Despite an increasing number of studies on caterpillar (Insecta: Lepidoptera) gut microbiota, bacteria have been emphasized more than fungi. Therefore, we lack data on whether fungal microbiota is resident or transient and shaped by factors similar to those of bacteria. We sampled nine polyphagous caterpillar species from several tree species at multiple sites to determine the factors shaping leaf and gut bacterial and fungal microbiota as well as the extent to which caterpillars acquire microbiota from their diet. We performed 16S and ITS2 DNA metabarcoding of the leaves and guts to determine the composition and richness of the respective microbiota. While spatial variables shaped the bacterial and fungal microbiota of the leaves, they only affected fungi in the guts, whereas the bacteria were shaped primarily by caterpillar species, with some species harboring more specific bacterial consortia. Leaf and gut microbiota significantly differed; in bacteria, this difference was more pronounced. The quantitative similarity between leaves and guts significantly differed among caterpillar species in bacteria but not fungi, suggesting that some species have more transient bacterial microbiota. Our results suggest the complexity of the factors shaping the gut microbiota, while highlighting interspecific differences in microbiota residency within the same insect functional group.

Department of Biology and Ecology Faculty of Science University of Ostrava Chittussiho 10 710 00 Ostrava Czech Republic

Institute of Microbiology Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Prague Czech Republic

Zobrazit více v PubMed

Futuyma DJ, Agrawal AA. Macroevolution and the biological diversity of plants and herbivores. Proc. Natl. Acad. Sci. 2009;106:18054–18061. doi: 10.1073/pnas.0904106106. PubMed DOI PMC

Frago E, Dicke M, Godfray HCJ. Insect symbionts as hidden players in insect–plant interactions. Trends Ecol. Evol. 2012;27:705–711. doi: 10.1016/j.tree.2012.08.013. PubMed DOI

Gurung K, Wertheim B, Salles JF. The microbiome of pest insects: It is not just bacteria. Entomol. Exp. Appl. 2019;167:156–170. doi: 10.1111/eea.12768. DOI

Douglas AE. Multiorganismal insects: Diversity and function of resident microorganisms. Annu. Rev. Entomol. 2015;60:17–34. doi: 10.1146/annurev-ento-010814-020822. PubMed DOI PMC

Engel P, Moran NA. The gut microbiota of insects—diversity in structure and function. FEMS Microbiol. Rev. 2013;37:699–735. doi: 10.1111/1574-6976.12025. PubMed DOI

Giron D, et al. Chapter seven—influence of microbial symbionts on plant-insect interactions. In: Sauvion N, Thiéry D, Calatayud P-A, et al., editors. Advances in Botanical Research. Academic Press; 2017. pp. 225–257.

Chen B, et al. Biodiversity and activity of the gut microbiota across the life history of the insect herbivore Spodoptera littoralis. Sci. Rep. 2016;6:29505. doi: 10.1038/srep29505. PubMed DOI PMC

Vacher C, et al. The phyllosphere: Microbial jungle at the plant–climate interface. Annu. Rev. Ecol. Evol. Syst. 2016;47:1–24. doi: 10.1146/annurev-ecolsys-121415-032238. DOI

Griffin EA, Carson WP. Tree endophytes: cryptic drivers of tropical forest diversity. In: Pirttilä AM, Frank AC, editors. Endophytes of Forest Trees: Biology and Applications. Springer International Publishing; 2018. pp. 63–103.

Peñuelas J, Rico L, Ogaya R, Jump AS, Terradas J. Summer season and long-term drought increase the richness of bacteria and fungi in the foliar phyllosphere of Quercus ilex in a mixed Mediterranean forest. Plant Biol. 2012;14:565–575. doi: 10.1111/j.1438-8677.2011.00532.x. PubMed DOI

Laforest-Lapointe I, Paquette A, Messier C, Kembel SW. Leaf bacterial diversity mediates plant diversity and ecosystem function relationships. Nature. 2017;546:145–147. doi: 10.1038/nature22399. PubMed DOI

Kembel SW, et al. Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest. Proc. Natl. Acad. Sci. USA. 2014;111:13715–13720. doi: 10.1073/pnas.1216057111. PubMed DOI PMC

Kembel SW, Mueller RC. Plant traits and taxonomy drive host associations in tropical phyllosphere fungal communities. Botany. 2014;92:303–311. doi: 10.1139/cjb-2013-0194. DOI

