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Microbial Communities of Stored Product Mites: Variation by Species and Population

. 2021 Feb ; 81 (2) : 506-522. [epub] 20200827

Language English Country United States Media print-electronic

Document type Journal Article

Grant support
GA19-09998S Grantová Agentura České Republiky
19-14-00004 Russian Science Foundation

Links

PubMed 32852571
DOI 10.1007/s00248-020-01581-y
PII: 10.1007/s00248-020-01581-y
Knihovny.cz E-resources

Arthropod-associated microorganisms are important because they affect host fitness, protect hosts from pathogens, and influence the host's ability to vector pathogens. Stored product mites (Astigmata) often establish large populations in various types of food items, damaging the food by direct feeding and introducing contaminants, including their own bodies, allergen-containing feces, and associated microorganisms. Here we access the microbial structure and abundance in rearing diets, eggs, feces fraction, and mite bodies of 16 mite populations belonging to three species (Carpoglyphus lactis, Acarus siro, and Tyrophagus putrescentiae) using quantitative PCR and 16S ribosomal RNA (rRNA) gene amplicon sequencing. The mite microbiomes had a complex structure dominated by the following bacterial taxa (OTUs): (a) intracellular symbionts of the genera Cardinium and Wolbachia in the mite bodies and eggs; (b) putative gut symbionts of the genera Solitalea, Bartonella, and Sodalis abundant in mite bodies and also present in mite feces; (c) feces-associated or environmental bacteria of the genera Bacillus, Staphylococcus, and Kocuria in the diet, mite bodies, and feces. Interestingly and counterintuitively, the differences between microbial communities in various conspecific mite populations were higher than those between different mite species. To explain some of these differences, we hypothesize that the intracellular bacterial symbionts can affect microbiome composition in mite bodies, causing differences between microbial profiles. Microbial profiles differed between various sample types, such as mite eggs, bodies, and the environment (spent growth medium-SPGM). Low bacterial abundances in eggs may result in stochastic effects in parent-offspring microbial transmission, except for the intracellular symbionts. Bacteria in the rearing diet had little effect on the microbial community structure in SPGM and mite bodies. Mite fitness was positively correlated with bacterial abundance in SPGM and negatively correlated with bacterial abundances in mite bodies. Our study demonstrates critical host-microbe interactions, affecting all stages of mite growth and leading to alteration of the environmental microbiome. Correlational evidence based on absolute quantitation of bacterial 16S rRNA gene copies suggests that mite-associated microorganisms are critical for modulating important pest properties of mites by altering population growth.

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Chambers J, Bhushy Thind B, Dunn JA, Pearson DJ (1999) The importance of storage mite allergens in occupational and domestic environments. In: Robinson W, Rettich F, Rambo GW (eds) Proceedings of the 3rd International Conference on Urban Pests: Prague, 19–22 July 1999. Czech University of Agriculture, Prague, pp 559–569

Zhao Y, Abbar S, Amoah B, Phillips TW, Schilling MW (2016) Controlling pests in dry-cured ham: a review. Meat Sci 111:183–191. https://doi.org/10.1016/j.meatsci.2015.09.009 PubMed DOI

Athanassiou CG, Palyvos NE, Eliopoulos PA, Papadoulis GT (2001) Distribution and migration of insects and mites in flat storage containing wheat. Phytoparasitica 29:379–392. https://doi.org/10.1007/BF02981856 DOI

Hughes AM (1976) The mites of stored food and houses: technical bulletin 9 of the Ministry of Agriculture, Fisheries and Food2nd edn. Her Majesty’s Stationery Office, London

Hubert J, Erban T, Nesvorna M, Stejskal V (2011) Emerging risk of infestation and contamination of dried fruits by mites in the Czech Republic. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 28:1129–1135. https://doi.org/10.1080/19440049.2011.584911 PubMed DOI

Green WF, Woolcock AJ (1978) Tyrophagus putrescentiae: an allergenically important mite. Clin Allergy 8:135–144. https://doi.org/10.1111/j.1365-2222.1978.tb00458.x PubMed DOI

