https://doi.org/10.4081/jear.2022.10316

Infestation of Apis mellifera workers and larvae with Varroa destructor affects gut bacterial diversity

Vol. 54 No. 1 (2022)
Submitted: 6 December 2021
Accepted: 1 February 2022
Published: 1 August 2022
Apis mellifera, midgut bacteria, Varroa destructor, 16S rRNA
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Authors

F.F. Abuldahab
Entomology Department, Faculty of Science, Benha University, Benha, Egypt.
M.F. Abd-ElAziz
Entomology Department, Faculty of Science, Benha University, Benha, Egypt.
Y.A. El-Sayed
Entomology Department, Faculty of Science, Benha University, Benha, Egypt.
M.S. Abdallah
Entomology Department, Faculty of Science, Benha University, Benha, Egypt.
S.H. Mahmoud
dr.shaymaahussein@science.menofia.edu.eg
Zoology Department, Faculty of Science, Menoufia University, Menoufia, Egypt.

The present work aims to investigate the midgut bacteria of honey bee (Apis mellifera lamarckii) workers and larvae infested with Varroa destructor. To achieve this task, uninfested and infested honey bee larvae and workers with the parasitic mites were obtained from natural bees apiaries in Qaliubiya Governorate, Egypt. Aerobic bacteria were isolated from the midgut of uninfested and infested A. mellifera on nutrient agar medium and identified using a culture-dependent isolation along with 16S rRNA sequences. The results revealed that both uninfested and V. destructor infested workers and larvae harbored gut bacteria belonging to Gamma-proteobacteria strains. The uninfested workers contained Enterobacter cloacae and Serratia nematodiphila, while V. destructor infested workers harbored Enterobacter species. Uninfested larvae contained E. hormaechei and Klebsiella variicola. V. destructor infested larvae harbored K. oxytoca as a result of infection. We concluded that the honeybee microbiome composition is likely to be impacted through isolates variation by stage and diversity disruption of isolates by V. destructor infection.

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AHN J.H., HONG I.P., BOK J.I., 2012 - Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. - J Microbiol. 50: 735–745. DOI: https://doi.org/10.1007/s12275-012-2188-0
ALATAWY M., AL-ATTAS S.G., ASSAGAF A.I., AL-SHEHRI A., ALGHAMDI K.M., BAHIELDIN A., 2020 - Gut microbial communities of adult honey bee workers (Apis mellifera). - Biosci. Biotech. Res. Asia. 17: 353-362. DOI: https://doi.org/10.13005/bbra/2838
ANAND A.A.P., VENNISON S.J., SANKAR S.G., PRABHU D.I.G., VASAN P.T., RAGHURAMAN T., GEOFFREY C.J., VENDAN S.E., 2010 - Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion. – J. Insect Sci. 10: 107. DOI: https://doi.org/10.1673/031.010.10701
ANDERSON K.E., SHEEHAN T.H., MOTT B.M., MAES P., SNYDER L., SCHWAN M.R. WALTON A., JONES B.M., CORBY-HARRIS V., 2013 - Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera). – PLoS One. 8: e83125. DOI: https://doi.org/10.1371/journal.pone.0083125
ANJUM S.I., SHAH A.H., AURONGZEB M., KORI J., AZIM M.K., ANSARI M.J., BIN L., 2018 - Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan. - Saudi J Biol Sci. 25: 388-392. DOI: https://doi.org/10.1016/j.sjbs.2017.05.008
ARIAS-CORDERO E., PING L., REICHWALD K., DELB H., PLATZER M., BOLAND W., 2012 - Comparative evaluation of the gut microbiota associated with the below- and above ground life stages (larvae and beetles) of the forest cockchafer, Melolontha hippocastani. - PLoS One. 7: e51557. DOI: https://doi.org/10.1371/journal.pone.0051557
