Effect of microplastic pollution on the gut microbiome of anecic and endogeic earthworms

Abstract Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.


Introduction
Earthworms are well known as soil ecosystem engineers and soil health indicators .T hey ar e r elated to differ ent nutrient cycles, nitr ogen miner alization, phosphorus plant absor ption, and litter decomposition.The products of their activities are generally beneficial to other soil inv ertebr ates and to the plants, enhancing their growth and productivity (Blouin et al. 2013 ).Evidence also shows that earthworms contribute toward the formation of the structure of soil microbial community, either directly or indirectly (Brown andDoube 2004 , Medina-Sauza et al. 2019 ).
Based on feeding habits, earthworms are categorized in three groups: epigeic , anecic , and endogeic.Epigeic earthworms live on the soil surface and feed from litter.Endogeic earthworms live in the soil, produce horizontal tunnels, and feed on mineral soil and decomposed material.Anecic earthworms move between soil layers making v ertical burr ows and feed on the litter that they dr a g into their burrows (Capowiez et al. 2014 ).
The div erse micr obiome of earthworm hosts' gut plays an important role in their nutrient metabolism, immunity, and physiology (Liu et al. 2018 ).Mor eov er, the worm casts contain bacteria from the gut that contribute to several soil ecosystem functions such as nitrogen fixation (Drake et al. 2006 ) or vermiremediation processes , i.e .dissipation of organic contaminants in the soil (Rodriguez-Campos et al. 2014 ).The food source was found to affect the earthworm gut microbiome, but the core microbiomes r emained lar gel y unc hanged (Liu et al. 2018, Sa pk ota et al. 2020 ), indicating that the earthworms are k e y contributors of functional bacteria to the soil microbial communities, despite their lifestyle (Wang et al. 2022 ).
Anthropogenic soil pollution caused by the disposal of waste from industrial and urban sources or a gricultur al pesticides can interfere with soil microbiome and earthworm gut microbiome.An emerging type of pollution is the increasing load of microplastics (MPs; defined as plastics < 5 mm) in both aquatic and terr estrial envir onments (EC, Scientific Opinion 6/ 2019 ).Wher eas problems with MPs for functioning of aquatic ecosystems are intensiv el y studied, their envir onmental impacts in the terr estrial ecosystem remain largely unexplored.A recent study revealed that both polyethylene-based and bio-based MPs can hav e str ong effects on the assembly of the rhizosphere communities (Wesselink et al. 2023 ).According to the literature, a gricultur al land may contain more MPs than oceans.Initial quantifications suggest that bac kgr ound concentr ations of MP might be as high as 1%-7% in a gricultur al and industrial soils (Rillig 2012 ).
The effect of MPs on earthworm behavior and on earthworm's gut microbiome is largely unknown.Guo et al. ( 2023 ), in a liter atur e r e vie w, discov er ed that most of the highl y cited articles describe the toxic effects of MPs on earthworm's growth and metabolism, including o xidati v e str ess and DNA dama ge.Onl y a few studies focused on the shifts of the composition of earthworm's gut microbiome after ingestions of plastic (Zhu et al. 2018, Cheng et al. 2021 ), usually in the presence of another pollutant like heavy metals (Wang et al. 2019, Yang et al. 2022 ) or polycyclic ar omatic hydr ocarbons (Xu et al. 2021 ).Ho w e v er, these studies wer e mostl y performed on earthworms with an epigeic lifestyle and there is a knowledge gap on the effect of MPs on the gut microbiome of endogeic and anecic earthworms.
Based on the abo ve , we in vestigated the hypothesis that the presence of MPs in soil will be reflected in the bacterial communities of the gut of both endogeic and anecic earthworms .T he main aim of this study was to e v aluate the effect of MP pollution (in a r elativ el y low concentration of 0.2% low-density polyethylene) on the growth and survival of the earthworms Aporrectodea caliginosa (endogeic) and Lumbricus terrestris (anecic) and on the composition of the gut bacterial communities (gut microbiome) using 16S rRNA gene amplicon sequencing.

