Pontoscolex corethrurus: A homeless invasive tropical earthworm?

The presence of earthworm species in crop fields is as old as agriculture itself. The earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) are associated with the emergence of agriculture and sedentism in the region Amazon and Maya, respectively. Both species have shifted their preference from their natural habitat to the cropland niche. They contrast in terms of intensification of agricultural land use (anthropic impact to the symbiotic soil microbiome). P. corethrurus inhabits conventional agroecosystems, while B. pearsei thrives in traditional agroecosystems, i.e., P. corethrurus has not yet been recorded in soils where B. pearsei dwells. The demographic behavior of these two earthworm species was assessed in the laboratory over 100 days, according to their origin (OE; P. corethrurus and B. pearsei) food quality (FQ; soil only, maize stubble, Mucuna pruriens), and soil moisture (SM; 25, 33, 42%). The results showed that OE, FQ, SM, and the OE x FQ interaction were highly significant for the survival, growth, and reproduction of earthworms. P. corethrurus showed a lower survival rate (> mortality). P. corethrurus survivors fed a diet of low-to-intermediate nutritional quality (soil and stubble maize, respectively) showed a greater capacity to grow and reproduce; however, it was surpassed by the native earthworm when fed a high-quality diet (M. pruriens). Besides, P. corethrurus displayed a low cocoon hatching (emergence of juveniles). These results suggest that the presence of the invasive species was associated with a negative interaction with the soil microbiota where the native species dwells, and with the absence of natural mutualistic bacteria (gut, nephridia, and cocoons). These results are consistent with the absence of P. corethrurus in milpa and pasture-type agricultural niches managed by peasants (agroecologists) to grow food regularly through biological soil management. Results reported here suggest that P. corethrurus is an invasive species that is neither wild nor domesticated, that is, its eco-evolutionary phylogeny needs to be derived based on its symbionts.

Introduction

Although humans have produced novel niches prior to the advent of agriculture, the innovation of domestication led to changes in the life cycle of one or a few species, and the local microenvironments were manipulated, especially soil biota [1-4]. The artificial landscapes that resulted from these practices (anthropocentric ecology) were exported as agricultural packages from the centers of origin [1, 2, 4]. Thus, over a relatively short period in the history of mankind, the expansion of agriculture has brought about the remodeling of biodiversity as one of the most significant anthropogenic impacts on terrestrial ecosystems [1, 2, 5].

Agriculture has given rise to uniform and predictable disturbed ecological niches (invasible habitats), which have proven highly beneficial for non-domesticated species or weeds [1, 2, 6], and some earthworm species. Blakemore [7] has suggested that the origins of cosmopolitan (invasive) earthworms at family level are associated with domestication centers of plants and animals; that is, the presence of earthworms in crop fields is as old as agriculture itself [7-9]. The terms of the Millennium Ecosystem Assessment highlight the catalytic role of earthworms regarding two environmental services [10], namely the formation of soil and biogeochemical cycles, both of which are prerequisites for other environmental services [10-11].

Most of the studies focused on earthworms have used species adapted to crops, and most of them are currently considered as invasive [11]. It has been documented that 3% of the diversity of earthworms are invasive species [12]. As an example, European earthworms are frequently mentioned as the main cause of an irreversible change in the diversity and functioning of ecosystems in North America (Wisconsin glaciation areas) that were previously free from earthworms 12 thousand years ago [13-15]. However, there is a deeply rooted positive attitude toward earthworms in human populations in North America, acknowledging their beneficial effects on agricultural soils and urban gardens [10, 16].

Among the invasive tropical earthworms, the endogeic species Pontoscolex corethrurus was collected and described in crop fields in Blumenau, Brazil 160 years ago [17-18]; it has a broad distribution range and is the most studied tropical species [19-20]. Native species also move across a region in a similar way to invasive species, in addition to natural displacements [5, 7, 21]. The native endogeic earthworm Balanteodrilus pearsei was first collected and described from Gongora cave in Okcutzcab, Yucatan 81 years ago [22]; it is distributed in the east and southeast of Mexico and Belize [19]; it dwells in natural and agricultural environments and is the most studied species native to Mexico. Most studies conducted with both species point to a positive influence of their biological activity on soil [20, 23], i.e., they do not meet the definition of pest [24]. For this reason, we use the term invasive with reference to the biogeographical status of the species, regardless of its impact on soil [24-25].

Similar to weeds [6, 8, 9, 26], it can be suggested that P. corethrurus and B. pearsei have shifted their preference from their natural habitat to agricultural environments, spreading geographically beyond their place of origin, and are currently key elements of agricultural environments. The presence of P. corethrurus and B. pearsei is associated with the development of pre-Columbian cultivation techniques in the Amazon [2, 27, 28, 29] and Maya [2, 30, 31] regions, respectively. For example, it is believed that P. corethrurus facilitated the formation of fertile soils in the Amazon area named "Terra Preta do Indo" [32, 33, 34, 35, 36, 37]. Both species have adapted to niches that emerged from agriculture [38], but contrast regarding the intensification of agricultural land use and/or the diversion of each from natural habitats (anthropic manipulation of soil). P. corethrurus is commonly found in conventional agrecosystems (use of fertilizers, herbicides, pesticides, and tillage), as well as in industrial (polluted with heavy metals, petroleum hydrocarbons, and others) and urban areas [20, 39, 40, 41]. B. pearsei inhabits soils managed under an agroecological approach (little human impact of the soil microbiome), such as traditional agroecosystems (no use of industrial inputs) and in natural ecosystems [40, 42, 43]. P. corethrurus has been found coexisting with native species in some agroecosystems [41, 44, 45], but there are no records of its coexistence with B. pearsei so far [40, 42, 43].