Faeth SH, Hammon KE. Fungal endophytes in oak trees: Long-term patterns of abundance and associations with leafminers. Ecology. 1997;78:810–819. doi: 10.1890/0012-9658(1997)078[0810:FEIOTL]2.0.CO;2. DOI

Broderick NA, Raffa KF, Goodman RM, Handelsman J. Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods. Appl. Environ. Microbiol. 2004;70:293–300. doi: 10.1128/AEM.70.1.293-300.2004. PubMed DOI PMC

Pinto-Tomás AA, et al. Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets. Environ. Entomol. 2011;40:1111–1122. doi: 10.1603/EN11083. PubMed DOI

Ravenscraft A, Berry M, Hammer T, Peay K, Boggs C. Structure and function of the bacterial and fungal gut microbiota of Neotropical butterflies. Ecol. Monogr. 2019;89:e01346. doi: 10.1002/ecm.1346. DOI

Hammer TJ, Sanders JG, Fierer N. Not all animals need a microbiome. FEMS Microbiol. Lett. 2019;366:117. doi: 10.1093/femsle/fnz117. PubMed DOI

Mason CJ, et al. Diet influences proliferation and stability of gut bacterial populations in herbivorous lepidopteran larvae. PLoS ONE. 2020;15:e0229848. doi: 10.1371/journal.pone.0229848. PubMed DOI PMC

Montagna M, et al. Evidence of a bacterial core in the stored products pest Plodia interpunctella: The influence of different diets. Environ. Microbiol. 2016;18:4961–4973. doi: 10.1111/1462-2920.13450. PubMed DOI

Phalnikar K, Kunte K, Agashe D. Disrupting butterfly caterpillar microbiomes does not impact their survival and development. Proc. R. Soc. B Biol. Sci. 2019;286:20192438. doi: 10.1098/rspb.2019.2438. PubMed DOI PMC

Somerville J, Zhou L, Raymond B. Aseptic rearing and infection with gut bacteria improve the fitness of transgenic diamondback moth, Plutella xylostella. Insects. 2019;10:89. doi: 10.3390/insects10040089. PubMed DOI PMC

González-Serrano F, et al. The gut microbiota composition of the moth brithys crini reflects insect metamorphosis. Microb. Ecol. 2020;79:960–970. doi: 10.1007/s00248-019-01460-1. PubMed DOI

Goharrostami M, JalaliSendi J. Investigation on endosymbionts of Mediterranean flour moth gut and studying their role in physiology and biology. J. Stored Prod. Res. 2018;75:10–17. doi: 10.1016/j.jspr.2017.11.003. DOI

Vilanova C, Baixeras J, Latorre A, Porcar M. The generalist inside the specialist: Gut bacterial communities of two insect species feeding on toxic plants are dominated by Enterococcus sp. Front. Microbiol. 2016;7:1005. doi: 10.3389/fmicb.2016.01005. PubMed DOI PMC

Minard G, Tikhonov G, Ovaskainen O, Saastamoinen M. The microbiome of the Melitaea cinxia butterfly shows marked variation but is only little explained by the traits of the butterfly or its host plant. Environ. Microbiol. 2019;21:4253–4269. doi: 10.1111/1462-2920.14786. PubMed DOI PMC

Shapira M. Gut microbiotas and host evolution: Scaling up symbiosis. Trends Ecol. Evol. 2016;31:539–549. doi: 10.1016/j.tree.2016.03.006. PubMed DOI

Chen B, et al. Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives. ISME J. 2018;12:2252–2262. doi: 10.1038/s41396-018-0174-1. PubMed DOI PMC

Mason CJ, Raffa KF. Acquisition and structuring of midgut bacterial communities in gypsy moth (Lepidoptera: Erebidae) larvae. Environ. Entomol. 2014;43:595–604. doi: 10.1603/EN14031. PubMed DOI

Paniagua Voirol LR, Frago E, Kaltenpoth M, Hilker M, Fatouros NE. Bacterial symbionts in Lepidoptera: Their diversity, transmission, and impact on the host. Front. Microbiol. 2018;9:556. doi: 10.3389/fmicb.2018.00556. PubMed DOI PMC

Laforest-Lapointe I, Messier C, Kembel SW. Host species identity, site and time drive temperate tree phyllosphere bacterial community structure. Microbiome. 2016;4:27. doi: 10.1186/s40168-016-0174-1. PubMed DOI PMC