BABENDREIER D., JOLLER D., ROMEIS J., BIGLER F., WIDMER F., 2007 - Bacterial community structures in honey bee intestines and their response to two insecticidal proteins. FEMS Microbiol Ecol. 59: 600-610. DOI: https://doi.org/10.1111/j.1574-6941.2006.00249.x
BOUCIAS D.G., CAI Y., SUN Y., LIETZE V.U., SEN R., RAYCHOUDHURY R., SCHARF M.E., 2013 - The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition. Mol. Ecol. 22: 1836-1853. DOI: https://doi.org/10.1111/mec.12230
BRODERICK N.A., RAFFA K.F., GOODMAN R.M., HANDELSMAN J., 2004 - Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture independent methods. - Appl Environ Microbiol. 70: 293–300. DOI: https://doi.org/10.1128/AEM.70.1.293-300.2004
BUCHLER R., 2015 - Varroa tolerance in honey bees occurrence, characters and breeding. - Bee World. 75: 54–70. DOI: https://doi.org/10.1080/0005772X.1994.11099201
CHANDLER D., SUNDERLAND K.D., BALL B.V., DAVIDSON G., 2011 - Prospective biological control agents of Varroa destructor n. sp., an important pest of the European honeybee, Apis mellifera. - Biocontrol Sci Technol. 11: 429-448. DOI: https://doi.org/10.1080/09583150120067472
CHANTAWANNAKUL P., DE GUZMAN L.I., LI J., WILLIAMS G.R., 2016 - Parasites, pathogens, and pests of honeybees in Asia. - Apidologie. 47: 301–3243. DOI: https://doi.org/10.1007/s13592-015-0407-5
CHO G.S., STEIN M., BRINKS E., RATHJE J., LEE W., SUH S.H., FRANZ C.M.A.P., 2020 - Serratia nevei sp. nov. and Serratia bockelmannii sp. nov., isolated from fresh produce in Germany and reclassifica-tion of Serratia marcescenssubsp. sakuensis Ajithkumar et al. 2003 as a later heterotypic synonym of Serratia marcescens subsp. - Marcescens. Syst Appl Microbiol. 43: 126082. DOI: https://doi.org/10.1016/j.syapm.2020.126055
CORBY-HARRIS V., MAES P., ANDERSON K.E., 2014 - The bacterial communities associated with honey bee (Apis mellifera) foragers. - PLoS One. 9: e95056. DOI: https://doi.org/10.1371/journal.pone.0095056
COX-FOSTER D.L., CONLAN S., HOLMES E.C., PALACIOS G., EVANS J.D., MORAN N.A., QUAN P.L., BRIESE T., HORNIG M., GEISER D.M., MARTINSON V., VANENGELSDORP D., KALKSTEIN A.L., DRYSDALE A., HUI J., ZHAI J., CUIN L., HUTCHISON S.K., FREDRIK SIMONS J., EGHOLM M., PETTIS J.S., LIPKIN W.I., 2007 - A metagenomic survey of microbes in honey bee colony collapse disorder. - Science. 318: 283-287. DOI: https://doi.org/10.1126/science.1146498
DILLON R.J., DILLON M., 2004 - The gut bacteria of insects: nonpathogenic interactions. - Annu. Rev. Entomol. 49: 71-92. DOI: https://doi.org/10.1146/annurev.ento.49.061802.123416
DILLON R.J., WEBSTER G., WEIGHTMAN A.J., CHARNLEY A.K., 2010 - Diversity of gut microbiota increases with aging and starvation in the desert locust. - Antonie Van Leeuwenhoek. 97: 69-77. DOI: https://doi.org/10.1007/s10482-009-9389-5
DISAYATHANOOWAT T., YOSHIYAMA M., KIMURA K., CHANTAWANNAKUL P., 2012 - Isolation and characterization of bacteria from the midgut of the Asian honey bee (Apis cerana indica). - J. Apic. Res. 51: 312-319. DOI: https://doi.org/10.3896/IBRA.1.51.4.04
ELATEEK S.Y., ABDEL-RAZIK A.B., IBRAHIM S.A., 2020 - Initial clustering of some Egyptian nematicides Serratia isolates based on their 16S rRNAs gene sequences. - Egypt. J. Genet. Cytol. 49: 59-68.