A previous study of coexistence under controlled conditions showed no competitive interaction between P. corethrurus and B. pearsei, i.e., both can coexist [23]. However, the question to address is, why P. corethrurus has not invaded the agroecological niche of B. pearsei? Therefore, this work compared the demographic behavior of P. corethrurus vs. B. pearsei assuming that the survival rate of the invasive species decreases in soil populated by the native species.

Materials and methods

Ethics statement

No permits were required for the collection and laboratory trials. Soil and earthworms were provided by farmers with free of charge. The experimental procedure used in this study is detailed elsewhere [23].

Soil

Soil was collected from a maize field (MM) rotated with the tropical legume velvet bean (Mucuna prurien var. utilis) located near the village Tamulté de las Sabanas (18°08´N, 92°47´W), 30 km east of Villahermosa, Tabasco, Mexico. The silty clay loam soil (41.5% sand; 26.8% clay; 31.6% silt) was air-dried in the shade at room temperature and sieved through a 2 mm mesh. The main chemical characteristics of this soil were: 2.7% organic matter; 0.14% total N; 11.4 C/N; pH (H2O) of 6.3.

Earthworms

Two tropical endogeic earthworm species were used in this study: B. pearsei (native) and P. corethrurus (invasive). B. pearsei was collected from the MM field, whereas P. corethrurus was collected from pastures at Huimanguillo (79 km southwest Villahermosa, 17°48´N, 93°28´W), given its absence in the former site. All earthworms (120 for each species) were collected two weeks prior to the beginning of the experiment.

Food quality

The effects of foof quality were assessed by using two different types of plant litter of contrasting nutritional quality: M. pruriens (52.4% C, 2.25% N, 23.3 C/N, and 9.67% ash) and maize stubble (52% C, 0.84% N, 61.9% C/N, and 10.3% ash). Both materials were obtained from the MM field, oven-dried at 60°C for 48 h, and sieved (1 mm).

Experiment

Growth, sexual maturity, reproduction (cocoons and juveniles), and mortality of B. pearsei and P. corethrurus were investigated during 100 days using a factorial design with three factors: origin of earthworms (OE), soil moisture (SM), and food quality (FQ). SM involved 3 levels, corresponding to the permanent wilt point (25%), field capacity (42%), and an intermediate level (33%). FQ included three levels: 300 g soil only (S), 294 g soil + 6 g maize stubble (MS), and 294 g soil + 6g M. pruriens (MP); the amounts added correspond to those commonly found in both maize monocultures and cultures rotating maize and M. pruriens. The earthworm species used belong to two different classes based on origin: Native (B. pearsei) and Invasive (P. corethrurus).

The combination of the three factors and three levels produced nine treatments with five replicates per treatment. Each replicate consisted of a plastic container (12×12×8 cm) containing 300 g dried soil of the corresponding food-soil mixture and soil moisture; two individuals of B. pearsei and two of P. corethrurus were transferred to each container (Table 1).

Table 1

F-values and significance levels (ANOVA) of the interaction of three factors on growth and reproduction of the tropical endogeic earthworm Pontoscolex corethrurus and Balanteodrilus pearsei at 100 days of culture in soil with low anthropic impact.

Independent variable	Biomass adult		Sexual maturity		Cocoon				Juveniles
					Number		Biomass		Number		Biomass
	F	P	F	P	F	P	F	P	F	P	F	P
Origen Earthworm (OE)	29.7	0.0000	22.3	0.0000	4.4	0.0390	2238.8	0.0000	25.1	0.0000	836.9	0.0000
Soil Moisture (SM)	20.4	0.0000	9.2	0.0002	22.2	0.0000	31.7	0.0000	14.8	0.0000	35.2	0.0000
Food quality (FQ)	191.7	0.0000	299.4	0.0000	109.0	0.0000	12.5	0.0004	67.0	0.0000	11.6	0.0006
OE×SM	0.03	0.9739	4.2	0.0164	4.4	0.0161	9.3	0.0000	5.0	0.0095	13.5	0.0000
OE×FQ	5.2	0.0068	3.2	0.0438	11.6	0.0000	3.5	0.0609	25.2	0.0000	10.8	0.0010
FQ×SM	7.3	0.0000	2.1	0.0897	13.4	0.0000	5.9	0.0029	11.4	0.0000	21.7	0.0000
OE×FQ×SM	1.1	0.3626	2.5	0.0442	6.4	0.0002	2.5	0.0859	6.2	0.0002	15.3	0.0000

Earthworms were washed, dried on paper towels, weighed, and assigned randomly to each treatment. The baseline weight of the 45 replicates from the nine treatments was statistically similar in B. pearsei and P. corethrurus (76.06 ± 26.1 mg, n = 90 and 66.04 ± 31.1 mg, n = 90, respectively). Containers were incubated at 26 ± 1 ºC. Body weight, mortality, clitellum appearance (sexual maturity), and number and biomass of cocoons and juveniles of B. pearsei and P. corethrurus were recorded at 10-day intervals, and soil was replaced. Before use, fresh soil (including the corresponding food-soil mixture and moisture) was preincubated for 8 days at 26°C in order to trigger litter substrate decomposition. Each cocoon produced was incubated in a petri dish at 26°C; incubation time as well as number and weight of all juveniles hatched were recorded.

Statistical analysis

Cocoon and juvenile weight, and growth were evaluated through Analysis of Varienace (ANOVA). Mortality, sexual maturity, number of cocoons, and number of juveniles were analyzed using generalized linear models, specifically the Poisson distribution wich is widely used for modelling count data. Differences between means were evaluated with Tukey's HSD. All statistical analyses were perfomed using the Statistica software.