Meyer KM, Leveau JHJ. Microbiology of the phyllosphere: A playground for testing ecological concepts. Oecologia. 2012;168:621–629. doi: 10.1007/s00442-011-2138-2. PubMed DOI PMC

Gomes T, Pereira JA, Benhadi J, Lino-Neto T, Baptista P. Endophytic and epiphytic phyllosphere fungal communities are shaped by different environmental factors in a Mediterranean ecosystem. Microb. Ecol. 2018;76:668–679. doi: 10.1007/s00248-018-1161-9. PubMed DOI

Rastogi G, et al. Leaf microbiota in an agroecosystem: Spatiotemporal variation in bacterial community composition on field-grown lettuce. ISME J. 2012;6:1812–1822. doi: 10.1038/ismej.2012.32. PubMed DOI PMC

Whitaker MRL, Salzman S, Sanders J, Kaltenpoth M, Pierce NE. Microbial communities of lycaenid butterflies do not correlate with larval diet. Front. Microbiol. 2016;7:1920. doi: 10.3389/fmicb.2016.01920. PubMed DOI PMC

Zheng Y, et al. Midgut microbiota diversity of potato tuber moth associated with potato tissue consumed. BMC Microbiol. 2020;20:58. doi: 10.1186/s12866-020-01740-8. PubMed DOI PMC

Griffin EA, Harrison JG, McCormick MK, Burghardt KT, Parker JD. Tree diversity reduces fungal endophyte richness and diversity in a large-scale temperate forest experiment. Diversity. 2019;11:234. doi: 10.3390/d11120234. DOI

Kim M, et al. Distinctive phyllosphere bacterial communities in tropical trees. Microb. Ecol. 2012;63:674–681. doi: 10.1007/s00248-011-9953-1. PubMed DOI

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

Višňovská D, et al. Caterpillar gut and host plant phylloplane mycobiomes differ: A new perspective on fungal involvement in insect guts. FEMS Microbiol. Ecol. 2020;96:fiaa116. doi: 10.1093/femsec/fiaa116. PubMed DOI

Voříšková J, Baldrian P. Fungal community on decomposing leaf litter undergoes rapid successional changes. ISME J. 2013;7:477–486. doi: 10.1038/ismej.2012.116. PubMed DOI PMC

Pochon X, Zaiko A, Fletcher LM, Laroche O, Wood SA. Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications. PLoS ONE. 2017;12:e0187636. doi: 10.1371/journal.pone.0187636. PubMed DOI PMC

Schlechter RO, Miebach M, Remus-Emsermann MNP. Driving factors of epiphytic bacterial communities: A review. J. Adv. Res. 2019;19:57–65. doi: 10.1016/j.jare.2019.03.003. PubMed DOI PMC

Seabloom EW, et al. Effects of nutrient supply, herbivory, and host community on fungal endophyte diversity. Ecology. 2019;100:e02758. doi: 10.1002/ecy.2758. PubMed DOI

Berlec A. Novel techniques and findings in the study of plant microbiota: Search for plant probiotics. Plant Sci. 2012;193–194:96–102. doi: 10.1016/j.plantsci.2012.05.010. PubMed DOI

Unterseher M, Reiher A, Finstermeier K, Otto P, Morawetz W. Species richness and distribution patterns of leaf-inhabiting endophytic fungi in a temperate forest canopy. Mycol. Prog. 2007;6:201–212. doi: 10.1007/s11557-007-0541-1. DOI

Gilbert GS, Reynolds DR, Bethancourt A. The patchiness of epifoliar fungi in tropical forests: Host range, host abundance, and environment. Ecology. 2007;88:575–581. doi: 10.1890/05-1170. PubMed DOI

Stone BWG, Jackson CR. Canopy position is a stronger determinant of bacterial community composition and diversity than environmental disturbance in the phyllosphere. FEMS Microbiol. Ecol. 2019;95:fiz032. doi: 10.1093/femsec/fiz032. PubMed DOI

Copeland JK, Yuan L, Layeghifard M, Wang PW, Guttman DS. Seasonal community succession of the phyllosphere microbiome. Mol. Plant. Microbe Interact. 2015;28:274–285. doi: 10.1094/MPMI-10-14-0331-FI. PubMed DOI