ENGEL P., MARTINSON V.G., MORAN N.A., 2012 - Functional diversity within the simple gut microbiota of the honey bee. - Proc Natl Acad Sci USA 109: 11002-11007. DOI: https://doi.org/10.1073/pnas.1202970109
ENGEL P., MORAN N.A., 2013 - The gut microbiota of insects diversity in structure and function. - FEMS Microbiol. Rev. 37: 699-735. DOI: https://doi.org/10.1111/1574-6976.12025
EVANS J.D., SCHWARZ R.S., 2011 - Bees brought to their knees: microbes affecting honey bee health. - Trends Microbiol. 19: 614-620. DOI: https://doi.org/10.1016/j.tim.2011.09.003
GASPER J., TERENTJEVA M., KANTOR A., IVANISOVA E., KNAZOVICKA V., KLUZ M., KACANIOVA M., 2017 - Enterobacteriaceae in gut of honey bee (Apis mellifera) and the antibiotic resistance of the isolates. - J. Anim. Sci. Biotechnol. 50: 69-72.
GILLIAM M., 1997 - Identification and roles of non-pathogenic microflora associated with honey bees. - FEMS Microbiol- Lett. 155: 1-10. DOI: https://doi.org/10.1016/S0378-1097(97)00337-6
GILLIAM M., LORENZ B.J., RICHARDSON G.V., 1988 - Digestive Enzymes and microorganisms in honey bees (Apis melifera L.) influence of Streptomycin, Age, Season and Pollen. - Microbios. 55: 95-114.
GOUVEIA C., MARISE D., ASENSI M.D, ZAHNER V., RANGEL E.F., DE Oliveira S.M.P., 2008 - Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae). – Neotrop. Entomol. 37: 597-601. DOI: https://doi.org/10.1590/S1519-566X2008000500016
GUNATHILAKA N., RANASINGHE K., AMARASINGHE D., RODRIGO W., MALLAWARACHCHI H., CHANDRASENA N., 2020 - Molecular characterization of culturable aerobic bacteria in the midgut of field-caught Culex tritaeniorhynchus, Culex gelidus, and Mansonia annulifera mosquitoes in the Gampaha district of Sri Lanka. - BioMed Res. Int. 1-13. DOI: https://doi.org/10.1155/2020/8732473
GUO C., SUN L., KONG D., SUN M., ZHAO K., 2014 - Klebsiella variicola, anitrogen fixing activity endophytic bactrium isolated from the gut of Odontotermes formosanus. – Afr. J. Microbiol. Res. 8: 1322-1330. DOI: https://doi.org/10.5897/AJMR2013.6570
HORTON M.A., OLIVER R., NEWTON I.L., 2015 - No apparent correlation between honey bee forager gut microbiota and honey production. - Peer J. 3: e1329. DOI: https://doi.org/10.7717/peerj.1329
HRONCOVA Z., KILLER J., HAKL J., TITERA D., HAVLIK J., 2019 - In-hive variation of the gut microbial composition of honey bee larvae and pupae from the same oviposition time. - BMC Microbiol. 19: 110. DOI: https://doi.org/10.1186/s12866-019-1490-y
HUBERT J., BICIANOVA M., LEDVINKA O., KAMLER M., LESTER P.J., NESVORNA M., KOPECKY J., ERBAN T., 2017 - Changes in the bacteriome of honey bees associated with the parasite Varroa destructor, and pathogens Nosema and Lotmaria passim. - Microb Ecol. 73: 685–698. DOI: https://doi.org/10.1007/s00248-016-0869-7
INGLIS G.D., YANKE L.J., GOETTEL M.S., 1998 - Anaerobic bacteria isolated from the alimentary canals of alfalfa leafcutting bee larvae. - Apidologie. 29: 327-332. DOI: https://doi.org/10.1051/apido:19980403
JEYAPRAKASH A., HOY M.A., ALLSOPP M.H., 2003 - Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences. - J. Invertebr. Pathol. 84: 96-103. DOI: https://doi.org/10.1016/j.jip.2003.08.007