Results

At 100 days of culture, significant effects were observed between the origin of earthworms (OE), food quality (FQ) and soil moisture (SM), and the interaction between these three factors on sexual maturity, number of cocoons, and number and biomass of juveniles (Table 1).

Mortality

At the end of the culture, the invasive earthworm (P. corethrurus) had a 21.1% mortality rate in the soil treatment (33% and 25% SM), while that of the native earthworm (B. pearsei) had only a 1.1% mortality rate in the soil treatment (only 42% SM). In the M. pruriens and maize stubble treatments (25%, 33% and 42% SM) no mortality was observed in both earthworm species.

Growth

Growth of the endogeic earthworms clearly varied in response to EO, FQ, SM, and the EO⊆CF and SM⊆CF interactions (Table 1). At 100 days of culture, the growth of the invasive and native species (P. corethrurus and B. pearsei, respectively) was higher when food quality increased (Fig 1). In the three FQ levels (soil, maize stubble, and M. pruriens) the exotic species showed a faster growth (1.6, 9.4, and 12.3 mg/day, respectively) relative to the native species (0.34, 4.8, and 10.4 mg/day).

Fig 1

Biomass of the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture using three diets of different nutritional quality in soil with low anthropic impact.

Vertical lines represent standard error.

Reproduction

Sexual maturity (clitellum)

When fed M. pruriens, the onset of sexual maturity in P. corethrurus and B. pearsei occurred at 30 days; when fed maize stubble, sexual maturity was obseved at 30 and 70 days in P. corethrurus and B. pearsei, respectively.

At 100 days of culture, OE, FQ, SM, and the OE ⊆ CF ⊆ SM interaction significantly affected clitellum development (Table 1). The invasive and native earthworms reached sexual maturity in the treatments with M. pruriens (100% and 86.6%) and maize stubble (96.7% and 70.0%), respectively (Fig 2). No individuals reached sexual maturity after 100 days in the soil treatments; however, in the soil treatment with 33% SM, one earthworm of P. corethrurus (6.7%) reached sexual maturity at 80 days.

Fig 2

Sexual maturity (formation of the clitellum) in the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact.

Vertical lines represent standard error.

Cocoon production

B. pearsei and P. corethrurus displayed biparental and uniparental sexual reproduction, respectively. On M. pruriens treatments (25%, 33% and 42% SM), cocoon production started when B. pearsei and P. corethrurus reached a mean biomass of 773.5 ± 146.8 mg and 644.7 ± 71.1 mg (average of 25%, 33% and 42% SM), respectively. On maize stubble treatments, it started when B. pearsei and P. corethrurus reached a mean body weight of 593.0 ± 80.9 mg and 598.5 ± 95.2 mg (average of 25%, 33% and 42% SM), respectively. Cocoon production in P. corethrurus was observed in soil (6.7%), maize stubble (53.3%), and M. pruriens (86.7%) treatments, but in B. pearsei it was observed only in maize stubble (33.3%) and M. pruriens (86.7%) treatments.

Mean cocoon production was significantly influenced by EO, CF, SM, and the interaction between these three factors (Table 1). After 100 days of culture, peak mean cocoon production in B. pearsei and P. corethrurus was observed in M. pruriens treatments, with 59.7 ± 40.8 and 35.5 ± 21.5 cocoons (average of 25%, 33% and 42% SM treatments), respectively (Fig 3). When fed maize stubble, B. pearsei and P. corethrhrus produced 7.9 ± 3.2 and 14.4 ± 9.2 cocoons (average of 25%, 33% and 42% SM treatments), respectivelly. Finally, when fed soil only (33% SM), P. corethrurus (448 mg body weight) produced only two cocoons.

Fig 3

Number of cocoons produced by the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact.

Vertical lines represent standard error.

Cocoon biomass varied significantly in response to EO, FQ, SM and the OE x SM and FQ x SM interactions (Table 1). Average cocoon biomass produced by B. pearsei and P. corethrurus with SM treatments (25%, 33% and 42%) was 10.2 ± 1.4 mg and 27.7 ± 3.7 mg, respectively.

Juvenile production

The mean cocoon incubation time was similar among treatments (P > 0.05). In general, mean cocoon incubation time was 20.4 ± 5.2 days (B. perasei) and 30.3 ± 2.2 days (P. corethrhrus), with one individual hatching per cocoon in all cases. Of the total number of cocoons produced by B. pearsei and P. corethrurus in M. pruriens and corn stubble treatments, the average number of hatched juveniles was 64.7 ± 16.6% and 29.5 ± 7.0% (average of 25%, 33% and 42% SM treatments) and 59.5 ± 24.7 and 24.0 ± 10.6 (average of 25%, 33% and 42% SM treatments), respectively.

The number of hatched juveniles of B. pearsei and P. corethrurus varied significantly with OE, CF, SM, and the interaction between these three factors (Table 1; Fig 4). The mean number of hatched juveniles of B. pearsei and P. corethrurus increased in adults fed M. pruriens, as well as with increasing soil moisture (mean 59.7±40.8 and 35.5±21.5 individuals, respectively), and corn stubble (mean 7.9 ± 3.3 and 14.6 ± 9.2 individuals).

Fig 4

Number of juveniles hatched from cocoons produced by the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact.

Vertical lines represent standard error.

At hatching, in the M. pruriens and corn stubble treatments, mean biomass of P. corethrurus juveniles (21.2 ± 1.0 and 18.6 ± 7.4 mg, respectively) was higher vs. B. pearsei juveniles (8.5 ± 0.7 and 8.5 ± 1.3 mg, respectively).