Stone BWG, Jackson CR. Seasonal patterns contribute more towards phyllosphere bacterial community structure than short-term perturbations. Microb. Ecol. 2020 doi: 10.1007/s00248-020-01564-z. PubMed DOI PMC

Truchado P, Gil MI, Reboleiro P, Rodelas B, Allende A. Impact of solar radiation exposure on phyllosphere bacterial community of red-pigmented baby leaf lettuce. Food Microbiol. 2017;66:77–85. doi: 10.1016/j.fm.2017.03.018. PubMed DOI

Wang X, et al. Variability of gut microbiota across the life cycle of Grapholita molesta (Lepidoptera: Tortricidae) Front. Microbiol. 2020;11:1366. doi: 10.3389/fmicb.2020.01366. PubMed DOI PMC

Toju H, Fukatsu T. Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: Relevance of local climate and host plants. Mol. Ecol. 2011;20:853–868. doi: 10.1111/j.1365-294X.2010.04980.x. PubMed DOI

Yun J-H, et al. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl. Environ. Microbiol. 2014;80:5254–5264. doi: 10.1128/AEM.01226-14. PubMed DOI PMC

Sánchez NE, Pereyra PC, Luna MG. Spatial patterns of parasitism of the solitary parasitoid Pseudapanteles dignus (Hymenoptera: Braconidae) on Tuta absoluta (Lepidoptera: Gelechiidae) Environ. Entomol. 2009;38:365–374. doi: 10.1603/022.038.0208. PubMed DOI

Santos AMC, Quicke DLJ. Large-scale diversity patterns of parasitoid insects. Entomol. Sci. 2011;14:371–382. doi: 10.1111/j.1479-8298.2011.00481.x. DOI

Mereghetti V, Chouaia B, Montagna M. New insights into the microbiota of moth pests. Int. J. Mol. Sci. 2017;18:2450. doi: 10.3390/ijms18112450. PubMed DOI PMC

Floater GJ. Estimating movement of the processionary caterpillar Ochrogaster zunifer Herrich-Schäffer (Lepidoptera: Thaumetopoeidae) between discrete resource patches. Aust. J. Entomol. 1996;35:279–283. doi: 10.1111/j.1440-6055.1996.tb01403.x. DOI

Turčáni M, Patočka J. Does intraguild predation of Cosmia trapezina L. (Lep.: Noctuidae) influence the abundance of other Lepidoptera forest pests? J. For. Sci. 2011;57:472–482. doi: 10.17221/99/2010-JFS. DOI

Hikisz J, Soszynska-Maj A. What moths fly in winter? The assemblage of moths active in a temperate deciduous forest during the cold season in Central Poland. J. Entomol. Res. Soc. 2015;17:59–71.

Bell JR, Bohan DA, Shaw EM, Weyman GS. Ballooning dispersal using silk: World fauna, phylogenies, genetics and models. Bull. Entomol. Res. 2005;95:69–114. doi: 10.1079/BER2004350. PubMed DOI

Griffin EA, Carson WP. The ecology and natural history of foliar bacteria with a focus on tropical forests and agroecosystems. Bot. Rev. 2015;81:105–149. doi: 10.1007/s12229-015-9151-9. DOI

Qian X, et al. Mainland and island populations of Mussaenda kwangtungensis differ in their phyllosphere fungal community composition and network structure. Sci. Rep. 2020;10:952. doi: 10.1038/s41598-020-57622-6. PubMed DOI PMC

Herren CM, McMahon KD. Keystone taxa predict compositional change in microbial communities. Environ. Microbiol. 2018;20:2207–2217. doi: 10.1111/1462-2920.14257. PubMed DOI

Humphrey PT, Whiteman NK. Insect herbivory reshapes a native leaf microbiome. Nat. Ecol. Evol. 2020;4:221–229. doi: 10.1038/s41559-019-1085-x. PubMed DOI PMC

Müller T, Müller M, Behrendt U, Stadler B. Diversity of culturable phyllosphere bacteria on beech and oak: The effects of lepidopterous larvae. Microbiol. Res. 2003;158:291–297. doi: 10.1078/0944-5013-00207. PubMed DOI

Hrcek J, Miller SE, Quicke DLJ, Smith MA. Molecular detection of trophic links in a complex insect host-parasitoid food web. Mol. Ecol. Resour. 2011;11:786–794. doi: 10.1111/j.1755-0998.2011.03016.x. PubMed DOI