Sanchez-Borges M, Fernandez-Caldas E (2015) Hidden allergens and oral mite anaphylaxis: the pancake syndrome revisited. Curr Opin Allergy Clin Immunol 15:337–343. https://doi.org/10.1097/ACI.0000000000000175 PubMed DOI

Hubert J, Stejskal V, Athanassiou CG, Throne JE (2018) Health hazards associated with arthropod infestation of stored products. Annu Rev Entomol 63:553–573. https://doi.org/10.1146/annurev-ento-020117-043218 PubMed DOI

Cunnington AM (1965) Physical limits for complete development of the grain mite, Acarus siro L. (Acarina, Acaridae), in relation to its world distribution. J Appl Ecol 2:295–306. https://doi.org/10.2307/2401481 DOI

Hibberson CE, Vogelnest LJ (2014) Storage mite contamination of commercial dry dog food in south-eastern Australia. Aust Vet J 92:219–224. https://doi.org/10.1111/avj.12185 PubMed DOI

Robertson PL (1946) Tyroglyphid mites in stored products in New Zealand. T Roy Soc N Z 76:185–207

Smrz J, Catska V (1987) Food selection of the field population of Tyrophagus putrescentiae (Schrank) (Acari, Acarida). J Appl Entomol 104:329–335. https://doi.org/10.1111/j.1439-0418.1987.tb00533.x DOI

Collins DA (2012) A review on the factors affecting mite growth in stored grain commodities. Exp Appl Acarol 56:191–208. https://doi.org/10.1007/s10493-012-9512-6 PubMed DOI

Kavallieratos NG, Athanassiou CG, Guedes RNC, Drempela JD, Boukouvala MC (2017) Invader competition with local competitors: displacement or coexistence among the invasive khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), and two other major stored-grain beetles? Front Plant Sci 8:1837. https://doi.org/10.3389/fpls.2017.01837 PubMed DOI PMC

Shah JA, Vendl T, Aulicky R, Stejskal V (2020) Frass produced by the primary pest Rhyzopertha dominica supports the population growth of the secondary stored product pests Oryzaephilus surinamensis, Tribolium castaneum, and T. confusum. Bull Entomol Res (in press). https://doi.org/10.1017/S0007485320000425

Mason CJ, Campbell AM, Scully ED, Hoover K (2019) Bacterial and fungal midgut community dynamics and transfer between mother and brood in the Asian longhorned beetle (Anoplophora glabripennis), an invasive xylophage. Microb Ecol 77:230–242. https://doi.org/10.1007/s00248-018-1205-1 PubMed DOI

Erban T, Rybanska D, Harant K, Hortova B, Hubert J (2016) Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases. Front Physiol 7:53. https://doi.org/10.3389/fphys.2016.00053 PubMed DOI PMC

Hubert J, Nesvorna M, Sopko B, Smrz J, Klimov P, Erban T (2018) Two populations of mites (Tyrophagus putrescentiae) differ in response to feeding on feces-containing diets. Front Microbiol 9:2590. https://doi.org/10.3389/fmicb.2018.02590 PubMed DOI PMC

Tovey ER, Chapman MD, Platts-Mills TAE (1981) Mite faeces are a major source of house dust allergens. Nature 289:592–593. https://doi.org/10.1038/289592a0 PubMed DOI

Stewart GA (1982) Isolation and characterization of the allergen Dpt 12 from Dermatophagoides pteronyssinus by chromatofocusing. Int Arch Allergy Appl Immunol 69:224–230. https://doi.org/10.1159/000233175 PubMed DOI

Stewart GA, Lake FR (1991) Thompson PJ (1991) Faecally derived hydrolytic enzymes from Dermatophagoides pteronyssinus: physicochemical characterisation of potential allergens. Int Arch Allergy Appl Immunol 95:248–256. https://doi.org/10.1159/000235437 PubMed DOI

Batard T, Hrabina A, Bi XZ, Chabre H, Lemoine P, Couret M-N, Faccenda D, Villet B, Harzic P, Andre F, Goh SY, Andre C, Chew FT, Moingeon P (2006) Production and proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus and Dermatophagoides farinae extracts for allergy vaccines. Int Arch Allergy Immunol 140:295–305. https://doi.org/10.1159/000093707 PubMed DOI

