the samples tested negative for N. apis suggesting that the species is not in the country, it is not very prevalent or there is a non-reported polymorphism (at high frequency) that results in false negative result. Assuming that only N. apis was originally present, it seems plausible that N. ceranae has replaced N. apis at some time before 1990. Some recent findings seem to support the idea that N. ceranae has replaced N. apis in different parts of the world. In the southeast of Provincia de Buenos Aires (Argentina), close to the border with Uruguay, only 2 of the 14 localities sampled had bees co-infected with N. ceranae and N.
apis, while N. ceranae was found in all the remaining localities (Sarlo et al., 2008). Furthermore, in a time series of infected bee samples from Finland, it was found that N. ceranae increased its frequency in relation to N. apis since its detection in 1998, and in the present, colonies are infected exclusively by N. ceranae or coinfected by both species (Paxton et al., 2007). In our case, the samples analyzed are not enough to determine when and how this replacement would have occurred. In Uruguay, the impact of N. ceranae in colony loss is not clear. The DILAVE has records of Nosemosis from samples of bees sent by beekeepers since 1964. Until 2007, 61,916 samples were analyzed (no. samples/year = 127–3487) (Fig. 2). According to such records, the highest incidence of Nosemosis was observed from 1964 to 1967 with over 40% of positive samples. Values decreased in the following years, until reaching an average value lower than 10% for the period 1978–1983. The second period of highest incidence was from 1998 to 2002 with over 35% of positive samples. Since then, values have remained under 30%. It can be observed that Nosemosis has not increased steadily since 1990 (since when the presence of N. ceranae is certain). Such behavior of the disease is very different from the epidemiological pattern found in Spain between 1999 and 2005, characterized by a noticeable increase of 111 positive samples, surpassing 90% of the total (Martín-Hernández et al., 2007) or that colonies even die within a year from being infected, if not treated with fumagillin (Higes et al., 2008). Some episodes of high colony losses have happened in Uruguay in autumn and winter, but only in some years and affecting preferentially some regions of the country. One of the characteristics of the infected apiaries is the depopulation of colonies with abandonment of the brood and the reserves of honey and pollen. This
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Positive samples (%)
60
50
40
30
20
10
0 64 966 968 970 972 974 976 978 980 982 984 986 988 990 992 994 996 998 000 002 004 006 2 2 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1
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Year Fig. 2. Proportion of samples infected with Nosemosis in Uruguay from 1964 to 2007. The oldest sample diagnosed as Nosema ceranae in this study was sampled 1990 or earlier.
aspect of the symptoms resembles very much those described in colonies infected by N. ceranae in Europe (Higes et al., 2008) and in the USA (Oldroyd, 2007). Nevertheless, there is not a marked presence of Nosema in these colonies, and losses seem to be more strongly associated with the difficulty in controlling the mite Varroa destructor (e.g. problems of resistance to acaricids) because when mites are efficiently eliminated from colonies, winter losses are minimized. Nosemosis does appear in colonies moved to forestations of Eucalyptus spp. in autumn. If colonies are not retrieved immediately after the flowering period has finished, beekeepers have important colony losses. In such cases, Nosemosis can be the main agent responsible for the death of colonies (Invernizzi et al., 2005). The fact that the high virulence of N. ceranae in some parts of the world (Martín-Hernández et al., 2007; Higes et al., 2008) has not been reported in Uruguay, at least as observed by beekeepers, may be due to several factors. First, it is possible that N. ceranae act in association with different viruses, an analogous situation to that of the mite V. destructor (Shen et al., 2005; Chen and Siede, 2007). The complex Nosema virus could vary between regions, affecting bees differentially. Second, different strains of N. ceranae might differ in virulence. Williams et al. (2008) found two haplotypes in samples from North America, Europe and Asia, which might be characterized by different levels of virulence. Other factors that might affect the virulence of N. ceranae are bee race and the time they have been exposed to the pathogen. All these aspects remain to be further studied. This is the first diagnosis at the species level of the genus Nosema in Uruguay, which is key for any posterior epidemiological study. Acknowledgments We are grateful to Dr. Mariano Higes for providing samples of bees infected with N. ceranae and N. apis. We would like to thank beekeepers who provided samples. We thank Gabriela Wlasiuk for improving this manuscript and the three anonymous reviewers for their comments. This study was funded by Instituto Nacional de Investigación Agropecuaria (INIA).
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