Glyphosate-based herbicide affects the composition of microbes associated with Colorado potato beetle (Leptinotarsa decemlineata)

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2020

Glyphosate-based herbicide affects the composition of microbes associated

with Colorado potato beetle (Leptinotarsa decemlineata)

Gómez-Gallego, Carlos

Oxford University Press (OUP)

Tieteelliset aikakauslehtiartikkelit

http://dx.doi.org/10.1093/femsle/fnaa050

https://erepo.uef.fi/handle/123456789/8172

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Glyphosate-based herbicide affects the composition of microbes associated with Colorado potato beetle (Leptinotarsa decemlineata)

One-sentence Summary: This study showed for the first time that soil-mediated glyphosate affects the microbiota associated with a non-targeted organism in two different life stages by increasing relative abundance of Agrobacterium populations and reducing the relative abundance of other bacterial groups.

Carlos Gómez-Gallego^1,2*, Miia J. Rainio^3*†, M. Carmen Collado^2,4, Anastasia Mantziari², Seppo Salminen², Kari Saikkonen⁵, Marjo Helander³

1 Institute of Public Health and Clinical Nutrition, School of Medicine, University of Eastern Finland, Kuopio, Finland

2 Functional Foods Forum, University of Turku, Turku, Finland

3 Department of Biology, University of Turku, Turku, Finland

4 Department of Biotechnology, Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC), Valencia, Spain

5 Biodiversity Unit, University of Turku, Turku, Finland

*Authors contributed equally to this article. Listed in alphabetical order.

Key words: Colorado potato beetle, dysbiosis, herbicides, insects, microbiota, Roundup Abstract

Here we examined whether glyphosate affects the microbiota of herbivores feeding on non-target plants. Colorado potato beetles (Leptinotarsa decemlineata) were reared on potato plants grown in pots containing soil treated with glyphosate-based herbicide (GBH) or untreated. Per the manufacturer’s safety recommendations, the GBH soil treatments were done two weeks prior to planting the potatoes.

Later, two-day-old larvae were introduced to the potato plants and then collected in two phases, 4th instar larvae and adults. The larvae’s internal microbiota and the adults’ intestinal microbiota were examined by 16S rRNA gene sequencing. The beetles’ microbial composition was affected by the GBH treatment and the differences in microbial composition between the control and insects exposed to GBH were more pronounced in the adults. The GBH treatment increased the relative abundance of

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Agrobacterium in the larvae and the adults. This effect may be related to the tolerance of some Agrobacterium species to glyphosate or to glyphosate-mediated changes in potato plants. On the other hand, the relative abundance of Enterobacteriaceae, Rhodobacter, Rhizobium and Acidovorax in the adult beetles and Ochrobactrum in the larvae were reduced in GBH treatment. These results demonstrate that glyphosate can impact microbial communities associated with herbivores feeding on non-target crop plants.

Introduction

The insect gut is a complex ecosystem inhabited by a large variety of microbes (Colman et al. 2012) that play important roles in physiology, well-being and behaviour (Xia et al. 2018). The gut microbiota significantly affects food digestion (Warnecke et al. 2007), nutrition (Engel and Moran 2013), immune function (Ryu et al. 2010), amino acid metabolism (Mardinoglu et al. 2015) and pathogen and parasite defence (Dillon et al. 2005; Engel and Moran 2013). The gut microbiota also contributes to the detoxification of plant allelochemicals (Broderick et al. 2004; Hammer and Bowers 2015) and xenobiotic compounds (Claus et al. 2016; Dowd and Shen 1990; Karasov and Martinez del Rio 2007).

The highly diverse insect gut microbiota varies according to insect species, developmental stage and diet and environmental conditions (Brauman et al. 2001; Yun et al. 2014). For example, drugs, antibiotics, food additives and environmental pollution such as pesticides, heavy metals and persistent organic pollutants can affect gut microbiota (Jin et al. 2017), and insect microbiota is not an exception.

These changes in microbial composition may have additive and cumulative effects as they can disrupt the detoxification capability of microbiota against environmental contamination, thus affecting the physiology, health, reproduction and survival of the host (Claus et al. 2016).

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Health concerns regarding the effects of pesticides on animals have increased lately (Hallmann et al.