Prepar a tion of soil with MPs
Lo w-density poly ethylene (LDPE) (Riblon, TER Hell Plastic GmbH) MPs wer e pr epar ed accor ding to Huerta Lw anga et al. ( 2016) with some modifications: LDPE was frozen with liquid nitrogen and fr a gmented manuall y into irr egular sha pes, between 100 and 150 μm, simulating the size found in soils of agricultural areas where plastic is frequently used (Huerta Lwanga et al. 2023 ).The MP particles wer e thor oughl y mixed into sandy soil (26.6% brown sand, 24% silver sand, and 50% loamy silt with 0.2% organic matter) at a final concentration of 0.2% w/w (Huerta Lwanga et al. 2023 ) and 20% moistur e content, equiv alent to 3% higher than field ca pacity.MP concentr ation was c hosen on account of (i) being envir onmentall y r ele v ant for soils under human pr essur e (de Souza Machaldo et al. 2019 ) and (ii) producing a blocking effect on water filtration in soils (Liu et al. 2022 ).Two hundr ed gr ams of sandy soil and sandy soil with LDPE MPs were placed in eight glass pots, r espectiv el y, totaling 16 pots (10.5 cm × 10.3 cm).

Earthworm microcosm experiment
Lumbricus terrestris and A. caliginosa , which correspond to anecic and endogeic lifestyles, r espectiv el y, wer e obtained fr om Wormenwekerij Wasse Company (Beilen, The Netherlands) and organic fields of Br abant, r espectiv el y.The worms were starved for 24 h before the start of the experiment, so as to have empty guts.Adult earthw orms w er e installed separ atel y per glass pot to a total initial biomass per pot of 8.18 ± 1.2 g for L. terrestris and 1.75 ± 0.07 g for A. calaginosa , which corresponded to a final number of two anecic worms per pot and three to five endogeic worms per pot.(Fig. 1 ).Then the pots were incubated at 15 • C, in darkness, since the earthworms ar e photosensitiv e .T he soil moisture content was maintained at 20 ± 2% (av er a ge envir onmental conditions observed in the Netherlands during autumn) by weighting the pots at the beginning of the trial and by the use of a time-domain reflectometry (TDR) moisture sensor.Earthworm incubation time was 55 da ys , in order to assure intestinal bacteria adaptation to the new conditions (minimum 49 days; Chao et al. 2021 ).

Earthworm measurements
The weight of the earthworms was recorded before placement in the glass pots and at the end of the incubation period, for the earthworms that were still alive.Once the weight was recorded, the earthw orms w er e fr ozen at −18 • C for assessment of the gut microbiome.If the earthworms were found dead, they were immediatel y fr ozen and their w eight w as not recor ded for av oiding contamination, since their body was very fragile .T he gain and loss of weight of the earthworms that were alive till the end of the incubation period was calculated according to Equation ( 1 ), where K gr is the growth rate , Mor g 1 is the initial individual weight, Morg 2 is the individual weight at the end of the incubation period, and t is the experimental time. (1)

Sta tistical anal ysis
Significant differences of the earthworms' growth rate among tr eatments wer e e v aluated with the t -test, once the normality of the data was verified, or the nonparametric Mann-Whitney U test (or Wilcoxon rank-sum test) if the data did not follow the normal distribution.

Earthworm's gut microbiome DNA extraction and sequencing
In sterile conditions (UV cabinet), earthworms were opened according to Huerta Lwanga et al. ( 2018) for extracting the gut content, which was immediately frozen at −18 • C. DN A w as extr acted fr om the earthw orm gut samples using the DNeasy Po w-erSoil Pro Kit (Qiagen) following the manufacturer's instructions.Extracted DNA quality was assessed via electr ophor esis on 0.8% a gar ose gel and DNA concentr ation was determined with a NanoDr op spectr ophotometer (Thermo Fisher Scientific).Subsequently, DN A w as subjected to amplicon sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene using Illlumina MiSeq 300 pair ed-end tec hnology in the sequencing center Base-Clear B.V., Leiden, Netherlands .T he amplification was performed with the primer set 341f 5 -CCTA CGGGNGGCWGCA G-3 and 785r 5 -GA CTA CHV GGGTATCTAATCC-3 (Thijs et al. 2017 ).

