Intracellular endosymbiont, Wolbachia spp., have been previously reported in many different orders of insects, but not in the European honeybee sub species Apis mellifera carnica. We first report the presence of Wolbachia spp. in the honeybee sub spp. Apis mellifera carnica of the order Hymenoptera. Using standard polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in honeybee Queen and workers collected from different parts of Germany. Results of the study revealed 100 infection status of Wolbachia in Apis mellifera carnica collected from different parts of Germany, confirming that the Wolbachia spp. were invariably present in the honeybee Queen and the workers screened. Â© IDOSI Publications, 2011

Brueckner, D.

Junier, P.

Pattabhiramaiah, M.

Reddy, M.S.

Witzel, K.P.

Intracellular endosymbiont, Wolbachia spp., have been previously reported in many different orders of insects, but not in the European honeybee sub species Apis mellifera carnica. We first report the presence of Wolbachia spp. in the honeybee sub spp. Apis mellifera carnica of the order Hymenoptera. Using standard polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in honeybee Queen and workers collected from different parts of Germany. Results of the study revealed 100% infection status of Wolbachia in Apis mellifera carnica collected from different parts of Germany, confirming that the Wolbachia spp. were invariably present in the honeybee Queen and the workers screened

Witzel, K.

Reddy, M.

MPG.PuRe

World Applied Sciences Journal 15 (11): 1503-1506, 2011ISSN 1818-4952© IDOSI Publications, 2011Corresponding Author: Mahesh Pattabhiramaiah, Honeybee Research Unit, University of Bremen, FB 2, 28334, Bremen, Germany. 1503Prevalence of Wolbachia in the European Honeybee, Apis mellifera carnicaMahesh Pattabhiramaiah, Dorothea Brueckner, Karl Paul Witzel, Pilar Junier and M.S. Reddy1   2   3    4    5Honeybee Research Unit, University of Bremen, FB 2, 28334, Bremen, Germany1,2Max Planck Institute for Evolutionary Biology, Ploen, Germany3,4Department Of Zoology, Centre for Apiculture Studies, Bangalore University,5Jnana Bharathi, Bangalore-560056, IndiaAbstract: Intracellular endosymbiont, Wolbachia spp., have been previously reported in many different ordersof insects, but not in the European honeybee sub species Apis mellifera carnica. We first report the presenceof Wolbachia spp. in the honeybee sub spp. Apis mellifera carnica of the order Hymenoptera. Using standardpolymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp.in honeybee Queen and workers collected from different parts of Germany. Results of the study revealed 100%infection status of Wolbachia in Apis mellifera carnica collected from different parts of Germany, confirmingthat the Wolbachia spp. were invariably present in the honeybee Queen and the workers screened.Key words: Wolbachia % Apis mellifera carnica % 16S ribosomal RNA gene % PCRINTRODUCTION Although routinely transmitted vertically, WolbachiaThe  "-proteobacterium  Wolbachia  pipientis is even between different orders of insects. These verticallyprobably the most common endosymbiont in the world, transmitted symbionts may thus affect the expression andinfecting an estimated 25- 76% of all insect species, as regulation of reproductive conflict in insect societies.well  as  many  other  arthropod and filarial nematode Rather little effort has been undertaken to estimate thespecies [1, 2]. This alphaproteobacteria was first total diversity of this endosymbiont associated with thesedescribed by Hertig & Wolbach (1924) in the mosquito social insects. The limits of their distribution are currentlyCulex pipiens. Later, Hertig (1936) named them unknown. The studies available today suggest thatWolbachia pipientis. surveys of this kind are rewarding because, a number ofWolbachia pipientis is well known for inducing a interesting specific associations between social insectsvariety of reproductive abnormalities in the diverse and microbial symbionts have recently been discoveredarthropod hosts which include- altering sex ratio, and analyzed for their co-evolutionary interactions withfeminization, induction of cytoplasmic incompatibility, molecular tools. Despite their common occurrence andinducing thelytoky [5-8], male killing and parthenogenesis major effects on host biology, little is currently knownin at least 40 species of Hymenoptera [8] to enhance its about the molecular mechanisms that mediate theown inheritance from mother to daughter. Wolbachia has interactions between Wolbachia and their invertebratebeen found in numerous species of Hymenoptera, hosts.including parasitoids and ants [2, 