2014; Jin et al. 2012; Jin et al. 2015) due in part to the antimicrobial activity of pesticides such as glyphosate (N-(phosphonomethyl)glycine), which can change the gut microbiota (Claus et al. 2016; Jin et al. 2017; Krüger, et al. 2013; Motta, et al. 2018; Shehata et al. 2013). Glyphosate is the most commonly used herbicide worldwide given its cost effectiveness and its non-selectivity in killing herbaceous plants (Helander et al. 2012; Myers et al. 2016; Woodburn 2000). Glyphosate has been proclaimed to be safe for non-target organisms due to its low accumulation rate and rapid inactivation in soils (Giesy et al. 2000; Vereecken 2005). However, new and accumulating evidence has demonstrated that glyphosate and its degradation metabolites can remain in soils for years and affect non-target organisms (Helander et al. 2018). For example, recent toxicological studies have shown that glyphosate, together with its metabolites (e.g., aminomethylphosphonic acid, AMPA) and adjuvants used in commercial products, can cause various toxic effects. These include changes in cell function, tissue, physiology, survival and behaviour in non-target species (reviewed in Claus et al. 2016;

Mesnage et al. 2015). While the consequences for microbial-mediated ecosystem functions and services are largely unknown, their occurrence cannot be ruled out. Regarding animal-associated microbes, Motta et al. (2018) showed that glyphosate-based herbicides can perturb the gut microbiota of honeybees by altering their gut community.

In addition, glyphosate is commonly considered to be harmless to animals (Duke 2008; Helander et al.

2012) because the function of glyphosate is based on the inactivation of the 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme. This enzyme belongs to the shikimate metabolic pathway, which appears only in plants and in some microbes that include fungi and bacteria (Bentley 1990; Haslam 1993; Helander et al. 2018). Class I EPSPS enzymes, found within plants, archaea, and some bacteria and fungi, have been shown to be glyphosate-sensitive, whereas Class II EPSPS enzymes are

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glyphosate-tolerant (Funke et al. 2007; Light et al. 2016; Priestman et al. 2005). Differences in microbial sensitivity to glyphosate may affect the microbial composition in habitats such as soil, plants and animal intestinal tracts that accumulate glyphosate (Van Bruggen et al. 2018).So far, the potential impacts of glyphosate on the bacterial communities in the gut environment have received little attention (Jin et al. 2017; Liu et al. 2017; Motta et al. 2018, Nielsen et al. 2018; Shehata et al. 2013). The known importance of gut microbiota for host health calls for a better, more-detailed understanding of the relationship between glyphosate and gut microbiota in non-target species.

In this study, we investigated the effects of indirect exposure a glyphosate-based herbicide (GBH) introduced via soils on the gut microbiota of a non-target herbivore using the Colorado potato beetle (Leptinotarsa decemlineata, Coleoptera, Chrysomelidae) as a model species. By using the larvae and adults, we were also able to examine whether the microbial composition, diversity and richness differ between the developmental stages. We hypothesized that changes in soil and potato plants and/or GBH residues accumulated in soil and plants may affect the gut microbiota of the Colorado potato beetles feeding on potatoes planted in soils treated with GBH two weeks before the potatoes were planted, and beetles introduced to the plants. We also expected the possible effects of GBH on gut microbiota to vary between the developmental stages due to differences in the gut microbiome in the larval and adult stages.

Materials and methods

Study design

To study the soil-mediated effects of GBH on Colorado potato beetles in a greenhouse experiment, we used soils that had been pre-treated with GBH. The soil was collected from a long-term field experiment established at the Botanical Garden of the University of Turku (60° 26’ N, 22°10’ E) in 2013. The experimental field was divided into alternating 12 control and 12 GBH treatment plots (23 m

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× 1.5 m), with 1.5 m buffer strips between the plots (see more details in Hagner et al. 2019 and Helander et al. 2019). The experimental soil was treated with an environmentally relevant dose of GBH in the form of RoundUp® Gold (glyphosate concentration 450 g l^-1, CAS: 38641-94-0, application rate 6.4 l ha^-1). The control soil received the same amount of tap water as the treated soil but without the GBH that was applied twice per year (specifically, in May 2014, 2015 and 2016 and October 2014 and 2015). The soil type in the field was medium clay with high organic matter content (>120 g kg^-1) with pH 5.9 (year 2016). The nutritional status (Ca, K, Mg, P) of the field site was determined prior to GBH treatment and the results have been reported in the study of Hagner et al. (2019). The mean annual and summer (May-July) temperatures in 2016 were 6.4°C and 16.6°C, respectively and the yearly precipitation 495 mm in 2016. In June 2016, the soil for the greenhouse experiment was collected from the field experiment two weeks after a GBH treatment to follow the manufacturer´s safety recommendations and divided into 34 pots (Ø 19cm; 17 controls, 17 GBH-treated). The pots were transferred into a licensed quarantine greenhouse in Ruissalo Botanical Garden for the bioassay.