JONG D.D., JONG P.H.D., GONCALVES L.S., LOSS W., 1982 - Damage to developing worker honeybees from infestation with Varroa Jacobsoni. - J. Apicultural Research. 21: 165-167. DOI: https://doi.org/10.1080/00218839.1982.11100535
KHAN K.A., ANSARI M.J., AL-GHAMDI A., NURU A., HARAKEH S., IQBAL J., 2017 - Investigation of gut microbial communities associated with indigenous honey bee (Apis mellifera jemenitica) from two different eco-regions of Saudi Arabia. - Saudi J. Biol. Sci. 24: 1061-1068. DOI: https://doi.org/10.1016/j.sjbs.2017.01.055
LUDVIGSEN J., RANGBERG A., AVERSHINA E., SEKELJA M., KREIBICH C., AMDAM G., RUDI K., 2015 - Shifts in the midgut/pyloric microbiota composition within a honey bee apiary throughout a season. - Microb. Environ. 30: 235-244. DOI: https://doi.org/10.1264/jsme2.ME15019
MARTIN S.J., 1994 - Ontogenesis of the mite Varroa jacobsoni Oud. in worker brood of the honeybee Apis mellifera L. under natural conditions. - Exp. Appl. Acarol. 18: 87-100. DOI: https://doi.org/10.1007/BF00055033
MARTINSON V.G., MOY J., MORAN N.A., 2012 - Establishment of characteristic gut bacteria during development of the honeybee worker. - Appl. Environ. Microbiol. 78: 2830-2840. DOI: https://doi.org/10.1128/AEM.07810-11
MARTINSON V.G., DANFORTH B.N., MINCKLEY R.L., RUEPPELL O., TINGEK S., MORAN N.A., 2011 - A simple and distinctive microbiota associated with honey bees and bumble bees. - Mol Ecol. 20: 619-628. DOI: https://doi.org/10.1111/j.1365-294X.2010.04959.x
MATTILA H.R., SEELEY T.D., 2007 - Genetic diversity in honey bee colonies enhances productivity and fitness. - Science 317: 362-4. DOI: https://doi.org/10.1126/science.1143046
MCMENAMIN A.J., GENERSCH E., 2015 - Honey bee colony losses and associated viruses. – Curr. Opin. Insect Sci. 8: 121–129. DOI: https://doi.org/10.1016/j.cois.2015.01.015
MILLER C.S., HANDLEY K.M., WRIGHTON K.C., FRISCHKORN K.R., THOMAS B.C., BANFIELD J.F., 2013 - Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments. - PloS One. 8: e56018. DOI: https://doi.org/10.1371/journal.pone.0056018
MOHR K.I., TEBBE C.C., 2006 - Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoida) at an oilseed rape field. – Eviron. Microbiol. 8: 258-272. DOI: https://doi.org/10.1111/j.1462-2920.2005.00893.x
MORAN N.A., HANSEN A.K., POWELL J.E., SABREE Z.L., 2012 - Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees. - PloS One 7: e36393. DOI: https://doi.org/10.1371/journal.pone.0036393
OLOFSSON T.C., VASQUEZ A., 2008 - Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honey bee Apis mellifera. - Curr Microbiol. 57: 356-363. DOI: https://doi.org/10.1007/s00284-008-9202-0
OTTO C.R., ROTH C.L., CARLSON B.L., SMART M.D., 2016 - Land-use change reduces habitat suitability for supporting managed honey bee colonies in the Northern Great Plains. - Proc Natl Acad Sci USA 113: 10430-10435. DOI: https://doi.org/10.1073/pnas.1603481113
PALMER C., BIK E.M., DIGIULIO D.B., RELMAN D.A., BROWN P.O., 2007 - Development of the human infant intestinal microbiota. - PLoS Biol. 5: e177. DOI: https://doi.org/10.1371/journal.pbio.0050177
POULSEN M., SAPOUNTZIS P., 2012 - Behind every great ant, there is a great gut. - Mol. Ecol. 21: 2054-2057. DOI: https://doi.org/10.1111/j.1365-294X.2012.05510.x
