Arsenic transformation mediated by gut microbiota affects the fecundity of Caenorhabditis elegans

https://doi.org/10.1016/j.envpol.2020.113991Get rights and content

Highlights

  • E. coli stably colonized in C. elegans gut.

  • As(III) was detected in guts colonized with E. coli BL21, As(III) and DMAs in guts with BL21M after exposed to As(V).

  • As(III) and DMAs(V) produced by gut microbes affected reproduction of C. elegans.

Abstract

Arsenic biotransformation has been discovered in guts of soil invertebrates. Reproduction of invertebrates is sensitive to arsenic contamination in soils. However, little is known about the impact of gut microbe-mediated arsenic biotransformation on the fecundity of invertebrates. Here, Caenorhabditis elegans was firstly pre-fed with Escherichia coli BL21 possessing the capability of reducing arsenate [As(V)] or BL21M having the ability to reduce As(V) and methylate arsenite [As(III)], then inoculated worms were transferred to inactive E. coli AW3110 (harboring no arsenic transformation gene)-seeded plates treated with As(V) at different concentrations. Quantification of gut microbes showed that both E. coli BL21 and BL21M stably colonized in the guts after worms were cultured on inactive E. coli AW3110-seeded plates for 72 h. The analysis of arsenic species indicated that there was As(III) in C. elegans guts colonized with E. coli BL21, As(III) and dimethylarsinic acid [DMAs(V)] in C. elegans guts with E. coli BL21M exposed to As(V) for 6 h. After treatment of 100 μM As(V), decrease in brood sizes was observed for worms that were colonized with E. coli BL21 or BL21M compared to that with AW3110 in the guts. The levels of vitellogenin (VTG), glutathione S-transferases (GST) and superoxide dismutase (SOD), closely linked to reproduction and antioxidation-linked indicators, were the highest in worms whose guts colonized with E. coli BL21, followed by worms colonized with E. coli BL21M and worms colonized with inactive E. coli AW3110 exposed to As(V). Our results indicated the toxic impact of As(III) and DMAs(V) produced by gut microbes on reproduction of C. elegans. The work provides novel insight into the interplay between arsenic biotransformation mediated by gut microbes and the host fecundity in soils.

Introduction

Arsenic is widely distributed throughout the earth’s crust in a complex with pyrite that can be readily dissociated from the mineral and enters into soils and water (Finnegan and Chen, 2012). Arsenic uptake and metabolism by microorganisms result in high bioavailability to other organisms (Zhu et al., 2017). Arsenic exposure generally disrupts plant metabolism, affects the growth of soil fauna, and induces the germline apoptosis in nematodes (Wang et al., 2019). Long-term exposure to arsenic is associated with human cancers, diabetes, cardiovascular disorders and reproductive defects (Zhu et al., 2014). Toxicity of arsenic to biota depends largely on its species. Relative toxicities of several arsenicals are as follows, monomethylarsenous acid [MAs(III)] = dimethylarsenous acid [DMAs(III)] > As(III) > As(V) > monomethylarsonic acid [MAs(V)] > DMAs(V) > trimethylarsinic oxide (TMAO) (Sharma and Sohn, 2009). Arsenic biotransformation is majorly mediated by microorganisms in the environment, including As(V) reduction, As(III) oxidation, arsenic methylation, demethylation, and arsenic transport. Fungi, mouse, and humans have been found to methylate As(III) as well (Gosio, 1892; Lu et al., 2014; Gokulan et al., 2018; Chi et al., 2019).

Terrestrial invertebrates are important components of the terrestrial food web (Moriarty et al., 2009). Arsenic concentrations and species in the food chains determine arsenic toxicity to organisms at higher trophic level (Moriarty et al., 2009). Moriarty et al. (2009) find that As(V) and As(III) are the main arsenic species in the terrestrial invertebrates, while methylated arsenic, arsenobetaine, and arsenocholine are present only in trace amounts (Langdon et al., 2002; Moriarty et al., 2009). Various arsenic species in invertebrates may be from arsenic biotransformation mediated by invertebrates or their gut microbiota and enrichment via food chains (Langdon et al., 2002; Moriarty et al., 2009). Most of invertebrates including bark beetles, Drosophila melanogaster and Mamestra configurata can’t transform arsenic in vivo by themselves (Rizki et al., 2006; Morrissey et al., 2007; Reimer et al., 2010), whereas gut microbiota of them have been recently demonstrated to contribute significantly to As(V) reduction in the earthworm Metaphire sieboldin (Wang et al., 2019). In addition, it is well documented that the gut microbiota of mice play an important role in arsenic transformation and its toxic effects on the host (Chi et al., 2019). Nevertheless, limited literature has studied how the gut microbiota of invertebrates involve in the arsenic transformation. Some works demonstrate the importance of arsenic metabolism mediated by gut microbiota to the physical conditions of the hosts (Tremaroli and Bäckhed, 2012), however, interactions between arsenic biotransformation mediated by specific gut microbes and the health of terrestrial invertebrates remain to be elucidated.