Organic cv. ‘Ditta’ potatoes were planted in the pots with the GBH-treated and control soils, and the pots were then fully randomized into the greenhouse. The plants were grown in ambient June-July day- length in southwest Finland (about 17-19 h daylight) under a 20 °C/15 °C day/night temperature.

After 3.5 weeks of plant growth, small 1^st instar larvae were introduced to the potato plants (five larvae per plant), which were covered by light-permeable fabric bags. Nine days later, 37 (20 controls, 17 GBH-treated) 4^th instar larvae were collected for microbiota analyses. The remaining larvae were grown until they dropped from the plant and burrowed into the soil to pupate. Once all larvae had burrowed into the soil, the potato shoots were cut. When the adults emerged, 30 (12 controls, 18 GBH- treated) were collected and used for microbiota analyses to study the possible carry-over effects of GBH. The larvae and adult beetle samples were stored at -80 ºC until DNA extraction. For the

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microbiota analyses, we used pooled samples of the larvae (three pools per treatment group, five larvae in each pool) and adult beetles (four pools per treatment group, three beetles in each pool) to increase the sample volume.

We used the US (Vermont) Colorado potato beetle population collected from the field (44°43´N, 73°20`W) in 2010, which has been since grown in laboratory conditions in the University of Jyväskylä (for a more detailed description of the laboratory conditions, see Lehmann et al. 2015, Margus 2018).

Briefly, prior to the experiment the eggs of the Colorado potato beetles were collected into the petri dishes (diameter 92 mm, Sarstedt, Germany) lined with moisturized filter paper and kept in the climate chambers (Growth Chamber FH-1300, Hi-Point, Taiwan) at 23 °C, under a long day regime (18h of light and 6 h of dark, with 1 h of dim light). The newly hatched larvae were fed ad libitum with fresh leaves and stems of potato (variety Challenger) until they were transferred to the greenhouse experiment. The license for rearing quarantine pest species in laboratory conditions was received from the Finnish Food Authority, Finland (permission 3861/541/2007).

DNA isolation and 16S gene sequencing

The surface of the larvae was cleaned and sterilized by immersion in 70% ethanol for 1 min with gentle, slow stirring. The ethanol was removed by immersion in sterile PBS for 1 min, also with gentle stirring. Five larvae from the same group were pooled in a 2-ml screwcap tube containing 0.25 g of glass sand (Ø 0.1 mm) and four pieces of glass beads (Ø 1.5 mm) for desegregation and DNA extraction, as described previously by Kumar et al. (2016), using the InviMag® stool DNA kit (Invitek, Germany) with agitation in a FastPrep® bead beater (FP120-230, Bio 101 ThermoSavant, Holbrook, NY, USA). The adult beetles were cleaned in ethanol in the same manner as the larvae. The adult beetles were dissected, and the gut (midgut and hindgut) was removed to a sterile petri dish containing

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a sterile buffer. Three guts from the animals in the same group were randomly pooled in a 2-ml tube, and the DNA was extracted as previously described for the larvae.

The total DNA concentrations were measured using a Qubit® 2.0 Fluorometer (Life Technology, Carlsbad, CA, USA) and normalized. A specific 16S rRNA gene region (V3-V4 region) was amplified following the 16S rDNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Cod.

15044223 Rev. A). The primers were selected from Klindworth et al. (2013). The full length primer sequences were: 16S rDNA gene Amplicon PCR Forward (Illumina adapter sequence underlined) =

5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG3' and 16S

rDNA gene Amplicon PCR Reverse (Illumina adapter sequence underlined) = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3'. After 16S rDNA gene amplification, the multiplexing step was performed using Nextera XT Index Kit (FC- 131-2001). One μl of the PCR product was run on a Bioanalyzer DNA 1000 chip to verify the size, the expected size on a Bioanalyzer trace is ~550 bp. The libraries were sequenced using a 2 x 300 bp paired-end run (MiSeq Reagent kit v3) on a MiSeq-Illumina platform (FISABIO sequencing service, Valencia, Spain) according to the manufacturer’s instructions. Obtained reads were searched for residual adaptors using the program Trimmomatic (Bolger et al., 2014), Artifacts and quality check as well as quality trimming were performed using the program prinseq-lite (Schmieder et al., 2011). R1 and R2 reads were then joined using overlapping reads with the program FLASH (Magoč and Salzberg, 2011). The quality filtered sequences were checked for chimera, and the non-chimeric sequences were processed using a QIIME pipeline (version 1.9.0) (Caporaso et al. 2010). The sequences were clustered at 97% of identity into operational taxonomic units (OTUs), and representative sequences were taxonomically classified based on the Greengenes 16S rRNA gene database (version 13.8).