PROENCA D.N.S., GRASS C.E., MORAIS G., PAULA V., 2012 - Draft genome sequence of Serratia sp. Strain M24T3, isolated from pinewood disease nematode Bursaphelenchus xylophilus. - J Bacteriol. 194: 3764. DOI: https://doi.org/10.1128/JB.00670-12
RAHUL K.S., PATIL C.D., HEMANT B., PATIL S.V., 2014 - Nematicidal activity of microbial pigment from Serratia marcescens. - Nat. Prod. Res. 28: 1399-404. DOI: https://doi.org/10.1080/14786419.2014.904310
RAJAGOPAL R., 2009 - Beneficial interactions between insects and gut bacteria. - Indian J Microbiol. 49: 114-119. DOI: https://doi.org/10.1007/s12088-009-0023-z
RAYMANN K., MORAN N.A., 2018 - The role of the gut microbiome in health and disease of adult honey bee workers. – Curr. Opin. Insect. Sci. 26: 97-104. DOI: https://doi.org/10.1016/j.cois.2018.02.012
ROBERTS J.M.K., ANDERSON D.L., DURR P.A., 2017 - Absence of deformed wing virus and Varroa destructor in Australia provides unique perspectives on honeybee viral landscapes and colony losses. - Sci Rep. 7: 1-11. DOI: https://doi.org/10.1038/s41598-017-07290-w
SABREE Z.L., HANSEN A.K., MORAN N.A., 2012 - Independent studies using deep sequencing resolve the same set of core bacterial species dominating gut communities of honey bees. - PloS One. 7: e41250. DOI: https://doi.org/10.1371/journal.pone.0041250
SACCA M.L., LODESANI M., 2020 - Isolation of bacterial microbiota associated to honey bees and evaluation of potential biocontrol agents of Varroa destructor. – Benef. Microbes. 11: 641-654. DOI: https://doi.org/10.3920/BM2019.0164
SALEM H., KREUTZER E., SUDAKARAN S., KALTENPOTH M., 2013 - Actinobacteria as essential symbionts in firebugs and cotton stainers (Hemiptera, Pyrrhocoridae). - Environ. Microbiol. 15: 1956-1968. DOI: https://doi.org/10.1111/1462-2920.12001
SAMBROOK J., 2001 - Molecular cloning: a laboratory manual. Cold Spring Harbor, N Y: Cold Spring Harbor Laboratory Press.
SAMMATARO D., GERSON U., NEEDHAM G., 2000 - Parasitic Mites of Honey Bees: Life History, Implications, and Impact. - Annu. Rev. Entomol. 45: 519-548. DOI: https://doi.org/10.1146/annurev.ento.45.1.519
SHIL R.K., MOJUMDER S., SADIDA F.F., UDDIN M., SIKDAR D., 2014 - Isolation and identification of cellulolytic bacteria from the gut of three phytophagus insect species. - Braz. Arch. Biol. Technol. 57: 927-932. DOI: https://doi.org/10.1590/S1516-8913201402620
STANLEY J., SAH K., JAIN S.K., BHATT J.C., SUSHIL S.N., 2015 - Evaluation of pesticide toxicity at their field recommended doses to honeybees, Apis cerana and A. mellifera through laboratory, semi-field and field studies. - Chemosphere. 119: 668-674. DOI: https://doi.org/10.1016/j.chemosphere.2014.07.039
TAJABADI N., MARDAN M., MANAP M.Y., MUSTAFA S., 2013 - Molecular identification of Lactobacillus spp. isolated from the honey comb of the honey bee (Apis dorsata) by 16S rRNA gene sequencing. - J. Apic. Res. 52: 235-241. DOI: https://doi.org/10.3896/IBRA.1.52.5.10
TAJABADI N., MARDAN M., SHUHAIMI M., ABDUL MANAP M.Y., 2011 - Isolation and identification of Enterococcus sp. from honey stomach of honeybee based on biochemical and 16S rRNA sequencing analysis. - Int. J. Probiotics Prebiotics. 6: 95-100.