Bateman C, Šigut M, Skelton J, Smith KE, Hulcr J. Fungal associates of the Xylosandrus compactus (Coleoptera: Curculionidae, Scolytinae) are spatially segregated on the insect body. Environ. Entomol. 2016;45:883–890. doi: 10.1093/ee/nvw070. PubMed DOI

Toju H, Tanabe AS, Yamamoto S, Sato H. High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE. 2012;7:e40863. doi: 10.1371/journal.pone.0040863. PubMed DOI PMC

Chelius MK, Triplett EW. The diversity of archaea and bacteria in association with the roots of Zea mays L. Microb. Ecol. 2001;41:252–263. doi: 10.1007/s002480000087. PubMed DOI

Redford AJ, Bowers RM, Knight R, Linhart Y, Fierer N. The ecology of the phyllosphere: Geographic and phylogenetic variability in the distribution of bacteria on tree leaves. Environ. Microbiol. 2010;12:2885–2893. doi: 10.1111/j.1462-2920.2010.02258.x. PubMed DOI PMC

Bolyen, E. et al. QIIME 2: Reproducible, Interactive, Scalable, and Extensible Microbiome Data Sciencehttps://peerj.com/preprints/27295 (2018) 10.7287/peerj.preprints.27295v2.

Rivers AR, Weber KC, Gardner TG, Liu S, Armstrong SD. ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis. F1000Research. 2018;7:1418. doi: 10.12688/f1000research.15704.1. PubMed DOI PMC

Callahan BJ, et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Bokulich NA, et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome. 2018;6:90. doi: 10.1186/s40168-018-0470-z. PubMed DOI PMC

Quast C, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–D596. doi: 10.1093/nar/gks1219. PubMed DOI PMC

Nilsson RH, et al. The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res. 2019;47:D259–D264. doi: 10.1093/nar/gky1022. PubMed DOI PMC

UNITE Community. UNITE QIIME Release for Fungi 2. (2019).

Davis NM, Proctor DM, Holmes SP, Relman DA, Callahan BJ. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6:226. doi: 10.1186/s40168-018-0605-2. PubMed DOI PMC

R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2020).

Ter Braak, C. J. F. ter & Smilauer, P. Canoco reference manual and user’s guide: software for ordination, version 5.0. (2012).

Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC Bioinform. 2011;12:385. doi: 10.1186/1471-2105-12-385. PubMed DOI PMC

Chrostek E, Pelz-Stelinski K, Hurst GDD, Hughes GL. Horizontal transmission of intracellular insect symbionts via plants. Front. Microbiol. 2017;8:2237. doi: 10.3389/fmicb.2017.02237. PubMed DOI PMC

Fox J, Weisberg S. An R Companion to Applied Regression. SAGE Publications; 2018.

Oksanen, J. et al. vegan: Community Ecology Package. (2020).

Anderson MJ. Distance-based tests for homogeneity of multivariate dispersions. Biometrics. 2006;62:245–253. doi: 10.1111/j.1541-0420.2005.00440.x. PubMed DOI

Renkonen O. Statistisch-ökologische Untersuchungen über die terrestrische Käferwelt der finnischen Bruchmoore. Ann. Zool. Soc. Zool.-Bot. Fenn. Vanamo. 1938;6:1–231.

Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 2015;67:1–48. doi: 10.18637/jss.v067.i01. DOI

Roberts, D. W. labdsv: Ordination and Multivariate Analysis for Ecology (2019).

Cáceres MD, Legendre P. Associations between species and groups of sites: Indices and statistical inference. Ecology. 2009;90:3566–3574. doi: 10.1890/08-1823.1. PubMed DOI

Dufrêne M, Legendre P. Species assemblages and indicator species: The need for a flexible asymmetrical approach. Ecol. Monogr. 1997;67:345–366.

Benjamini Y, Hochberg Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B Methodol. 1995;57:289–300.

Concentration-dependent effect of plant secondary metabolites on bacterial and fungal microbiomes in caterpillar guts

Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are species-specific in larvae but similar in nutritive mycelia

Seasonal Shifts in Bacterial and Fungal Microbiomes of Leaves and Associated Leaf-Mining Larvae Reveal Persistence of Core Taxa Regardless of Diet