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Statistical analysis

The microbial differences between the two developmental stages and the two treatment groups were analysed using Calypso version 8.56 with data normalised by cumulative sum scaling (CSS), which corrects the bias in the assessment of differential abundance introduced by total-sum normalisation. For the multivariate analysis of bacterial genera, a discriminant analysis of principal components (DAPC) was run considering groups based on developmental stage and GBH treatment. An ANOVA test was used to analyse the differences between the relative abundances of microbial groups in the Colorado potato beetle and between the Shannon diversity index, species richness and Chao1 index.The analyses were performed with samples rarefied to a read depth of 12934.

Results and discussion

Colorado potato beetle larvae and adults were sampled for sequencing, resulting in 1037737 total read counts with a mean of 54617 sequences per sample. A total of 4206 OTUs were obtained.

Our results showed that 1) GBH can affect the microbiota of the Colorado potato beetle and 2) the microbiota differs between the larvae and adults. Contrary to many other studies of various insect species (Arias-Cordero et al. 2012; Hammer et al. 2014; Martinson et al. 2012), we found that the bacterial α-diversity tended to be higher in adults (p=0.065) and the bacterial richness was statistically significantly (p=0.016) higher in adults than in larvae (Fig. 1), even though only the gut tissue was used for the adults as opposed to the whole body for larvae. This should be confirmed in future studies isolating properly only gut bacteria in larvae. A total of 12 bacterial phyla and unclassified bacteria were detected (Fig. 2A). The main phyla in the larvae were Proteobacteria (93%), Firmicutes (4%) and Bacteroidetes (3%). In the adult beetles, the predominant phyla were Proteobacteria (77%), Actinobacteria (11%), Bacteroidetes (6%) and the TM7division group (5%). At the genus level (Fig.

2B), the most abundant groups found in the larvae were Enterobacter (58%), Acinetobacter (9%) and Methylobacterium (4%). In the adult beetles, the three most abundant genera were the same—

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Enterobacter (19%), Acinetobacter (10%) and Methylobacterium (9%)—followed by Sphingomonas (7%) and Agrobacterium (6%), which may be derived from the soil.

Proteobacteria clearly dominates the Colorado potato beetle microbiota in the larvae and adults (Fig. 2A). This result parallels that of a previous study on the species conducted based on culture methods (Muratoglu et al. 2011). In that study, most of the bacteria isolated belonged to the Proteobacteria phyla. A similar pattern has been detected in other insects (Colman et al. 2012; Yun et al. 2014), including some other Coleopteran species (Kim et al. 2017). Several genera from the Proteobacteria phyla have been described as being linked to digestion, detoxification processes, fermentation (Engel and Moran 2013) and the production of the components of aggregation pheromones (Dillon and Charnley 2002).

At the genus level, Enterobacter was the most abundant group, followed by Acinetobacter. Both belong to a class of Gammaproteobacteria (Proteobacteria phylum) (Fig. 2B). Some Enterobacter and Acinetobacter species were previously found in Colorado potato beetles by Muratoglu et al. (2011) and Chung et al. (2017). The third dominant genus in the Colorado potato beetle was Methylobacterium, which belongs to the class of Alphaproteobacteria. Methylobacterium shows great phenotypic plasticity and is commonly found in soil, water (Lindow and Brandl 2003) and plants, where it appears as an epiphyte or endophyte (Dourado, et al. 2015; Holland 1997). Methylobacterium has been found in insects such as Apion ulicis (Coleoptera), Epiphyas postvittana (Lepidoptera), Cydia ulicetana (Lepidoptera) and Sericothrips staphylinus (Thysanoptera) (Yamoah et al. 2008).

The detected differences in microbiota between the larvae and adults can be related to the changes that occur during the metamorphosis from larvae to pupa and adult (Moll et al. 2001; Yun et al. 2014) or to differences in diet (Hammer et al. 2014) and living environments. However, in our current study,

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different tissues were used for the two life stages, so it is difficult to determine what impact that had on the composition of the microbial community. In addition, it has been proposed that bleach-based surface sterilization can be a more effective method to remove bacteria that may adhere to the outside cuticle (Binetruy et al., 2019) and it should be considered in future studies. In the case of the Colorado potato beetle, the larvae and adults occupy the same niches and feed on potato leaves in nature.