TAMURA K., STECHER G., PETERSON D., FILIPSKI A., KUMAR S., 2013 - MEGA6: molecular evolutionary genetics analysis version 6.0. - Mol. Biol. Evol. 30: 2725-2729. DOI: https://doi.org/10.1093/molbev/mst197
THAKUR A., DHAMMI P., SAINI H.S., KAUR S., 2015 - Pathogenicity of bacteria isolated from gut of Spodoptera litura (Lepidoptera: Noctuidae) and fitness costs of insect associated with consumption of bacteria. - J. Invertebr. Pathol. 127: 38-46. DOI: https://doi.org/10.1016/j.jip.2015.02.007
THAOCHAN N., DREW R.A.I., HUGHES J.M., VIJAYSEGARAN S., CHINAJARIYAWONG A., 2010 - Alimentary tract bacteria isolated and identified with API-20E and molecular cloning techniques from Australian tropical fruit flies, Bactrocera cacuminata and B. tryoni. - J. Insect Sci. 10: 131. DOI: https://doi.org/10.1673/031.010.13101
TOWNSEND R.J., CALLAGHAN M., JOHN-SON V.W., JACKSON T.A., 2003 - Compatibility of microbial control agents Serratia entomophila and Beauveria bassiana with selected fertilizers. - N. Z. Plant Protect. 56: 118-122. DOI: https://doi.org/10.30843/nzpp.2003.56.6051
VASSART M., THEVENON J., COTTO F., 1988 - Health study of the Neo-Caledonian apiary. - Bull. Tech. Apic. 15: 195-202.
VOJVODIC S., REHAN S.M., ANDERSON K.E., 2013 - Microbial gut diversity of Africanized and European honey bee larval instars. - PLoS One. 8: e72106. DOI: https://doi.org/10.1371/journal.pone.0072106
WINSTON M.L., 1987 - The Biology of the Honey Bee. Camb. Harvard Uni. Press. 281.
YADAV K.K., BORA A., DATTA S., CHANDELK., GOGOI H.K., PRASAD G.B.K.S., VEER V., 2015 - Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India. - Paras. Vectors. 8: 641. DOI: https://doi.org/10.1186/s13071-015-1252-0
YOSHIIYAMA M., KIMURA K., 2009 - Bacteria in the gut of the Japanese honey bee, A. cerana japonica, and their antagonistic effect against P. larvae, the causal agent of American foulbrood. - J. Invertebr. Pathol. 102: 91-96. DOI: https://doi.org/10.1016/j.jip.2009.07.005
YUN J., ROH S.W., WHON T.W., JUNG M., KIM M., PARK D., YOON C., NAM Y., KIM Y., CHOI J., KIM J., SHIN N., KIM S., LEE W., BAE J., 2014 - Insects gut bacterial diversity determined by host environmental habitat, diet, developmental stage and phylogeny. - Appl. Environ. Microbiol. 80: 5254-5264. DOI: https://doi.org/10.1128/AEM.01226-14
ZHANG C.X., LIU J.R., XU M.X., SUN J., YANG S.Y., AN X., GAO G.F., LIN M.S., LAI R., 2008 - Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes. - J. Invertebr. Pathol. 98: 153-168. DOI: https://doi.org/10.1016/j.jip.2008.02.011

How to Cite

Abuldahab, F., Abd-ElAziz, M. ., El-Sayed, Y. ., Abdallah, M. ., & Mahmoud, S. . (2022). Infestation of <em>Apis mellifera</em> workers and larvae with <em>Varroa destructor</em> affects gut bacterial diversity. Journal of Entomological and Acarological Research, 54(1). https://doi.org/10.4081/jear.2022.10316
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