Nematodes are the most abundant soil metazoa and serve an important role in the food web of soil ecosystems (Bernard, 1992). Worms can be germ-free through bleaching hatchlings and then assembled diverse gut microbiota after germ-free eggs exposed to different microbial environments (Berg et al., 2016). C. elegans is an emerging model organism for studying environmental toxicology and host-gut microbiota interaction due to its small size, easiness of laboratory maintenance, and short life cycle (Sulston et al., 1983). Germline development and reproduction of C. elegans are very sensitive to toxicant exposure (Monteiro et al., 2014), and it helps to unravel the mechanism of interaction between host physiological activity and gut microbial metabolism in the host under contaminated environments. Besides, exposure to arsenic induces oxidative stress resulting from the imbalance between radical-generating and antioxidant defense system, which represents a stressful and toxic situation of chemicals for C. elegans (Liao and Yu, 2005). Hence, reproduction and oxidative stress can be biomarkers for the healthy state of C. elegans (Liao and Yu, 2005).

Escherichia coli strains are common microbes in the gut of C. elegans and one of the most intensively used engineering bacteria (Zhang et al., 2000). The ars operon of wild type E. coli strains confers arsenical resistance via reducing As(V) and extruding As(III) (Tseng et al., 2007). E. coli strains as facultative anaerobes can potentially colonize in the gut of C. elegans and involve in arsenic transformation under anoxic environment. To unveil the effect of arsenic reduction and methylation mediated by gut microbiota on the host fecundity, three types of E. coli strains were used in this study. E. coli AW3110, which loses the ability to reduce As(V) to As(III) (Carlin et al., 1995), was used as the control. E. coli BL21 containing arsRBC was served as ArsC donor to reduce As(V). E. coli BL21M bearing arsRBC and As(III) S-adenosine methyltransferase gene (arsM) from Synechocystis sp. PCC 6803 can generate As(III) and methylated arsenic from As(V) (Xue et al., 2017). Our objectives were to i) investigate the colonization of different of E. coli strains in the gut of C. elegans; ii) identify arsenic transformation occurred in the gut of C. elegans; iii) explore the fecundity and oxidative stress level of C. elegans after being exposed to different arsenic species produced by various gut microbes.

Section snippets

C. elegans, bacterial strains, and plasmids

C. elegans strain (Bristol) N2 was maintained on nematode growth media (NGM) seeded with E. coli OP50 at 25 °C (Brenner, 1974). E. coli strains were grown aerobically in Luria Broth (LB) medium at 37 °C supplemented with 30 μg mL−1 chloramphenicol (for E. coli AW3110) or 50 μg mL−1 kanamycin (E. coli BL21M) as required. Information about all strains and plasmids are presented in Table S1.

Single colonies of E. coli AW3110, BL21 or BL21M were inoculated into 5 mL of LB medium consisting of the

Reproduction of C. elegans exposed to As(V)

There were no differences in brood sizes between worms pre-fed with E. coli BL21, BL21M, and inactive E. coli AW3110 after being treated with 0, 10 and 50 μM As(V) (Fig. 1a and b). Brood sizes of worms pre-fed with E. coli BL21 and BL21M were significantly (P < 0.05) lower than those pre-fed with E. coli AW3110 under exposure to 100 μM As(V) (Fig. 1a and b). No difference in brood sizes between worms pre-fed with E. coli BL21 (135.78 ± 7.70 eggs per worm) and those pre-fed with E. coli BL21M

Discussion

Interplay between host and gut microbiota in a variety of animals demonstrates the significant importance of gut microbiota to the host’s health, development, resource utilization and immune protection (Clemente et al., 2012; Erkosar and Leulier, 2014). To ensure arsenic biotransformation mediated by the gut microbiota, germ-free eggs were pre-grown on live E. coli BL21 or BL21M-seeded plates up to L4 larva stage, then transferred to inactive E. coli AW3110 and exposed to As(V). The stable

Conclusion

In this study, we employed E. coli strains BL21 and BL21M as model bacteria. Both strains successfully colonized in the gut of C. elegans and contributed a lot to As(V) reduction and DMAs formation in the gut of worms treated with As(V). The generation of As(III) and DMAs by gut microbes significantly induced the increases of VTG, GST and SOD levels which are connected with reproduction and oxidative stress defense of worms. Toxicity test further showed that arsenic biotransformation mediated

CRediT authorship contribution statement

Guo-Wei Zhou: Methodology, Investigation, Formal analysis, Visualization, Writing - original draft. Xiao-Ru Yang: Writing - review & editing, Visualization. Fei Zheng: Investigation, Writing - review & editing. Zi-Xing Zhang: Resources, Writing - review & editing. Bang-Xiao Zheng: Writing - review & editing. Yong-Guan Zhu: Conceptualization, Project administration, Writing - review & editing. Xi-Mei Xue: Methodology, Investigation, Formal analysis, Visualization, Project administration, Writing

Declaration of competing interest

We declare that we have participated sufficiently in the work to take public responsibility for the appropriateness of the experimental design and method, and the collection, analysis, and interpretation of the data. We have reviewed the final version of the manuscript and all authors have agreed to all the contents in the manuscript, including the data as presented. The authors declare no competing financial interest.

Neither the entire paper nor any part of its content has been published or

Acknowledgements

We thank Dr. Ya-Mei Wang (Xiamen University) for kindly providing the wild-type C. elegans strain N2 (Bristol) obtained from Caenorhabditis Genetics Center funded by the NIH National Center for Research Resources. This work was supported by the National Natural Science Foundation of China (41877422) and Science and Technology Project of Fujian Province (2017Y0082).

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