However, in the present study, the newly hatched adults were collected before they started to feed on potato leaves. On the other hand, we cannot rule out the possibility of soil microbiota affecting the beetles during their two weeks pupal stage in the soil. The pupa may further directly expose to GBH residues at their pupal stage, which could at least partly affect the bacterial composition of the adult Colorado potato beetles. The possible direct changes in soil microbiota or indirect changes in potatoes caused by the GBH treatment did not appear to explain the diversity of the Colorado potato beetle gut microbiota.

Neither the larvae nor the adults showed significant differences in the Shannon α-diversity index and microbial richness between the GBH treatment and control groups (Fig. 3), although in the larvae a slightly lower bacterial diversity and richness was recorded for the GBH-treated group compared to the controls (p = 0.11 and p = 0.074, respectively). However, the GBH treatment did partly explain the microbial composition, especially at the genus level. In the discriminant multivariate test (DAPC analysis) (Fig. 4), the beetles were separated according to treatment group and developmental stage. A clear separation in the OTU level was observed between the developmental stages, which explained 42% of the microbiota variation. In contrast, the GBH exposure explained only 10% of the variation.

The DAPC plot showed a higher variation in microbial composition between the treatment groups at the OTU level in the adult beetles compared to the larvae, which may be due to the overall higher microbial diversity and richness of the adults compared to the larvae. However, the sample size of this

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study was relatively low, which may affect the power of the statistical test. Therefore, the results should be confirmed in future studies with higher sample size together with microbial activity.

At the genus level (Figure 5), the relative abundance of amplicons derived from Agrobacterium was significantly higher in the larvae and adults in the GBH-treatment group compared to the control group (p = 0.02 and p = 0.0032, respectively). It has been shown that some strains of Agrobacterium genus, such as the Agrobacterium tumefaciens strain CP4, contain a Class II EPSPS enzyme that is not inhibited by glyphosate (Padgette et al. 1995; Tian et al. 2012). This resistance may be associated with the higher Agrobacterium relative abundance in the beetles of GBH-treatment group, but a species- level bacterial identification would be needed to confirm the presence of A. tumefaciens (or other bacteria that contains the Class II EPSPS enzyme) in the beetle microbiota. In the larvae, the relative abundance of amplicons derived from unclassified Streptomycetaceae genus increased and the Ochrobactrum genus decreased in relation to GBH treatment (p = 0.026 and p = 0.011, respectively).

These changes were not detected in the adults. However, relative abundance of several other bacterial genera, such as unclassified Enterobacteriaceae (p = 0.02), Rhodobacter (p = 0.000099), Rhizobium (p

= 0.033) and Acidovorax (p = 0.048), were reduced in the adult beetles in GBH treatment compared to the controls. The lower relative abundance of these bacterial genera in the adults of GBH treatment group but not in the larvae may be related to the higher bacterial diversity and richness observed in the adults compared to the larvae. However, the differences in microbial sensitivity to GBH, depending on the EPSPS class they produce (Van Bruggen et al. 2018), may also partly explain the microbial differences at the genus level between the treatment groups.

The GBH-derived consequences of the changes in microbial composition of the Colorado potato beetle are not known and future studies are needed to clarify if the changes in taxonomic composition reflect changes in functionality. The observed microbial differences at the genus level between the treatment groups suggest a bacterial sensitivity to GBH. Therefore, further analyses at the species level are

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needed to determine the EPSPS classes and the sensitivity of those bacteria to GBH. The ingestion of GBH residues has been shown to affect, for example, the navigation system and behaviour of the honeybee (Herbert et al. 2014; Sol Balbuena et al. 2015) in ways that could have long-term negative effects on behaviour and survival. Van Bruggen et al. (2018) have pointed out the possibility of microbiota-mediated health effects associated with chronic indirect exposure to GBH due to the accumulation of GBH in the environment. For example, the gut microbiota dysbiosis associated with GBH has been shown to increase the susceptibility of cattle and poultry to certain diseases and pathogens (Ackermann et al. 2015; Krüger et al. 2013; Shehata et al. 2013). On the other hand, some of the determined bacterial genera, such as the candidate division TM7, Sphingomonas and Agrobacterium, may not be derived from the beetle´s microbiota and may simply be transiently associated with the insect, either through ingestion or adherence to the cuticular surface. As transient microorganism can impact resident communities by direct and indirect interactions, future studies should clarify how the GBH treatment affect both transient microorganisms and resident microbiota in composition and activity. Thus, complementary research is urgently needed to examine the relationship between GBH and gut microbiota and to determine the possible threshold levels of GBH residues affecting the microbiota of non-target species.