Yella L, Morgan MS, Arlian LG (2013) Population growth and allergen accumulation of Dermatophagoides farinae cultured at 20 and 25 °C. Exp Appl Acarol 60:117–126. https://doi.org/10.1007/s10493-012-9626-x PubMed DOI

Erban T, Hubert J (2008) Digestive function of lysozyme in synanthropic acaridid mites enables utilization of bacteria as a food source. Exp Appl Acarol 44:199–212. https://doi.org/10.1007/s10493-008-9138-x PubMed DOI

Hubert J, Doleckova-Maresova L, Hyblova J, Kudlikova I, Stejskal V, Mares M (2005) In vitro and in vivo inhibition of alpha-amylases of stored-product mite Acarus siro. Exp Appl Acarol 35:281–291. https://doi.org/10.1007/s10493-004-7834-8 PubMed DOI

Matsumoto K (1965) Studies on environmental factors for breeding of grain mites VII. Relationship between reproduction of mites and kind of carbohydrates in the diet. Med Entomol Zool 16:118–122. https://doi.org/10.7601/mez.16.118 (in Japanese with English summary) DOI

Naqib A, Poggi S, Wang W, Hyde M, Kunstman K, Green SJ (2018) Making and sequencing heavily multiplexed, high-throughput 16S ribosomal RNA gene amplicon libraries using a flexible, two-stage PCR protocol. In: Raghavachari N, Garcia-Reyero N (eds) Gene expression analysis: methods in and protocols. Humana Press, New York, pp 149–169. https://doi.org/10.1007/978-1-4939-7834-2_7 DOI

Sakai M, Matsuka A, Komura T, Kanazawa S (2004) Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots. J Microbiol Methods 59:81–89. https://doi.org/10.1016/j.mimet.2004.06.005 PubMed DOI

Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624. https://doi.org/10.1038/ismej.2012.8 PubMed DOI PMC

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. https://doi.org/10.1128/AEM.01541-09 PubMed DOI PMC

Edgar RC (2016) UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing. bioRxiv. https://doi.org/10.1101/081257 ; https://www.biorxiv.org/content/early/2016/10/15/081257 . Accessed 23 April 2020

Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998. https://doi.org/10.1038/nmeth.2604 PubMed DOI PMC

Hubert J, Nesvorna M, Kopecky J, Erban T, Klimov P (2019) Population and culture age influence the microbiome profiles of house dust mites. Microb Ecol 77:1048–1066. https://doi.org/10.1007/s00248-018-1294-x PubMed DOI

Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112–5120. https://doi.org/10.1128/AEM.01043-13 PubMed DOI PMC

Cole JR, Wang Q, Fish JA, Chai BL, McGarrell DM, Sun YN, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642. https://doi.org/10.1093/nar/gkt1244 PubMed DOI

Edgar RC (2016) SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv. https://doi.org/10.1101/074161 ; https://www.biorxiv.org/content/10.1101/074161v1 . Accessed 23 April 2020

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2 DOI

Kopecky J, Nesvorna M, Mareckova-Sagova M, Hubert J (2014) The effect of antibiotics on associated bacterial community of stored product mites. PLoS One 9:e112919. https://doi.org/10.1371/journal.pone.0112919 PubMed DOI PMC

Erban T, Klimov PB, Smrz J, Phillips TW, Nesvorna M, Kopecky J, Hubert J (2016) Populations of stored product mite Tyrophagus putrescentiae differ in their bacterial communities. Front Microbiol 7:1046. https://doi.org/10.3389/fmicb.2016.01046 PubMed DOI PMC

Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217. https://doi.org/10.1006/jmbi.2000.4042 PubMed DOI

Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang J-M, Taly J-F, Notredame C (2011) T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 39:W13–W17. https://doi.org/10.1093/nar/gkr245 PubMed DOI PMC

Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/10.1093/sysbio/syq010 PubMed DOI PMC

Rambaut A (2014) FigTree, a graphical viewer of phylogenetic trees: 2014-07-09 - v1.4.2. Molecular evolution, phylogenetics and epidemiology: research, software and publications of Andrew Rambaut and members of his research group. http://tree.bio.ed.ac.uk/software/figtree/ . Accessed 27 July 2015