Discussion

Domesticated, wild populations respond to changing selective pressures, which are reflected in their adaptation to agricultural niches [2, 46]. From an ecological perspective, the endogeic earthworm P. corethrurus resembles non-domesticated species or weeds given its strong profile (invading species) regarding growth rate, fertility, plasticity, interspecific competition, and environmental tolerance [7, 8, 9, 26, ]. This suggests that the four P. corethrurus ecotypes described by Taheri et al. [47] are likely the result of the selective forces imposed by cultivation, agricultural practices, and industrial and urban activities [20]. In the present study, soil in the habitat for B. pearsei was observed to restrain the presence of P. corethrurus.

The conversion of the Amazon forest to pastures led to the homogenization of soil biota [3, 48]. The potential resistance of soil (i.e., predators, low species richness, etc.) to earthworms has been documented [15, 49, 50]. For instance, the endogeic tropical earthworm Millsonia anomala from the savannah was unable to prosper in forest soil [49], similar findings have been reported with P. corethrurus from fallow (slash-and-burn) to mature forest [35]. Also, the shift in vegetation from grass to woody plants decreaced in the density and biomass of P. corethrurus [51]. Our results showed that the survival of P. corethrurus was lower in the environment where B. pearsei thrives, maybe due to a negative interaction with a more diverse edaphic microbiome [49, 50, 52], because it has been suggested that P. corethrhrus has a high ability to utilize soil organic resources as an energy source [39].

Earthworms harbor symbiotic microbiomes that are essential for their life history in the nephridia (excretory organs), and cocoons in tropical species such as P. corethrurus is poorly studied [53-58]. The microbiome is known to improve the nutritional status of low-quality diets [57-58]. For example, Topoliantz and Ponge [35] observed that the behaviour of two populations of P. corethrurus separated along the Maroni river (French Guiana, South America) differed significantly: fallow populations produced more cast on charcoal in the presence of forest soil, while the casting activity of the forest population was higher on soil regardless of the soil origin. Our findings show that P. corethrurus and B. pearsei differ in their diet preference (M. pruriens, corn stubble, and control), i.e., the invasive species displayed faster growth than the native species when nutritional quality improved. This suggests that P. corethrurus consumes and degrades a greater variety of organic materials given its greater ability (efficiency), evidenced by: a) producing endogenous cellulases [59-62]; b) its association with the gut microbiota [63-66]; c) gene expression (transcriptome) that contribute to the adaptation of its digestive system [65]; d) improving its digestion efficiency according to the type of cecum [59, 67]; and e) its association with nephridial bacteria [50, 68, 69].

It is known that in diets of low nutritional quality, mutualistic bacteria residing in earthworm nephridia (in 19 of 23 species studied) provide vitamins to its host, stimulate earlier sexual maturity, and contribute to pesticide detoxification [56, 57, 58, 60, 70, 71]. The results reported here showed that the invasive species of smaller size (biomass) fed on a lower nutritional diet (M. pruriens > corn stubble > soil) reached sexual maturity earlier than the native earthworm. This suggests that the nephridial symbionts of P. corethrurus are generalists, while those of B. pearsei are specialists.

Earthworms produce external cocoons that are colonized by bacteria from parents and soil [vertical and horizontal transmission, respectively 53, 58] and coul be used as biovectors for the introduction of benefical bateria [55]. In a new habitat, cocoons of invasive earthworms may be affected by the native microbiota, but they can survive if they carry a parental microbial inoculum. Our results show that P. corethrurus produced cocoons when fed either of the three diets, while B. pearsei fed the control diet (only soil) failed to produce cocoons. In contrast, cocoons of P. corethrurus had a low hatching rate (births), which was lower (diet with M. pruriens) compared to B. pearsei. These results suggest the absence and/or loss of parental symbionts bacteria, i.e., the loss of a parental care strategy to control predators, detoxify nitrogenous wastes, conserve nitrogen, and supply vitamins and essential cofactors to the offspring [55, 56, 57, 68, 69, 70, 72]. Thus, the likely symbiotic evolution of P. corethrurus with the microbiome (gut, nephridia and cocoons) should be explored as a source of biogeography and phylogenetic information [11, 57, 68, 70, 71, 73, 74]. That is, we could “…explain why P. corethrurus is rare or absent in undisturbed lands” [39].

The human-mediated translocation of species dates back to the Late Pleistocene [2, 5, 75]. Invasive plant species are usually divided in two groups according their residence time: archaeophytes were found from 1500 AD, and neophytes are found after this date [76]. This approach can contribute to elucidate the history of the invasion of P. corethrurus in Mexico. Until now, only two ecotypes have been recorded [47] and the criptic linage used in this study corresponds to L1 (the most widespread). The origin of P. corethrurus may be related to anthropogenic soil formation (“terras mulatas” and “terras pretas”). The domestication of manioc (bitter and sweet) and peach palm staple food that facilitated sedentary lifestyles in the Amazon region [5, 27, 28, 29, 32] has evolved to the point that we cannot recognize the predecessors of P. corethrurus, as evidenced by the recent designation of the P. corethrurus neotype from an anthropogenic environment [18] and temperate climate [77], and by the ambiguity used for assigning its place of origin [12, 78].

Based on the results reported here, we conclude that the invasive tropical earthworm P. corethrurus had lower survival and cocoons hatching rates (offspring) in the agro-ecological niche of the native endogeic earthworm, i.e., a finding consistent with the absence of P. corethrurus in parcels where maize- and M. pruriens crop rotation is practiced, as well as in pastures and other traditional tropical agroecosystems [40, 41, 42, 43, 44, 45]. This suggests that P. corethrurus is an invasive species that thrives far from its natural status, i.e., has no wild ancestry in the study area. Therefore, it is important to determine the preference of the four P. corethrurus ecotypes [47] in terms of soil type, cultivation, response to stressors and climate change.