Earthworm's gut microbiome analysis
Primers wer e r emov ed fr om the amplicon sequences with the "Cutada pt" tool (v ersion 4.3; Martin 2011 ) and the "dada2" pac ka ge (version 1.26.0;Callahan et al. 2016 ) of the R language for statistical computing (version 4.0.5;released 2021-03-31) (R Core Team 2020 ), following the instructions in D AD A2 ITS Pipeline Workflow (version 1.8).Initial quality control sho w ed that read 2 contained se v er al low-quality base pairs, which, when trimmed, led to a reduced number of high-quality reads and merged sequences compared to running the analysis with only read 1. T herefore , bacterial community analysis was performed with high-quality read 1 sequences.Read 1 sequence data ar e publicl y av ailable in the National Centre for Biotechnology Information (NCBI) under Bio-Project accession number PRJNA1051054.After primer r emov al, the r educed amplicon sequences wer e subjected to quality scr eening, c himer a r emov al, gener ation of the amplicon sequence variant (ASV) abundance matrices, and taxonomic classification with the dada2 pac ka ge.Silv a pr okaryotic small subunit (SSU) rRNA gene taxonomic training data formatted for "dada2", version 138.1, were used for the classification of the amplicon sequences.ASVs that were classified as mitoc hondria or c hlor oplasts and ASVs that wer e pr esent in onl y one sample wer e r emov ed fr om further anal ysis.Micr obial div ersity cov er a ge was assessed via r ar efaction curv es pr epar ed with the R pac ka ge "v egan" (v ersion 2.6.4;Oksanen et al. 2019 ) and the percentage of total species represented in a sample (Good 1953 ) was estimated with the R pac ka ge "entr opart" (v ersion 1.6.13;Marcon and Hérault 2015 ).The bacterial communities' α-diversity Figure 1.A schematic representation of the experimental setup.A total of 16 glass pots were filled with sandy soil and LDPE MPs were added into 8 of them at a concentration of 0.2% w/w.Adult earthworms belonging to A. caliginosa and L. terrestris were installed into the pots to a final number of three to five and two worms per pot, r espectiv el y.After incubating for 55 da ys , the gut of three worms from each treatment was collected and its bacterial community was analyzed.
F igure 2. Gro wth rate of endogeic earthworms in the absence and presence of LDPE MPs.Growth rates for each earthworm are presented for treatments without LDPE MPs (left) and for treatments with LDPE MPs (right).On the top right, the P -value of pairwise comparison test of the growth rates between the two MP treatments is presented.was e v aluated by calculating the observ ed ric hness (S), Pielou's e v enness index (Pielou 1966 ), Shannon index (Spellerberg and Fedor 2003 ), and Gini-Simpson index (Roswell et al. 2021 ) with the R pac ka ge "micr obiome" (v ersion 1.21.1;Lahti and Shetty 2012 ).The difference of the α-diversity indices between presence and absence of MPs for each earthworm was assessed with pairwise comparison using the t -test, if the indices in test were normally distributed, or the Wilcoxon rank-sum test if not.Canonical corr espondence anal ysis (CCA; ter Br aak and Verdonsc hot 1995 ) or r edundancy analysis (Israels 1984 ), accompanied by permutational anal ysis of v ariance, was used to estimate the β-diversity between tr eatments for eac h earthworm.The anal ysis to be performed was determined by the first axis length of detrended correspondence analysis (Lepš and Šmilauer 2003 ), which was carried out on Hellinger-transformed abundance matrices (Legendre and Galla gher 2001 ).Afor ementioned anal yses wer e conducted with the R pac ka ge "v egan".Lastl y, differ ential abundance anal ysis was performed with the nonparametric Kruskal-Wallis test after transforming the ASV abundances with robust centered log ratio (rclr) (Martino et al. 2019 ) of the "decostand" command of the "vegan" R pac ka ge.