Nesvorna M, Bittner V, Hubert J (2019) The mite Tyrophagus putrescentiae hosts population-specific microbiomes that respond weakly to starvation. Microb Ecol 77:488–501. https://doi.org/10.1007/s00248-018-1224-y PubMed DOI

Hammer O (2018). Past 3.x - the past of the future: current version (October 2018): 3.21. Natural History Museum, University of Oslo, Oslo, Norway. https://folk.uio.no/ohammer/past/ . Accessed 23 April 2020

R Development Core Team (2016) R: a language and environment for statistical computing, reference index version 3.3.1. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org . Accessed 23 April 2020

Oksanen J (2019) Vegan: an introduction to ordination: processed with vegan 2.5-6 in R version 3.6.1 (2019-07-05) on August 31, 2019. https://cran.r-project.org/web/packages/vegan/vignettes/intro-vegan.pdf . Accessed 23 April 2020

Mair P, Wilcox R (2019) Package ‘WRS2’. June 6, 2019. https://cran.r-project.org/web/packages/WRS2/WRS2.pdf . Accessed 23 April 2020

Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecol 26:32–46. https://doi.org/10.1111/j.1442-9993.2001.01070.pp.x DOI

Zeleny D (2019) Analysis of community ecology data in R. DavidZeleny.net . https://www.davidzeleny.net/anadat-r/doku.php/en:start . Accessed 23 April 2020

PopGen (2018) Detecting multilocus adaptation using Redundancy Analysis (RDA). Population genetics in R. https://popgen.nescent.org/2018-03-27_RDA_GEA.html . Accessed 23 April 2020

Mair P, Wilcox R (2019) Robust statistical methods in R using the WRS2 package. J Stat Softw 20:1–32. https://doi.org/10.18637/jss.v000.i00 ; https://dornsife.usc.edu/assets/sites/239/docs/WRS2.pdf . Accessed 23 April 2020

Zele F, Santos I, Olivieri I, Weill M, Duron O, Magalhaes S (2018) Endosymbiont diversity and prevalence in herbivorous spider mite populations in South-Western Europe. FEMS Microbiol Ecol 94:fiy015. https://doi.org/10.1093/femsec/fiy015 DOI

Kopecky J, Perotti MA, Nesvorna M, Erban T, Hubert J (2013) Cardinium endosymbionts are widespread in synanthropic mite species (Acari: Astigmata). J Invertebr Pathol 112:20–23. https://doi.org/10.1016/j.jip.2012.11.001 PubMed DOI

Erban T, Klimov P, Molva V, Hubert J (2020) Whole genomic sequencing and sex-dependent abundance estimation of Cardinium sp., a common and hyperabundant bacterial endosymbiont of the American house dust mite, Dermatophagoides farinae. Exp Appl Acarol 80:363–380. https://doi.org/10.1007/s10493-020-00475-5 PubMed DOI

Hubert J, Kopecky J, Nesvorna M, Perotti MA, Erban T (2016) Detection and localization of Solitalea-like and Cardinium bacteria in three Acarus siro populations (Astigmata: Acaridae). Exp Appl Acarol 70:309–327. https://doi.org/10.1007/s10493-016-0080-z PubMed DOI

Kopecky J, Nesvorna M, Hubert J (2014) Bartonella-like bacteria carried by domestic mite species. Exp Appl Acarol 64:21–32. https://doi.org/10.1007/s10493-014-9811-1 PubMed DOI

Hubert J, Erban T, Kopecky J, Sopko B, Nesvorna M, Lichovnikova M, Schicht S, Strube C, Sparagano O (2017) Comparison of microbiomes between red poultry mite populations (Dermanyssus gallinae): predominance of Bartonella-like bacteria. Microb Ecol 74:947–960. https://doi.org/10.1007/s00248-017-0993-z PubMed DOI

Hubert J, Kopecky J, Perotti MA, Nesvorna M, Braig HR, Sagova-Mareckova M, Macovei L, Zurek L (2012) Detection and identification of species-specific bacteria associated with synanthropic mites. Microb Ecol 63:919–928. https://doi.org/10.1007/s00248-011-9969-6 PubMed DOI