Results fitting linear model of the earthworm biomass.

(PDF)

Click here for additional data file.

Results fitting logistic model of sexual maturity of the earthworms.

(PDF)

Click here for additional data file.

Results fitting zero inflated poisson of the earthworms cocoons.

(PDF)

Click here for additional data file.

5 Jul 2019

PONE-D-19-15357

Pontoscolex corethrurus: a Homeless Invasive Tropical Earthworm?

PLOS ONE

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1]The authors have done an interesting study determining the reason as to why P. corethrurus is unable to invade the agricultural niche of B. pearsei?

2]The design of experiment is very appropriate and accurate.

3]But there are some loop holes that need to be plugged for eg: the figure numbers need to be checked and rectified

4]English language needs to be checked by someone with good command over language for clarification in the manuscript

5]Diiscussion should be written more precisely and directly analysing the results obtained in the experiments performed.

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Additional Editor Comments:

1]The authors have done an interesting study determining the reason as to why P. corethrurus is unable to invade the agricultural niche of B. pearsei?

2]The design of experiment is very appropriate and accurate.

3]But there are some loop holes that need to be plugged for eg: the figure numbers need to be checked and rectified

4]English language needs to be checked by someone with good command over language for clarification in the manuscript

5]Diiscussion should be written more precisely and directly analysing the results obtained in the experiments performed.

6]Other comments have been high lighted in the manuscript and also by the reviewers ,

7]All the comments must be answered to and necessary rectifications made in the manuscript based on the reviewers comments

Therefore manuscript can be accepted for publication only after the major revesion.

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Reviewer #1: Yes

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Reviewer #1: The study asks an interesting question given at the end of the introduction—why hasn’t P. corethrurus invaded the agricultural niche of B. pearsei? The experiment seems well designed. However, there are some items in the test that need to be corrected (e.g. figure numbers), some revisions for clarity are needed, and the Discussion needs revisions to make it more directly relevant to the experiment that was conducted. These are explained below.

Page 4 line 3, 100-120 of the species?

Last sentence page 4, confusing—are you switching back to P. corethrurus here?

Page 6, second to last line, 61.9 C/N does not have a percent—it’s a ratio

Page 8, mortality, please state the mortality results more systematically, instead of a few selected treatments.

Page 8-9. Figure 1 does not show growth rates, it shows biomass at 100 days with three food sources. In the second sentence it should say ‘At 100 days the biomass of invasive and native species…’ The last sentence appears to actually present growth rate data. Biomass at 100 days is related to the growth rate, but is not the same thing.

Middle of page 10. Fig. 4 does not show cocoon biomass.

Page 10-11, there are no Figs 5 and 6 in the manuscript that I have.

Discussion:

First sentence is incomplete; either say what happens when populations respond to domestication, or delete ‘When’ at the beginning of the sentence.

Page 13-14, 275 and 176 species of bacteria?

Page 15, Levis et al 2018 (not 208).

Most of the Discussion (although very interesting) is not about what was studied, and connections between the experiment presented in the results section and discussion topics are weak. For example, does your P. corethrurus belong to one of the four agrotypes mentioned on page 16? Do you know which bacterial taxonomic groups are present in your P. corethrurus?

The statements in the concluding paragraph on page 17 have no direct evidence from your study. Again, it is interesting, and well written, but very speculative as it relates to the study.

There is almost a review paper about evolutionary/agro-history of the earthworm species embedded in the Discussion. Perhaps the authors should consider expanding this and publishing it separately as a review paper! However, for purposes of the manuscript under review, the connections to the experiment are too weak, and this review material should be greatly shortened to one or two paragraphs. In place of most of this material there should be more discussion points related directly to the experimental evidence—for example comparison to survival rates, attainment of sexual maturity, biomass and cocoon production from other studies of these two earthworm species. Also, the conclusion should have a succinct summary about how the evidence from the experiment answers the question posed at the end of the introduction.

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Reviewer #1: No

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26 Jul 2019

Rebuttal Letter

General remarks

All the corrections indicated referees (Reviewer # 1 and Editorial Board Editor) was incorporated on the manuscript. In green, sentences and words eliminated; in red, sentences, words incorporated, and only in the text of the manuscript.

Specific points:

Introduction

Comments:

Page 4 line 3, 100-120 of the species?

Last sentence page 4, confusing—are you switching back to P. corethrurus here?
 Page Line

Response:

We agree with these comments. We include and delete the following information suggested:

Include:

It has been documented that 3 % of the diversity of earthworms are invasive species (Dupont et al. 2012).

Delete:

It has been documented that 3 % (100-120) of the diversity of earthworms are invasive species (Hendrix 2008), and stating that just a few of them had a negative impact on terrestrial agroecosystems would not be an exaggeration (Simberloff 2009; Hendrix et al. 2006)

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70-71

Results

Comments:

Page 8, mortality, please state the mortality results more systemaWcally, instead of a few selected treatments.
 Page Line

Response

We agree with these comments. We include and delete the following information suggested:

Include:

At 100 days of culture, significant effects were observed between the origin of earthworms (OE), food quality (FQ) and soil moisture (SM), and the interaction between these three factors on sexual maturity, number of cocoons, and number and biomass of juveniles (Table 1).

