Molecular detection of Nosema ceranae and Nosema ...
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Molecular detection of Nosema ceranae and Nosema ...
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11th "Seminar of Ecology – 2018" with International Participation 26-27 April 2018, Sofia
Molecular detection of Nosema ceranae and Nosema apis in Bulgarian honey bees
INTRODUCTION "The bee is so busy that she has no time to suffer.“ William Blake
• The Western honey bee (Apis mellifera L., Hymenoptera: Apidae) is a species of crucial importance, because it is main pollinators in the natural environment and it is a producer of bee products. • Nosema is genus of microsporidian intracellular parasite belonging to the kingdom Fungi causing Nosemosis of adult honey bees. • Only two main species of Nosema causing infection in Apis mellifera have been recognized-Nosema apis and Nosema ceranae. • The Nosema ceranae was suspected to one of the contributor to ``Colony Collapse Disorder'' (CCD) described in the United States and Europe.
Techniques for identifying Nosema species: • microscopic methods - Light microscopy, Giemsa and Toluidine staining, and Transmission electron microscopy were introduced. • N. ceranae spores seem to be more sculptured with deeper ornamentation than those of N. apis. • molecular methods - conventional or duplex PCR, PCR-RFLP, qPCR, species-specific PCR primers.
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AIM OF THE STUDY The main goal of the current study is to molecular detection of the presence and distribution of the two different Nosema spp. in the Bulgarian honey bee.
MATERIALS AND METHODS • Sample collection: 108 bee colonies from 3 regions of Bulgaria were tested. - Rousse district (North Bulgaria, N=44) - Sofia district (West Bulgaria, N=28) - Smolyan district (South Bulgaria, N=36) • sampling period – April-May 2017 Figure 1. Map showing samples location.
MATERIALS AND METHODS • DNA extraction • Gene selection
The small subunit (16S) ribosomal RNA gene for molecular identification.
• PCR amplification species-specific PCR primers. • Visualization of PCR products on 2% agarose gel electrophoresis • PCR purification • Sequence analysis
RESULTS • 57 samples out of 108 analyzed (52.8% successful amplifications) • 51 samples failed to produce a PCR product (47.2%) • No PCR products in the negative controls Sequence analysis The obtained sequences were deposited in GenBank under accession number MG657260.
Figure 2. 2 % agarose gels electrophoresis showing the results of duplex PCR reaction. PCR products correspond to N. ceranae; M – molecular marker 100 bp.
RESULTS
RESULTS
Figure 3. Phylogenetics analysis of Nosema ceranae isolates from Bulgaria (GenBank acc. number MG657260)
DISCUSSION • Nosema ceranae displaces Nosema apis in all investigated regions. • N. ceranae is more virulent and thus possesses a competitive advantage in compared to N. apis. It is an invasive species. • US honey bees may be less susceptible to N. ceranae infections than European bees or that the US isolates of the pathogen are less infective and less virulent than European isolates. • N. ceranae infection prevailed in honey bee colonies from the flat part of the country (Rousse and Sofia district), while in the mountainous part (Smolyan district, the Rodopa Mountains) the prevalence was the lowest.
DISCUSSION
Figure 4. Map showing sampling locations in Bulgaria. Nosema species distribution are represented in all Balkan countries
Why Nosema ceranae in Rodopa mountains is with the lowest intensities infection? • Hypothesis 1: Shifting the native subspecies A. m. macedonica with the introduced A. m. ligustica, A. m. carnica and A. m. caucasica. • Hypothesis 2: The Rodopa Mountains climate is rather colder than the other two investigated regions. This determine the later development of honey bee colonies (May-August).
DISCUSSION
Figure 5. Pre-2003 world distribution of N. apis (triangles) and N. ceranae (circle) in honey bees based on (i) our own RFLP analysis (filled symbols, N = 5 isolates; and (ii) sequenced partial rRNA from other sources (open symbols, N = 26 A. mellifera isolates and N = 2 A. cerana isolates. The host is A. mellifera except for *(A. cerana) and **(A. mellifera and A. cerana).
0022-2011/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.02.01
CONCLUSION This is the first report of the distribution of N. ceranae of honey bee colonies in Bulgaria. We found that N. ceranae is the dominant species in the Bulgarian honey bee. A local honey bee A. m. rodopica reared in the Rodopa Mountains seems to be more resistant in comparison to the introduced species. Because of this, local honey bees should be kept as a part of the genetic diversity and the related conservation activities. Shumkova et al. (2018), The first report of the prevalence of Nosema ceranae in Bulgaria. PeerJ 6:e4252; DOI:10.7717/peerj.4252
THANK YOU FOR ATTENTION Acknowledgments We thank the National Science Fund of the Bulgarian Ministry of Education and Science, Sofia (grant number 06/10 17.12.2016).
According to Albert Einstein “If the bee disappeared off the surface of the glob then man would only have four years of life left. No more bees, no more pollination, no more plants, no more animals, no more man”.