Earthw orm surviv al and gro wth r a te
All anecic earthw orms, regar dless of MP treatment, were found dead at the end of the incubation period (day 55); ther efor e, gr owth r ates wer e calculated onl y for the endogeic earthworms.In our experiment, the anecic earthworms lived for 25 days in the control and for 21 days in the treatment with LDPE MPs .T he anecic earthworms normally feed on soil and litter, and in order to provide similar conditions for both earthworm categories, both categories did not have litter on the soil surface .F eeding conditions are well known to influence earthworm's performance (Liu et al. 2018 ).
All in all, endogeic earthw orms lost w eight after 55 da ys , which is evident by the negative growth rates (Fig. 2 ), and LDPE MP treatment did not seem to have an effect on the weight loss of endogeic earthworms ( P -value .614)(Fig. 2 ).The lack of effect from the LDPE MPs could be due to the low concentration of the MPs used in the pr esent r esearc h.Se v er al studies hav e also shown that the adverse effect of MPs on earthworm growth and behavior is onl y pr esent in higher concentr ations of MPs.Cao et al. ( 2017)

Bacterial di v ersity in earthworm gut
The sequencing effort of the earthworms' gut microbiome yielded 19 956-28 874 amplicon sequences, which resulted in 11 849-20 476 high-quality sequences after quality control analysis ( Supplementary Table 1 ).The sample sizes correspond to the plateau of the r ar efaction curv es ( Supplementary Fig. 1 ), and the Good's cov er a ge index for all samples is 1 ( Supplementary Table 1 ), indicating adequate cov er a ge of the bacterial species richness.
The gut microbiome composition of anecic and endogeic earthw orms w as div erse, and v ariation was observ ed between the r eplicates of each earthworm.The bacterial communities of the L. terrestris gut were dominated by Firmicutes (70% ± 15.06), and more specifically by Peptostreptococcaceae , Clostridiaceae , and Enterobacteriaceae families, which, on av er a ge, accounted for 42.4% ± 25.1%, 14.1% ± 14.0%, and 7.1% ± 11.1% of the total microbial comm unity, r espectiv el y (Fig. 3 , Supplementary Table 2 ).Other highly abundant gr oups wer e Enterobacteriaceae and Micrococcaceae families, which accounted for 12.8% ± 15.1% and 1.4% ± 1.4%, respectiv el y.The gut bacterial communities of A. caliginosa comprised mostly of Firmicutes, Actinobacteriota, and Proteobacteria (also known as Pseudomonadota), with av er a ge r elativ e abundances in both treatments, of 40.8% ± 11.6%, 31.0%± 14.4%, and 18.6% ± 7.8%, r espectiv el y (Fig. 3 , Supplementary Table 2 ).Contrasting to anecic earthworms, Bacillaceae and Micrococcaceae families were highly abundant with 21% ± 3% and 6.8% ± 2.8% av er a ge r elativ e abundances in both treatments (Fig. 3 , Supplementary Table 2 ).The observed dominance of Proteobacteria, Firmicutes, and Actinobacteriota in both anecic and endogeic earthworms is in a gr eement with other studies.For example, Sa pk ota et al. ( 2020) sho w ed that the bacterial communities of the gut of earthworms with different lifestyles (endogeic and anecic) were dominated by Proteobacteria, Actinobacteria, and Firmicutes, whereas Liu et al. ( 2018 ) observed that only Proteobacteria were the dominant taxa of the gut microbial communities of E. fetida , an epigeic earthworm.

α-di v ersity of earthworms gut bacterial communities
Aporrectodea caliginosa hosted microbial communities with an aver a ge number of ASVs (observ ed ric hness) of 428.2 ± 188.3 in both treatments .T he bacterial composition was e v en and div erse, with Pielou's e v enness index at 0.86 ± 0.08 and Shannon's and Gini-Simpson's diversity indices at 5.10 ± 1.19 and 0.97 ± 0.04, r espectiv el y (Fig. 4 ).On the other hand, L. terrestris hosted a significantly less rich and diverse gut microbiome compared to the endogeic earthworm A. caliginosa.The observed number of ASVs was limited to 114.5 ± 89.3 species in both MP treatments and Pielou's e v enness was estimated at 0.63 ± 0.12 indicating that the members of the gut microbial communities do not share similar abundances.Shannon's and Gini-Simpson's diversity indices were 2.88 ± 0.91 and 0.86 ± 0.07, respectively (Fig. 4 ).Pairwise comparison sho w ed that the α-diversity indices of the gut micr obiome wer e notabl y differ ent betw een the tw o earthw orms ( P-value < .05,Fig. 4 ), although, no effect was evident of the MPs treatment in each earthworm ( P -value > .05,data not shown).The lack of effect of the presence of MPs could be attributed to the low amount of MPs in the soil.Similar results were obtained by Zhu et al. ( 2018 ) who tested the influence of various concentrations of pol ystyr ene MPs (0%, 0.025%, 0.5%, and 10% pol ystyr ene-oatmeal mixture) on the gut microbiome composition of the epigeic earthw orm Ench ytraeus crypticus .The diversity of the microbiome was not affected by the presence of polystyrene MPs at low concentrations, whereas it declined significantly at the highest tested concentration (10%) (Zhu et al. 2018 ).In the same manner, Cheng et al. ( 2021 ) demonstrated that the presence of 0.25% high-density pol yethylene (HDPE) or pol ypr opylene (PP) MPs did not affect significantl y the ric hness and div ersity of the bacterial communities of the anecic earthworm Metaphire guillelmi compared to the earthw orms gro wn without MPs.