Mortality

At the end of the culture, the invasive earthworm (P. corethrurus) had a 21.1 % mortality rate in the soil treatment (33 % and 25 % SM), while that of the native earthworm (B. pearsei) had only a 1.1 % mortality rate in the soil treatment (only 42 % SM). In the M. pruriens and maize stubble treatments (25 %, 33 % and 42 % SM) no mortality was observed in both earthworm species.

Delete:

At 100 days of culture, significant effects were observed between the origin of earthworms (OE), food quality (FQ), soil moisture (SM) and mortality, and the interaction between these three factors on sexual maturity, number of cocoons, and number and biomass of juveniles. The invasive earthworm (P. corethrurus) had a 21.1 % mortality rate in the soil treatment (33 % and 25 % SM). In contrast, the native earthworm (B. pearsei) had only a 1.1 % mortality rate in M. pruriens (only 42 % SM).

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Discussion

Comments:

Discussion should be wrigen more precisely and directly analysing the results obtained in the experiments performed.
 Page Line

Response:

We agree with these comments. We delete the following information suggested:

Included:

This suggets that the four P. corethrurus ecotypes described by Taheri et al. (2018a) are likely the result of the selective forces imposed by cultivation, agricultural practices, and industrial and urban activities (Taheri et al. 2018b).

Eliminated:

a finding consistent with the absence of P. corethrurus in parcels where maize- and M. pruriens crop rotation is practiced, as well as in pastures and other traditional tropical agroecosystems (Lavelle et al. 1981; Ortiz- Ceballos et al. 2004; Huerta et al. 2006; Marichal et al. 2010; Fragoso et al. 2016; Ortiz-Gamino et al. 2016).

Included:

, similar findings have been reported with P. corethrurus from fallow (slash-and-burn) to mature forest (Topoliantz and Ponge 2005).

, because it has been suggested that P. corethrhrus has a high ability to utilize soil organic resources as an energy source (Lavelle et al. 1987).

Delete:

Also, in a Glenrock soil (southern Australia), the survival of the European endogeic earthworm Aporrectodea trapezoides was found in association with soil bacteria (Davidson et al 2013; Menezes et al. 2018).

Include:

For example, Topoliantz and Ponge (2005) observed that the behaviour of two populations of P. corethrurus separated along the Maroni river (French Guiana, South America) differed significantly: fallow populations produced more cast on charcoal in the presence of forest soil, while the casting activity of the forest population was higher on soil regardless of the soil origin.

Delete:

Symbiosis, defined as the interaction between two different organisms living in close physical association, has been acknowledged as a source of evolutionary innovation that allows hosts to exploit otherwise inaccessible niches (Laland et al. 1999; Lund et al. 2010b; Aira et al. 2018). The hologenome (sum of the genetic information of the host and its microorganisms) theory of evolution is based on four generalization: a) all animals and plants establish symbiosis with microorganims; b) microorganisms can be transmitted between generations with fidelity; c) symbiosis affects the fitness of holobionts in their environment; d) genetic variation in holobionts can be enhanced by incorporating different symbiont populations and can change rapidly under enviromental stress (Zilber-Rosenberg and Rosenberg 2008).

Include:

Thus, the likely symbiotic evolution of P. corethrurus with the microbiome (gut, nephridia and cocoons) should be explored as a source of biogeography and phylogenetic information (Lund et al. 2010a; Brussaard et al. 2012; Davidson et al. 2013; Møller et al. 2015; Zwarycz et al. 2015; Schult et al. 2016; Marchán et al. 2018). That is, we could “…explain why P. corethrurus is rare or absent in undisturbed lands” (Lavelle et al. 1987).

Delete:

In Eisenia andrei and E. fetida, 275 and 176 bacteria were observed in their cocoons, respectively (Menezes et al. 2018); however, these were dominated by three vertically transmitted (parental) symbionts: Microbacteriaceae, Verminephrobacter, and Ca. Nephrothrix. For example, in a sterile environment, cocoons of M. anomala failed to develop, suggesting a functional relationship between nephridial bacteria of earthworms and soil microbiota (Gilot-Villenave 1994). Besides, it has been noted that during embryogenesis, the nephridial symbiont Verminephrobacter (Betaprototeobacteria) contributes to the biosynthesis of vitamins and the elimination of nitrogen wastes from cocoons, and conserving nitrogen (Schramm et al. 2003; Davidson and Stahl 2006; Lund et al. 2010b; Davidson et al. 2013; Møller et al. 2015).

Include:

Until now, only two ecotypes have been recorded (Taheri et al. 2018a) and the criptic linage used in this study corresponds to L1 (the most widespread). The origin of P. corethrurus may be related to anthropogenic soil formation (“terras mulatas” and “terras pretas”). The domestication of manioc (bitter and sweet) and peach palm staple food that facilitated sedentary lifestyles in the Amazon region (Lodge 1993; Glaser et al. 2000; Arroyo-Kalin 2010; Clement et al. 2015; Watling et al. 2018; Levis et al. 2018) has evolved to the point that we cannot recognize the predecessors of P. corethrurus, as evidenced by the recent designation of the P. corethrurus neotype from an anthropogenic environment (James et al. 2019) and temperate climate (Peel 2007), and by the ambiguity used for assigning its place of origin (Righi 1984; Dupont et al. 2012).

Delete:

Pyšek et al (2005) observed that archeophyte weeds are common in crops introduced at the beginning of agriculture (cereals), but are poorly represented in crops introduced relatively recently (rape, maize), where neophyte weeds are most numerous.

It is documented that an exchange of domesticated plants (manioc, maize, cacao and others), as well as recently introduced plants (livestock, pastures, coffee, sugar cane and others), took place between the Amazon area and Mesoamerica through agricultural packages that may have also included P. corethrurus as well as weeds, mice, insects, etc. (Denevan 1992; Boivin et al 2016; Levis et al. 208).