8-11]. It can also confer Because Wolbachia are fastidious bacteria thatfitness benefits on its host [12] and increase host cannot be cultured outside host cells, it can be detectedresistance to infection with a range of pathogens [13-17]. using modern molecular techniques such as PCR.Wolbachia's remarkable biology and its potential Molecular biological techniques are dramatically changingapplication for control of vector-borne diseases such as our view of microbial diversity in almost any environmentdengue fever, malaria or filariasis [16, 18] have led to that has so far been investigated. In a PCR-basednumerous genome sequencing projects aimed at screening study, Werren et al., [19] found that over 16%understanding Wolbachia's evolution and the genetic of insect species in their sample were infected withbasis of the phenotypes it induces. Wolbachia. In a study using similar methods, West et al.,has also undergone extensive intertaxon transmission,World Appl. Sci. J., 15 (11): 1503-1506, 20111504[20] revealed that 22% of British insects were infected. PCR Protocols: Standard PCR was performed by a hotThe latter screening study has indicated that the start method in a 25 µl reaction volume containing 1 µlprevalence may even be underestimated and that DNA sample, 1 µl forward and reverse primers, 5 µl 10XWolbachia infection levels are as high as 76% of all insect Buffer containing 15 mM of MgCl , 1 unit of Taqspecies [21]. polymerase (Roche) and 1 µl dNTPs (10mM). DeionizedAlthough the diversity of Wolbachia infection in the MilliQ water was added to a final volume of 25 µl. Thehoneybee spp. A. m. capensis and A. m. scutellata have PCR reaction mix was prepared in one batch and thenbeen well investigated [21] in African honeybee, there is added to each sample. A sample containing deionizeda lack of such investigation in the subspecies of European water in place of template DNA was included in allhoneybee Apis mellifera carnica of Germany. Until now, reactions as a negative control. PCR amplification wasthere are no reports on the existence of Wolbachia in the done on a Master Cycler Gradient (Eppendorf) under thesubspecies of European honeybee Apis mellifera carnica. following thermal profile: 95°C for 2min @ 1 cycle, 95°C forTo address this issue, PCR based screening was carried 30sec @ 30 cycles, 40°C for 30sec @ 30 cycles, 73°C forout for the first time in the Queen and workers of honey 3min @ 30cycles and an extension cycle of 73°C for 1minbee subspecies Apis mellifera carnica collected from @ 1cycle.Bremen and Ploen, Germany. Keeping all these in view, it The amplified products were detected by running awas thought to screen the bee species for the presence or 1.5 percent agarose gel (TAE buffer) with a 1kb molecularabsence of Wolbachia by means of PCR. weight  marker.  Gels  were  stained  with  ethidiumMATERIALS AND METHODS (Fig. 1 and Fig. 2).Colony Sources: A honeybee queen of the subspecies fresh reagents were used to avoid contamination.viz. Apis mellifera carnica and its workers were collected Negative controls (consisting of all components exceptfrom hives at the honeybee research unit, Bremen the DNA template) were conducted on each date to detectUniversity, Bremen and Ploen, Germany. The samples potential contamination, but positive controls were notwere collected in 95% ethanol and stored at -80°C in a carried out to reduce the likelihood of contamination.deep freezer prior to DNA extraction.DNA Extraction: DNA extraction and PCR amplificationwas carried out at the Max Planck Institute for The result of PCR (Fig. 1 and Fig. 2) using theEvolutionary Biology, Ploen, Germany. Single honeybee Wolbachia primers (Table 1) yielded 100% amplificationQueen and worker bees were used for DNA extraction. in all samples of Apis mellifera carnica collected fromThe whole abdomen was cut off from the bees using a different parts of Germany, yielding an amplicon of ~1100new single-edged razor blade for each bee and the internal bp which corresponds to Wolbachia. The result of thetissues were removed for extraction of genomic DNA. study showed that the amplifiable bacteria were invariablyTotal genomic DNA was extracted from the abdomen of present in the honeybee Queen (Fig. 1, Lane-2) and itsan individual bee using Aquapure Genomic DNA kit workers, collected from different colonies of Bremen,(catalog number 7326343) reagents following the Germany (Fig. 1, Lane 2-12). The primers invariablyprocedure suggested by the manufacturer and the amplified the 16s region of the prokaryotes which isgenomic  DNA  was  resuspended in 50 µl of sterile water. highly conserved for Wolbachia. Lane-13 showedFor PCR