Causes of dissimilarity of earthworms' gut microbiome
CCA r e v ealed that both species and MP treatments accounted for 29.2% of the total variation of the gut microbiomes (Fig. 5 ).On the first axis, which accounted for 70.2% of variation, it is evident that the samples ar e separ ated according to earthworm species, whereas on the second axis, which accounts for 29.8% of variation, the samples are separated according to MPs treatment (Fig. 5 ).Hence, the anecic and endogeic life form cause stronger dissimilarity in the gut microbiome.Our results are consistent with the study of Sa pk ota et al. ( 2020), in which they found that the earthworm life-form pr edominatel y sha pes the gut bacterial comm unity structure.
The shift of the ASVs' r elativ e abundance between the different microbiomes, was further explored on account of their distinct separation in the CC A analysis .Differential abundance analysis of the rclr-transformed abundances between each tested earthworm and MP treatment was performed with the nonparametric Kruskal-Wallis test.Clostridia were found at significantly higher r elativ e abundance in L. terrestris , such as Paraclostridium bifermentans (ASV 00002), Terrisporobacter glycolicus (ASV 00014 and 00027), P aeniclostridium sp .(ASV 00004), Anaerosporobacter (ASV 00037), Amnipila (ASV 01110), and Clostridium sensu stricto 1 (ASV 00015) and 13 (ASV 00012, 00026, and 00040), as well as the ASV 00035, a member of the Enterobacteriaceae family (Fig. 6 , Supplementary Fig. 2 ).The vast majority of the ASVs that were found in significantly higher relative abundance in L. terrestris (excluding ASV 00035) belonged to the Firmicutes phylum.Our findings are in a gr eement with the ones from Sapkota et al. ( 2020), who compared the OTUs of the gut bacterial communities between the anecic earthworm Lumbricus herculeus and the endogeic earthworms Allolobophora chlorotica , A. caliginosa , and Aporrectodea tuberculata and they observed that the majority of the OTUs that were in significantly higher abundances in L. herculeus are Firmicutes.Out of all the bacteria that were found in significantly higher relative abundance in L. terrestris ' gut, ASV0002 and ASV00035 were found to be in substantially higher r elativ e abundance in the absence of LDPE MPs, whereas the r elativ e abundance of ASVs 00012, 00014, 00015, 00026, 00027, 00037, and 00040 was increased in the presence of LDPE MPs (Fig. 6 , Supplementary Fig. 2 ).Unlike our findings, Cheng et al. ( 2021 )  the gut bacterial communities of the anecic earthworm Metaphire californica when exposed to pol yvin yl c hloride MPs, observ ed no significant differences comparing to untreated control.On the other hand, Zhu et al. ( 2018 ) in their study of the effect of differ ent pol ystyr ene MP concentr ations on the gut micr obiome of the epigeic earthworm E. crypticus , observed that the presence of 10% MPs resulted in increase in the relative abundance of members of the Bacteroidetes phylum and a decrease in members of the Proteobacteria and Planctomycetes phyla.Several ASVs were pr esent onl y in the endogeic species, A. caliginosa , with or without MP tr eatments suc h as se v en ASVs of the Actinobacteriota phylum (ASV 00314, 00426, 00521, 00583, 00602, 00603, and 00754), three ASVs that belong to Chroflexi orders KD4-96 and Anaerolinae (ASV 00139, 00409, and 00216), five ASVs classified in Bacillales (ASV 00089, 00154, 00158, 00166, and 00709), two ASVs in Paenibacilalles (ASV 00091 and 00132), five Alphaproteobacteria ASVs of the Rhizobiales order (ASV 00080, 00100, 00178, 00249, and 00292), and the ASVs 00266 ( Verrucomicrobiae / Chthoniobacter sp.), 00388 ( Acidobacteriota / Vicinamibacterales ), 00565 ( Planctom ycetes / Pir4 linea ge sp.), and 00711 ( Bur kholderia -Caballeronia -Parabur kholderia sp.).The composition of the bacterial communities of the endogeic earthworms have been less studied comparing to epigeic and anecic worms.Ne v ertheless, similar to our findings, Thakuria et al. ( 2010 ) in their study of bacteria that inhabit the walls of the gut of both endogeic and anecic earthworms, found that the endogeic earthworms A. callaginosa and A. longa contained species that belonged in the Proteobacteria and Actinobacteriota phyla.
All in all, the ASVs that were found in higher relative abundance in the presence of LDPE MPs belonged to the Firmicutes phylum for L. terrestris , and Proteobacteria, Plactomycetota, Actinobacteriota, and Nitr ospir ota for A. calaginosa .The shift of the bacterial communities that were induced by the MPs could lead to enrichment of communities with bacterial families that participate in the depolymerization of MPs.Arpia et al. ( 2021 )  In this study, we demonstrated that both the lifestyle of the worms and the presence of MPs, wer e r esponsible for shifts in the diversity and composition of the bacterial communities in the gut of anecic and endogeic earthworms, with lifestyle being a stronger influence.Pr e vious studies on the effect of MPs on earthworms' gut micr obiome wer e contr adictory, with some of them reporting str ong influence, wher eas others no significant effects .T he great div ersity of r esearc h findings , could ha v e r esulted fr om the div erse parameters that were studied, including the earthworms' species and lifestyles, the material of the plastics, their physicochemical pr operties (sha pe , size , and ad diti v es), and the exposur e concentration, as well as the incubation conditions (soil moisture content and temper atur e).Ther efor e, mor e r esearc h is needed in order to understand the mechanisms and effects of MP pollution in the gut microbiome , and o verall performance of earthworms in MP contaminated soil.