Delete:

Therefore, the four P. corethrurus ecotypes described by Taheri et al. (2018b) are likely the result of the selective forces imposed by cultivation, agricultural practices, and industrial and urban activities. That is, this species has evolved to develop resistance to herbicides, pesticides, heavy metals, and hydrocarbons, among others (Taheri et al. 2018b). The above may explain why the paradigm of Beddard (1900) " ...tropical earthworms invade only tropical regions (Neotropical) ..." is no longer valid, as P. corethrurus has been recorded across the five biogeographic regions: Nearctic (Gates 1954, Gates 1972, Blakemore 2009), Palearctic (Omodeo et al. 2003; Blakemore et al. 2006; Reynolds and Jones 2006, Sherlock and Carpenter 2009, Blakemore 2009, Cunha et al. 2014, Rota and Jong 2015; Taheri et al. 2018), Ethiopian (Plisko 2001), Indian (Tsai et al. 2000; Blakemore 2009; Singh et al. 2018; Subedi et al. 2018), and Australian (Blakemore 2009). The tropical microclimatic model of Lavelle et al. (1987) for the growth and development of P. corethrurus (20-30 °C) is also not applicable to all four P. corethrurus agrotypes (James et al. 2019), since a revision of databases and the literature revealed that this species has been collected in Neotropical latitudes with temperate climate (10- 20 °C; 1200 to 2000 m a.s.l.; Köppen 2011; Peel 2007; Volken and Brönnimann 2011) from México (Fragoso 2018; Juárez-Ramón and Fragoso 2014, Ortiz-Gamino et al. 2016), Lesser Antilles (Hendrix et al. 2006), Colombia (Gutiérrez-Sarmiento and Cardona 2014), Brasil (Müller 1857; Bunch et al. 2011; Ferreira et al. 2018; Steffen et al. 2018), Argentina (Mischis and Righi 1999; Mischis 2007), Madagascar (Chapuis-Lardy et al. 2010; Villenave et al. 2010), South África (Plisko 2001, Janion-Scheepers et al. 2016) e India (Singh et al. 2018; Subedi et al. 2018).

The origin of P. corethrurus may be related to anthropogenic soil formation (“terras mulatas” and “terras pretas”) and the domestication of manioc (bitter and sweet) and peach palm staple food that facilitated sedentary lifestyles in the Amazon region (Lodge 1993; Glaser et al. 2000; Arroyo-Kalin 2010; Clement et al. 2015; Watling et al. 2018; Levis et al. 2018), and has evolved to the point that we cannot recognize their wild predecessors, as evidenced by the recent designation of the P. corethrurus neotype from an anthropogenic environment (Müller 1857; James et al. 2019), and by the ambiguity used for assigning its place of origin (Righi 1984; Dupont et al. 2012).

Cryptic invasions occur frequently, often go unnoticed, and are hard to recognize. Also, it is known that cryptic species may display markedly different responses to a given stimulus or stressor (Liebeke et al. 2014; Schult et al. 2016; Morais and Reichard 2018). Therefore, it is important to classify the four P. corethrurus ecotypes (Taheri et al. 2018a; James et al. 2019) as separate evolutionary entities over the residence time, and determine the preference of each ecotype in terms of soil type, culture, and climate, and response to stimulus or stressors (pesticides, herbicides, heavy metals, etc.), among others. To this end, transcriptomes (Rad-Seq) may be used to elucidate the gene flow across cryptic lineages (Puga-Freitas et al. 2015; Schult et al. 2016; Morais and Reichard 2018). In addition, epigenetics could be used to investigate the functional adaptations of P. corethrurus lineages (Kille et al. 2013; Liu et al. 2017; Ponesakki et al. 2017; Fernández-Marchán et al. 2018). Last, potential symbionts may be found for each agrotype, including bacteria, nematodes, enchytraeids and other life forms associated with earthworms (Coates 1990; Fernández-Marchán et al. 2018).

Comment:

the conclusion should have a succinct summary about how the evidence from the experiment answers the question posed at the end of the introduction.

Response:

We agree with these comments. We delete the following information suggested

Include:

Based on the results reported here, we conclude that the lower survival and hatching rates of P. corethrurus cocoons (offspring) in the agro-ecological niche of B. pearsei; it is, a finding consistent with the absence of P. corethrurus in parcels where maize- and M. pruriens crop rotation is practiced, as well as in pastures and other traditional tropical agroecosystems (Lavelle et al. 1981; Ortiz-Ceballos et al. 2004; Huerta et al. 2006; Marichal et al. 2010; Fragoso et al. 2016; Ortiz-Gamino et al. 2016). This suggests that P. corethrurus is an invasive species that thrives far from its natural status, i.e., has no wild ancestry in the study area. Therefore, it is important to determine the preference the four P. corethrurus ecotypes (Taheri et al. 2018a) in terms of soil type, cultivation, response to stressors and climate change.

Delete:

Based on the results reported here, we conclude that the lower survival and hatching rates of P. corethrurus cocoons (offspring) are associated with its symbiotic bacteria, coupled with a higher diversity of the edaphic microbiome in the agro-ecological niche of B. pearsei. This suggests that P. corethrurus is an exotic species that thrives far from its natural status, i.e., has no wild ancestry in the study area. For this reason, the likely symbiotic eco-evolution of P. corethrurus with the microbiome in its gut, nephridia and cocoons should be explored as a source of biogeography and phylogenetic information (Lund et al. 2010a; Brussaard et al. 2012; Davidson et al. 2013; Møller et al. 2015; Zwarycz et al. 2015; Schult et al. 2016; Fernández- Marchán et al. 2018), i.e., going back to the roots (Philippot et al. 2013; Pérez-Jaramillo et al. 2016).