amplification 1 µl of genomic DNA was used. negative for PCR, indicating there was no contaminationWolbachia specific primers (eub primers in Table 1) in the PCR carried out.were used to amplify the 16S rRNA gene of the bacteria The result of PCR (Fig. 2, Lane 2-12) using thepresent  in  the  total genomic DNA, extracted from each Wolbachia primers (Table 1) yielded 100% amplificationof Queen and worker honeybee. in all samples of Apis mellifera carnica collected fromTable 1: 16S rRNA Wolbachia specific primersName of the primer SequenceEu27.F 5'- gagagtttgatcctggctcag-3'Eu1495.R 5'- ctacggctaccttgttacga-3'2bromide  and  bands visualized under UV illuminationClean laboratory practices, sealed pipette tips andRESULTS AND DISCUSSIONPloen, Germany, yielding an amplicon of ~1100 bp. Theresult of the study showed that the amplifiable bacteriawere invariably present in the honeybee workers,collected from Ploen, Germany. The primers invariablyamplified  the  16s   region   of  the  prokaryotes   which  is  World Appl. Sci. J., 15 (11): 1503-1506, 201115051 2 3 4 5 6 7 8 9 10 11 12 13 A PCR assay based on 16S primers was designed forFig. 1: PCR amplification using Wolbachia 16srRNA fromthe Queen, workers of the honeybee subspeciesApis mellifera carnica, collected from colonies ofhoneybee research unit, Bremen, GermanyLane 1 Mol. wt marker, 2 Honeybee Queen collected from honeybeeresearch unit, Bremen University, Germany.3-12 Worker honeybees collected from differentcolonies of honeybee research unit, BremenUniversity, Germany.13 Negative Control respectively1 2 3 4 5 6 7 8 9 10 11 12 13Fig. 2: PCR amplification using Wolbachia 16srRNA fromthe workers of honeybee subspecies Apismellifera carnica collected from different coloniesof Ploen, GermanyLane 1 Mol. wt marker, 2-12 Worker honeybees of the subspecies Apismellifera carnica collected from differentcolonies from Ploen, Germany. 13 Negative Control respectivelyhighly conserved. Lane-13 showed negative for PCR,clearly indicating there was no contamination in the PCRcarried out.the detection of W. pipientis in honeybee tissue and initialscreening of honeybees indicates that 100% of thesamples screened were positive for Wolbachia infectionand may induce parthenogenesis in the Hymenopteraninsects. The presence of Wolbachia in Apis melliferacarnica indicates a long-term and stable associationbetween the two and may increase fecundity or have noobvious effect at all.The  inability  to  grow  these  bacteria on definedcell-free medium has been the major factor underlyingthese uncertainties. We circumvented this problem byselective PCR amplification of the symbiont 16S rRNAgenes directly from infected insect tissue.The usual transmission of Wolbachia is vertical, fromthe mother to her offspring through the eggs, butoccasional horizontal transfers between individuals,which may or may not belong to the same species, seemto occur. It is evident from the PCR results (Fig. 1, Lane-2)that Wolbachia is present in honeybee Queen and in itsworkers (Fig. 1, Lane-3-12) indicating that verticaltransmission of Wolbachia is more common. Thehoneybee Queen and the workers of the sub spp. Apismellifera carnica were found to harbour a Wolbachiastrain closely related to the Wolbachia symbiont,showing that vertical transmission is taking place betweenthem. The finding of closely related symbiont in speciessharing only the same habitats suggests that verticaltransfers of Wolbachia could occur more often inhymenopteran insects.This obligatory symbiotic relationship is evident fromseveral reports of Marjorie et al., [22], who established thepresence of Wolbachia in honeybee species, Apismellifera capensis and Apis mellifera scutellata.Through this preliminary survey of naturallyoccurring Wolbachia infection types in Europeanhoneybee Apis mellifera carnica have been identified.However, Wolbachia infection in other species and racesof European and Asian honeybee needs to beinvestigated to understand the variation in Wolbachiainfection. Such studies will provide basic descriptiveinformation about the Wolbachia and its interactionbetween the hosts.REFERENCES1. Hilgenboecker, K., P. Hammerstein, P. Schlattmann,A. Telschow and J.H. Werren, 2008. How manyspecies are infected with Wolbachia? A statisticalanalysis of current data. Fems Microbiology Letters,281: 215-220.World Appl. Sci. J., 15 (11): 1503-1506, 201115062. Jeyaprakash, A. and M.A. Hoy, 2000. Long PCR 14. Hedges,  L.M.,  J.C.  Brownlie,    S.L.   O'Neill  andimproves Wolbachia DNA amplification: wsp K.N. Johnson, 2008. Wolbachia and virus protectionsequences found in 76% of sixty-three arthropod in insects. Sci., 322: 702.species. Insect Molecular Biol., 9: 393-405. 