Molva V, Bostlova M, Nesvorna M, Hubert J (2020) Do the microorganisms from laboratory culture spent growth medium affect house dust mite fitness and microbiome composition? Insect Sci 27:266–275. https://doi.org/10.1111/1744-7917.12636 PubMed DOI

Nesvorna M, Sopko B, Hubert J (2020) Cardinium and Wolbachia are negatively correlated in the microbiome of various populations of stored product mite Tyrophagus putrescentiae. Int J Acarol 46:192–199. https://doi.org/10.1080/01647954.2020.1752305 DOI

Erban T, Ledvinka O, Nesvorna M, Hubert J (2017) Experimental manipulation shows a greater influence of population than dietary perturbation on the microbiome of Tyrophagus putrescentiae. Appl Environ Microbiol 83:e00128–e00117. https://doi.org/10.1128/AEM.00128-17 PubMed DOI PMC

Lee J, Kim JY, M-h Y, Hwang Y, Lee I-Y, Nam S-H, Yong D, Yong T-S (2019) Comparative microbiome analysis of Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Tyrophagus putrescentiae. J Allergy Clin Immunol 143:1620–1623. https://doi.org/10.1016/j.jaci.2018.10.062 PubMed DOI

Hubert J, Kopecky J, Sagova-Mareckova M, Nesvorna M, Zurek L, Erban T (2016) Assessment of bacterial communities in thirteen species of laboratory-cultured domestic mites (Acari: Acaridida). J Econ Entomol 109:1887–1896. https://doi.org/10.1093/jee/tow089 PubMed DOI

Hammer TJ, Janzen DH, Hallwachs W, Jaffe SP, Fierer N (2017) Caterpillars lack a resident gut microbiome. Proc Natl Acad Sci U S A 114:9641–9646. https://doi.org/10.1073/pnas.1707186114 PubMed DOI PMC

Murillo P, Klimov P, Hubert J, OConnor BM (2018) Investigating species boundaries using DNA and morphology in the mite Tyrophagus curvipenis (Acari: Acaridae), an emerging invasive pest, with a molecular phylogeny of the genus Tyrophagus. Exp Appl Acarol 75:167–189. https://doi.org/10.1007/s10493-018-0256-9 PubMed DOI

Beroiz B, Couso-Ferrer F, Ortego F, Chamorro MJ, Arteaga C, Lombardero M, Castanera P, Hernandez-Crespo P (2014) Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification. Med Vet Entomol 28:287–296. https://doi.org/10.1111/mve.12052 PubMed DOI

Swe PM, Zakrzewski M, Waddell R, Sriprakash KS, Fischer K (2019) High-throughput metagenome analysis of the Sarcoptes scabiei internal microbiota and in-situ identification of intestinal Streptomyces sp. Sci Rep 9:11744. https://doi.org/10.1038/s41598-019-47892-0 PubMed DOI PMC

Salem H, Florez L, Gerardo N, Kaltenpoth M (2015) An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects. Proc Biol Sci 282:20142957. https://doi.org/10.1098/rspb.2014.2957 PubMed DOI PMC

Onchuru TO, Martinez AJ, Ingham CS, Kaltenpoth M (2018) Transmission of mutualistic bacteria in social and gregarious insects. Curr Opin Insect Sci 28:50–58. https://doi.org/10.1016/j.cois.2018.05.002 PubMed DOI

Zhu Y-X, Song Z-R, Song Y-L, Zhao D-S, Hong X-Y (2019) The microbiota in spider mite feces potentially reflects intestinal bacterial communities in the host. Insect Sci (in press). https://doi.org/10.1111/1744-7917.12716

Chandler JA, Lang JM, Bhatnagar S, Eisen JA, Kopp A (2011) Bacterial communities of diverse Drosophila species: ecological context of a host–microbe model system. PLoS Genet 7:e1002272. https://doi.org/10.1371/journal.pgen.1002272 PubMed DOI PMC

Zindel R, Ofek M, Minz D, Palevsky E, Zchori-Fein E, Aebi A (2013) The role of the bacterial community in the nutritional ecology of the bulb mite Rhizoglyphus robini (Acari: Astigmata: Acaridae). FASEB J 27:1488–1497. https://doi.org/10.1096/fj.12-216242 PubMed DOI

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