In conclusion, the present study suggested differences in the microbiota of the Colorado potato beetle between the two developmental stages and treatment groups (GBH vs. control). The adult beetles had a higher overall microbial diversity and richness compared to the larvae, but neither diversity nor richness were significantly affected by the GBH treatment. However, the microbial composition of the beetles was changed by the GBH treatment, and the difference was more pronounced in the adults than in the larvae. The indirect GBH exposure especially favoured the abundance of Agrobacterium in both developmental stages, which may be related to the tolerance of some Agrobacterium species to

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glyphosate. On the other hand, the reduction of unclassified Enterobacteriaceae, Rhodobacter, Rhizobium and Acidovorax in the adult beetles and Ochrobactrum in the larvae after GBH treatment suggested the sensitivity of those bacteria to GBH. That said, the consequences of glyphosate-induced changes in the microbiota and the function of those bacteria in the Colorado potato beetle remains unknown and needs further study at the bacterial-species level.

Funding

This study is funded by the Academy of Finland (grant no. 311077 to MH), the Tiina and Antti Herlin Foundation (MR) and the Alfred Kordelin Foundation (MR).

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgements

We thank Prof Leena Lindström and Aigi Margus from the University of Jyväskylä for providing the Colorado potato beetle larvae for this experiment. We also thank Maija Jortikka, Anna Pauna and Otto Saikkonen for their help during the experiment and Otto Selenius for the DNA extractions.

References

Ackermann W, Coenen M, Schrodl W et al. The influence of glyphosate on the microbiota and production of botulinum neurotoxin during ruminal fermentation. Curr Microbiol 2015;70: 374-82.

Arias-Cordero E, Ping L, Reichwald K et al. 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 2012;7: e51557.

Bentley R. The shikimate pathway - a metabolic tree with many branches. Crit Rev Biochem Mol Biol 1990;25:

307-84.

Binetruy, F., Dupraz, M., Buysse, M. et al. Surface sterilization methods impact measures of internal microbial diversity in ticks. Parasit Vectors 2019;12:268.

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics;

30 (15):2114-20.

Brauman A, Doré J, Eggleton P et al. Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits. FEMS Microbiol Ecol 2001;35: 27-36.

Broderick NA, Raffa KF, Goodman RM et al. 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.

Caporaso JG, Kuczynski J, Stombaugh J et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010;7: 335-336.

(15)

Claus SP, Guillou H, Ellero-Simatos S. The gut microbiota: a major player in the toxicity of environmental pollutants? volume 2: npj Biofilms and Microbiomes, 2016, 16003.

Chung SH, Scully ED, Peiffer M et al. Host plant species determines symbiotic bacterial community mediating suppression of plant defences. Sci Rep 2017; 7:39690.

Colman DR, Toolson EC, Takacs-Vesbach CD. Do diet and taxonomy influence insect gut bacterial communities? Mol Ecol 2012;21: 5124-37.

Dillon R, Charnley K. Mutualism between the desert locust Schistocerca gregaria and its gut microbiota. Res Microbiol 2002;153: 503-9.

Dillon RJ, Vennard CT, Buckling A et al. Diversity of locust gut bacteria protects against pathogen invasion.

Ecol Lett 2005;8: 1291-8.

Dourado MN, Neves AAC, Santos DS et al. Biotechnological and agronomic potential of endophytic pink- pigmented methylotrophic methylobacterium spp. Biomed Res Int 2015.

Dowd PF, Shen SK. The contribution of symbiotic yeast to toxin resistance of the cigarette beetle (Lasioderma serricorne). Entomol Exp Appl 1990;56: 241-8.

Duke SO, Powles SB. Glyphosate: A once-in-a-century herbicide. Pest Manag Sci 2008;64: 319-325.

Engel P, Moran NA. The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev 2013;37: 699-735.

Funke T, Healy-Fried ML, Han H et al. Differential inhibition of Class I and Class II 5-Enolpyruvylshikimate-3- phosphate synthases by tetrahedral reaction intermediate analogues. Biochemistry 2007;46: 13344-51.

Giesy JP, Dobson S, Solomon KR. Ecotoxicological risk assessment for Roundup (R) Herbicide. In: Ware GW (ed.) Rev Environ Contam Toxicol 167. New York: Springer, 2000, 35-120.