Figur e 3 .
Figur e 3. T he gut bacterial community composition at the family level of anecic ( L. terrestris ) and endogeic ( A. caliginosa ) earthworms when grown in the presence (LDPE MPs) and absence (No MPs) of MPs.

Figur e 5 .
Figur e 5. CC A of the gut bacterial communities of the anecic earthworm L. terrestris and the endogeic earthworm A. caliginosa when exposed to MPs (circle, •) or not (square, ).
Figure 6.Heatmaps of the log-transformed abundances of 25 bacterial ASVs in the gut of the anecic earthworm L. terrestris and the endogeic earthworm A. caliginosa whose abundance was significantly different between each tested earthworm and MP treatment.ASVs are clustered according to Euclidean distances .T he stars in the ASVs' annotation denote the le v el of significance ( * * * for P < .001,* * for P < .01,and * for P < .05).
in their liter atur e r e vie w on MP degr adation, displayed a set of bacteria that are able to degrade MPs, most of which belonged to the Firmicutes, Proteobacteria, and Actinobacteria phyla, and more specifically to the Bacillaceae , Enterobacteriaceae , Nocardiaceae , and Streptom ycetaceae families.Mor eov er, in a pr e vious study, Huerta Lwanga et al. ( 2018 ) isolated from the gut of L. terrestris six bacterial strains that when applied to LDPE MP contaminated soil, were able to reduce the concentration and the size of MPs.Two of the plasticdeteriorating bacteria were classified as Firmicutes ( Bacilaceae ) and the remaining four as Actinobacteria ( Microbacteriaceae , Nocardiaceae , Mycobacteriaceae , and Streptomycetaceae ) (Huerta-Lwanga et al. 2018 ).