15. Kambris, Z., P.E. Cook, H.K. Phuc and S.P. Sinkins,3. Hertig, M. and S.B. Wolbach, 1924. Studies on 2009. Immune activation by lifeshortening WolbachiaRickettsia-like microorganisms in insects. J. Medical and reduced filarial competence in mosquitoes. Sci.,Res., 44: 329-374. 326: 134-136.4. Hertig, M., 1936. The Rickettsia Wolbachia pipientis 16. Moreira, L.A., I. Iturbe-Ormaetxe, J.A. Jeffery, G.J. Lu,(gen. and sp. nov.) and associated inclusions in the A.T. Pyke, L.M. Hedges, B.C. Rocha, S. Hall-mosquito, Culex pipiens. Parasitol., 28: 453-486. Mendelin,   A.    Day,    M.    Riegler,   L.E.  Hugo,5. Rigaud, T. and F. Rousset, 1996. What generates the K.N. Johnson, B.H. Kay, E.A. McGraw, A.F. Vandiversity of Wolbachia-arthropod interactions? DenHurk, P.A. Ryan and S.L. O'Neill, 2009. ABiodiv Conserv, 5: 999-1013. Wolbachia symbiont in Aedes aegypti limits infection6. O’Neill, S.L., M.M. Pettigrew, S.P. Sinkins,  H.R. with Dengue, Chikungunya and Plasmodium. Cell,Braig, T.G. Andreadis and R.B. Tesh, 1997. In vitro 139: 1268-1278.cultivation of Wolbachia pipientis in an Aedes 17. Teixeira, L., A. Ferreira and  M.  Ashburner,  2008.albopictus  cell  line.  Insect Molecular Biol., 6: 33-39. The bacterial symbiont Wolbachia induces7. Werren, J.H., 1997. Biology of Wolbachia. Annual resistance to RNA viral infections in DrosophilaReview of Entomol., 42: 587-609. melanogaster. PLoS Biol., 6: 2753-2763.8. Cook, J.M. and R.D.J. Butcher, 1999. The 18. Sinkins, S.P. and  S.L.  O'Neill,  2000.  Wolbachia  astransmission  and   effects   of   Wolbachia  bacteria a vehicle to modify insect populations. Insectin  parasitoids.  Researches  on   Population   Ecol., Transgenesis, pp: 271-287.41: 15-28. 19. Werren, J.H., W. Zhang and L.R. Guo, 1995.9. Stouthamer, R., 1997. Wolbachia induced Evolution and phylogeny of Wolbachia:parthenogenesis,  In  S.C.  O’Neil,  J. Werren and reproductive  parasites of arthropods. Proc. R. Soc.A.A. Hoffman, (eds.), Influential passengers, B., 261: 55-63.inherited microorganisms and arthropod 20. West, S.A., M. Cook, J.H. Werren and H.C.J. Godfray,reproduction. Oxford University Press, 226: 102-124. 1998. Wolbachia in two insect host-parasitoid10. Shoemaker, D.D., K.G. Ross, L. Keller, E.L. Vargo and communities. Mol. Ecol., 7: 1457-1465.J.H. Werren, 2000. Wolbachia infections in native 21. Jeyaprakash, A., M.A. Hoy and M.H. Allsopp, 2003.and introduced populations of fire ants (Solenopsis Bacterial diversity in worker adults of Apis melliferaspp.). Insect Molecular Biol., 9: 661-673. capensis and Apis mellifera scutellata (Insecta:11. Wenseleers, T., F. Ito, S. Van Borm, et al., 1998. Hymenoptera) assessed using 16S rRNA sequences.Widespread occurrence of the micro-organism J. Invertebr. Pathol., 84: 96-103.Wolbachia in ants. Proceedings of the Royal Society 22. Marjorie, A. Hoy,  Ayyamperumal  Jeyaprakash,of London Series B, Biological Sci., 265: 1447-1452. Juan, M. Alvarez and Michael, H. Allsopp, 2003.12. Brownlie, J.C., B.N. Cass, M. Riegler, J.J. Witsenburg, Wolbachia  is  present  in  Apis  mellifera capensis,I. Iturbe-Ormaetxe, E.A. McGraw and S.L. O'Neill, A. m. scutellata and their hybrid in southern Africa.2009. Evidence for metabolic provisioning by a Apidologie, 34: 53-60.common invertebrate endosymbiont, Wolbachiapipientis, during periods of nutritional stress. Plos.Pathogens, pp: 5.13. Bian, G.W., Y. Xu, P. Lu, Y. Xie and Z.Y. Xi, 2010. Theendosymbiotic bacterium Wolbachia inducesresistance to Dengue virus in Aedes aegypti. PlosPathogens, pp: 6.

English

Prevalence of Wolbachia in the European Honeybee, Apis mellifera carnica

ePrints@Bangalore University

Prevalence of Wolbachia in the European honeybee, Apis mellifera carnica

Intracellular endosymbiont, Wolbachia spp., have been previously reported in many different orders
of insects, but not in the European honeybee sub species Apis mellifera carnica. We first report the presence
of Wolbachia spp. in the honeybee sub spp. Apis mellifera carnica of the order Hymenoptera. Using standard
polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp.
in honeybee Queen and workers collected from different parts of Germany. Results of the study revealed 100%
infection status of Wolbachia in Apis mellifera carnica collected from different parts of Germany, confirming
that the Wolbachia spp. were invariably present in the honeybee Queen and the workers screened

Prevalence of Wolbachia in the European honeybee, Apis mellifera carnica

Abstract

Similar works

Full text

Available Versions

MPG.PuRe

ePrints@Bangalore University

Max Planck Society eDoc Server

MPG.PuRe