Hagner M, Mikola J, Saloniemi I et al. Effects of a glyphosate-based herbicide on soil animal trophic groups and associated ecosystem functioning in a northern agricultural field. Sci Rep 2019; 9:8540.

Hallmann CA, Foppen RPB, van Turnhout CAM et al. Declines in insectivorous birds are associated with high neonicotinoid concentrations. Nature 2014;511: 341.

Hammer TJ, Bowers MD. Gut microbes may facilitate insect herbivory of chemically defended plants. Oecol 2015;179: 1-14.

Hammer TJ, McMillan WO, Fierer N. Metamorphosis of a butterfly-associated bacterial community. PLoS One 2014;9: e86995.

Haslam E. Shicimic Acid: Metabolism and Metabolites. Chichester, UK: Wiley, 1993.

Helander M, Pauna A, Saikkonen K & Saloniemi I. Glyphosate residues in soil affect crop plant germination and growth. Sci Rep 2019;9: 19653.

Helander M, Saloniemi I, Omacini M et al. Glyphosate decreases mycorrhizal colonization and affects plant-soil feedback. Sci Tot Environ 2018;642: 285-91.

Helander M, Saloniemi I, Saikkonen K. Glyphosate in northern ecosystems. Trends Plant Sci 2012;17: 569-74.

Herbert LT, Vazquez DE, Arenas A et al. Effects of field-realistic doses of glyphosate on honeybee appetitive behaviour. J Exp Biol 2014;217: 3457-64.

Holland MA. Methylobacterium and plants. Rec Res Dev Plant Physiol 1997;1: 207-13.

Jin Y, Liu J, Wang L et al. Permethrin exposure during puberty has the potential to enantioselectively induce reproductive toxicity in mice. Environ Int 2012;42: 144-51.

Jin Y, Liu Z, Peng T et al. The toxicity of chlorpyrifos on the early life stage of zebrafish: A survey on the endpoints at development, locomotor behavior, oxidative stress and immunotoxicity. Fish Shellfish Immun 2015;43: 405-14.

Jin YX, Wu SS, Zeng ZY et al. Effects of environmental pollutants on gut microbiota. Environ Pollut 2017;222:

1-9.

Karasov WH, Martinez del Rio C. Physiological ecology: how animals process energy, nutrients, and toxins.

Princeton: Princeton University Press, 2007.

Kim JM, Choi MY, Kim JW et al. Effects of diet type, developmental stage, and gut compartment in the gut bacterial communities of two Cerambycidae species (Coleoptera). J Microbiol 2017;55: 21-30.

Klindworth A, Pruesse E, Schweer T et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 2013; 41:e1.

(16)

Krüger M, Shehata AA, Schrödl W et al. Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum. Anaerobe 2013;20: 74-8.

Kumar H, du Toit E, Kulkarni A et al. Distinct patterns in human milk microbiota and fatty acid profiles across specific geographic locations. Front Microbiol 2016;7.

Lehmann P, Lyytinen A, Piiroinen S et al. Latitudinal differences in diapause related photoperiodic responses of European Colorado potato beetles (Leptinotarsa decemlineata). Evol Ecol 2015;29: 269-82.

Light SH, Krishna SN, Minasov G et al. An unusual cation-binding site and distinct domain–domain interactions distinguish Class II enolpyruvylshikimate-3-phosphate synthases. Biochem 2016;55: 1239-45.

Lindow SE, Brandl MT. Microbiology of the phyllosphere. Appl Environ Microbiol 2003;69: 1875-83.

Liu Q, Shao WT, Zhang CL et al. Organochloride pesticides modulated gut microbiota and influenced bile acid metabolism in mice. Environ Pollut 2017;226: 268-76.

Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies.

Bioinformatics. 2011; 27:2957-63.

Mardinoglu A, Shoaie S, Bergentall M et al. The gut microbiota modulates host amino acid and glutathione metabolism in mice. Mol Syst Biol 2015;11.

Margus, A. Adaptation to stressful environments: invasion success of the Colorado potato beetle (Leptinotarsa decemlineata)." JYU dissertations 2018;11.

Martinson VG, Moy J, Moran NA. Establishment of characteristic gut bacteria during development of the honeybee worker. Appl Environ Microbiol 2012;78: 2830-40.

Mesnage R, Defarge N, de Vendomois JS et al. Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem Toxicol 2015;84: 133-53.

Moll RM, Romoser WS, Modrakowski MC et al. Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol 2001;38: 29-32.