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Figures

Figures in the text:

Comments:

Page 8-9. Figure 1 does not show growth rates, it shows biomass at 100 days with three food sources. In the second sentence it should say ‘At 100 days the biomass of invasive and naWve species...’

We agree with these comments. We delete the following information suggested:

Include:

Fig. 1. Biomass of the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture using three diets of different nutritional quality in soil with low anthropic impact. Vertical lines represent standard error.

Delete:

Fig. 1. Growth rate of the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) after 100 days of culture using three diets of different nutritional quality in soil with low anthropic impact. Vertical lines represent standard error.

Comments:

Middle of page 10. Fig. 4 does not show cocoon biomass.
Page 10-11, there are no Figs 5 and 6 in the manuscript that I have.

We agree with these comments. We delete the following information suggested:

Include:

Fig. 1. Biomass of the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture using three diets of different nutritional quality in soil with low anthropic impact. Vertical lines represent standard error.

Fig. 2. Sexual maturity (formation of the clitellum) in the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact. Vertical lines represent standard error.

Fig. 3. Number of cocoons produced by the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact. Vertical lines represent standard error.

Fig. 4. Number of juveniles hatched from cocoons produced by the tropical endogeic earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) at 100 days of culture under the interaction of three diets of different nutritional quality and three moisture content levels in soil with low anthropic impact. Vertical lines represent standard error.

Delete:

We remove figures 4 (cocoon biomass) and 6 (juveniles biomass) from the text; that is, there were many figures.

References

We included and delete the following references: Page Line

Include:

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5. Lodge, D.M. (1993). Biological invasions: lessons for ecology. Trends in Ecology & Evolution 8(4): 133-137

6. Ortiz-Ceballos, A.I., Fragoso, C. (2004). Earthworm populations under tropical maize cultivation: the effect of mulching with Velvetbean. Biol. Fert. Soils 39, 438-445.

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8. Topoliantz, S., & Ponge, J.F. (2005). Charcoal consumption and casting activity by Pontoscolex corethrurus (Glossoscolecidae). Applied Soil Ecology 28: 217-224.

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5. Fragoso, G.C. (2018). Importancia de las lombrices de tierra (Oligochaeta) en el monitoreo de áreas prioritarias de conservación del centro, este y sureste de México. CONABIO. https://doi.org/10.15468/omvnpi accessed via GBIF.org on 2019-05-01

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Submitted filename: Response to Reviewers.docx

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9 Aug 2019

PONE-D-19-15357R1

Pontoscolex corethrurus: a Homeless Invasive Tropical Earthworm?

PLOS ONE

Dear Dr Ortiz-Ceballos,

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Tunira Bhadauria, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors need to be congratulated for sincerely incorporating the changes and suggestions in the revised manuscript. However there are some minor changes which further need to be incorporated into the text before manuscript can be accepted for the publication

1.Line 261 in revised manuscript spelling of which

2.Line 302 Year of reference 199

3.Line 332 sentence incomplete changes incorporated in text

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13 Aug 2019

August 12, 2019

PlosOne

Editor-in-Chief

Dear Editor:

Please find enclosed the manuscript titled (Tracked Changues version) Pontoscolex corethrurus: a Homeless Invasive Tropical Earthworm? Below is the rebuttal letter in which we list all the changes (point-by-point) we have made to the manuscript.

Thank you again your comments and assistance.

Sincerely yours

Angel I. Ortiz Ceballos

Rebuttal Letter

General remarks

All the corrections indicated referees (Editorial Board Editor) was incorporated on the manuscript. In green, sentences and words eliminated; in red, sentences, words incorporated, and only in the text of the manuscript.

Specific points:

Discussion

Comments:

…However there are some minor changes which further need to be incorporated into the text before manuscript can be accepted for the publication Page Line

Response:

We agree with these comments. We include and delete the following information suggested:

Delete (Line 261 in revised manuscript spelling of which):

, wich differ from those in the adjacent forest

Delete (Line 302 Year of reference 199):

(Daane et al. 199)

Include:

(Daane et al. 1999)

Delete (Line 332 sentence incomplete changes incorporated in text):

Based on the results reported here, we conclude that the lower survival and hatching rates of P. corethrurus cocoons (offspring) in the agro-ecological niche of B. pearsei is…

Include:

Based on the results reported here, we conclude that the invasive tropical earthworm P. corethrurus had lower survival and cocoons hatching rates (offspring) in the agro-ecological niche of the native endogeic earthworm, i.e., a finding…

Include:

Acknowledgments

We thank Mario M. Osorio-Arce, Angel Ramos-Sánchez and Efraín Hernández-Xolocotzi, promoters of agroecology in Mexico. In addition, we are grateful to anomymous reviewers and Diana Pérez-Staples for valuable comments and careful revision of the manuscript.

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Submitted filename: Response to Reviewers R1.docx

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28 Aug 2019

Pontoscolex corethrurus: a Homeless Invasive Tropical Earthworm?

PONE-D-19-15357R2

Dear Dr. Ortiz-Ceballos,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Tunira Bhadauria, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

i would like to congratulate the authors for having revised the manuscript as per the suggestions and comments and incorporation of the same at appropriate places in the text. I therefore recommend that the manuscript now can be accepted for publication in the journal.

Reviewers' comments:

6 Sep 2019

PONE-D-19-15357R2

Pontoscolex corethrurus: a Homeless Invasive Tropical Earthworm?

Dear Dr. Ortiz-Ceballos:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tunira Bhadauria

Academic Editor

PLOS ONE