Motta EVS, Raymann K, Moran NA. Glyphosate perturbs the gut microbiota of honey bees. Proc Nat Acad Sci 2018;115: 10305.

Muratoglu H, Demirbag Z, Sezen K. The first investigation of the diversity of bacteria associated with Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Biologia 2011;66: 288-93.

Myers JP, Antoniou MN, Blumberg B et al. Concerns overuse of glyphosate-based herbicides and risks associated with exposures: a consensus statement. Environ Health 2016;15: 19.

Nielsen LN, Roager HM, Casas ME et al. Glyphosate has limited short-term effects on commensal bacterial community composition in the gut environment due to sufficient aromatic amino acid levels. Environ Pollut 2018;233: 364-76.

Padgette SR, Kolacz KH, Delannay X et al. Development, identification, and characterization of a glyphosate- tolerant soybean line. Crop Sci 1995;35: 11.

Priestman MA, Funke T, Singh IM et al. 5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate. FEBS Lett 2005;579: 728-32.

Ryu J-H, Ha E-M, Lee W-J. Innate immunity and gut–microbe mutualism in Drosophila. Dev Comp Immunol 2010;34: 369-76.

Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics; 27:863-4.

Shehata AA, Schrodl W, Aldin AA et al. The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Curr Microbiol 2013;66: 350-8.

Sol Balbuena M, Tison L, Hahn ML et al. Effects of sub-lethal doses of glyphosate on honeybee navigation. J Exp Biol 2015.

Tian YS, Xu J, Xiong AS et al. Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from Halothermothrix orenii H168 in Escherichia coli and transgenic Arabidopsis. Appl Microbiol Biotechnol 2012;93: 241-50.

Van Bruggen AHC, He MM, Shin K et al. Environmental and health effects of the herbicide glyphosate. Sci Tot Environ 2018;616: 255-68.

Vereecken H. Mobility and leaching of glyphosate: a review. Pest Manag Sci 2005;61: 1139-51.

Warnecke F, Luginbühl P, Ivanova N et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 2007;450: 560.

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Woodburn AT. Glyphosate: production, pricing and use worldwide. Pest Manag Sci 2000;56: 309-12.

Xia XF, Sun BT, Gurr GM et al. Gut microbiota mediate insecticide resistance in the diamondback moth, Plutella xylostella (L.). Front Microbiol 2018;9.

Yamoah E, Jones EE, Weld RJ et al. Microbial population and diversity on the exoskeletons of four insect species associated with gorse (Ulex europaeus L.). Aust J Entomol 2008;47: 370-9.

Yun JH, Roh SW, Whon TW et al. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl Environ Microbiol 2014;80: 5254-64.

Figure 1. Bacterial diversity and richness at the OTU level of the Colorado potato beetles microbiota at the different developmental stages (larvae vs. adults). The Y-axis corresponds to the bacterial α-diversity (Shannon), richness and estimated richness (Chao1). Analysis were performed with samples rarefied to read depth of 12934.

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Figure 2. Bacterial compositions at the phylum (A) and genus (B) levels of the Colorado potato beetle microbiota. The group others at the phylum level includes: Planctomycetes, Chloroflexi, Acidobacteria, Chlamydiae, candidate phylum TM6 and Gemmatimonadetes.

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Figure 3. Differences in bacterial diversity and richness of the Colorado potato beetles microbiota at OTU level among different groups (bg: adult beetles exposed to GBH; bc: control adult beetles; lc: control larvae; lg:

larvae exposed to GBH. The Y-axis corresponds to the bacterial α-diversity (Shannon), richness and estimated richness (Chao1). * (p < 0.05), ** (p<0.01).

Figure 4. Discriminant analysis of principal components (DAPC) plots, showing the effect of the developmental stage of the Colorado potato beetle and exposure to GBH in microbiota at OTUs level. The 10000 most abundant bacterial OTUs were employed. A) DAPC plot revealed distinct clustering among groups (bg: adult beetles exposed to GBH; bc: non-exposed adult beetles; lc: larvae non-exposed; lg: larvae exposed to GBH), B) DAPC plot based on the developmental stage (red: larvae; blue: adults), C) DAPC plot based on the exposure to glyphosate (red: exposed; blue: non-exposed).

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Figure 5. Statistically significant differences at genus level after ANOVA analysis in microbiota of (A) the adults and (B) the larvae of the Colorado potato beetles between GBH treated group (blue) and control group (red). * (p < 0.05), ** (p<0.01),***(p<0.001).