Monocystis sp. As Possible Bias in the Use of Eisenia fetida for Ecotoxicological and Ecopathological Studies

TL;DR

This paper shows that a parasite in earthworms can affect their reproduction and may skew results in soil toxicity studies.

Contribution

The study reveals that Monocystis sp. infection in Eisenia fetida can bias ecotoxicological results by causing reproductive impairment.

Findings

Monocystis sp. infection reduces sperm production and alters seminal vesicles in Eisenia fetida.

Infected earthworms show immune responses like encapsulation and melanization in reproductive organs.

Parasite-induced changes in earthworms resemble pollutant effects, potentially biasing ecotoxicological studies.

Abstract

Earthworms are widely used to evaluate soil quality because they are sensitive to contaminants and easy to maintain under laboratory conditions. Little attention has been given to the initial health status of the animals that may affect the biological responses measured in ecotoxicological studies. In this study, we found that many individuals of E. fetida were naturally infected by Monocystis sp., a parasite known to inhabit the male reproductive organs of earthworms. The infection caused a reduction in the number of spermatozoa, disruption of the seminal vesicle, and encapsulation of the parasite. Therefore, the parasite can lead to reproductive impairment, which could be wrongly associated with the effects of pollutants. Our findings suggest that Monocystis sp. may bias soil-toxicity results and highlight the importance of screening laboratory earthworm cultures for parasites to ensure reliable ecotoxicological results.

Eisenia fetida is one of the soil invertebrates most used in ecotoxicological and ecopathological studies. To date, the potential contribution of naturally occurring parasites to the variability of ecotoxicological endpoints has been poorly investigated. In this study, we provide a detailed histological description of the male reproductive system of E. fetida and report the occurrence and histological alterations associated with Monocystis sp. infection in laboratory-reared individuals. Uninfected individuals exhibited complete spermatogenesis, with all developmental stages from spermatogonia to mature spermatozoa and normal sperm storage within the spermathecae. Meanwhile, infected earthworms displayed marked reproductive alterations, including reduced sperm production and diminished sperm retention within spermathecae. Multilayered encapsulations, inflammatory nodules and melanization were detected within the seminal vesicles, in contrast with the immunological evasion observed in Lumbricus terrestris. These findings suggest species-specific differences in immune response and indicate that Monocystis sp. infection can induce reproductive impairment and activate energy-consuming immune responses. Because these parasite-induced changes closely resemble pollutant-driven ecotoxicological effects, Monocystis infections may act as a potential bias in ecotoxicological studies. We therefore recommend implementing parasitological screening of laboratory cultures to ensure the reliability of studies employing E. fetida as a bioindicator.

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1. Introduction

Earthworms are widely used in ecotoxicological studies due to their ecological relevance, ease of culture, and sensitivity to environmental pollutants [1]. Among earthworm species, Eisenia fetida is one of the most commonly used in standardized assays [2,3]. In particular, reproductive endpoints such as sperm integrity, cocoon production, and histopathological analysis of reproductive tissues are frequently used to assess sublethal effects of soil contaminants and soil quality [4,5,6,7,8,9]. For all these reasons, the initial health status of the individuals undergoing experimentation is crucial.

Monocystis (Apicomplexa: Gregarinomorphea: Arthrogregarida) is one of the most studied genus of naturally occurring parasites in many lumbricid earthworms. After the ingestion, Monocystis sporocysts, containing several sporozoites, pass through the intestinal wall and reach the circulatory system. From here, *Monocystis * sp. sporozoites can colonize the seminal vesicles, where they mature into trophozoites and affect the development of spermatocytes [10]. As the infection progresses, paired gamonts fuse within a gametocyst, where successive divisions produce a zygote that forms a protective oocyst. Then, this structure develops into sporocysts containing 8 sporozoites, which are eventually released when the gametocyst breaks, allowing the parasite to disperse into the seminal fluid and the environment, perpetuating its life cycle [11]. The host–parasite interaction can cause structural and functional reproductive alterations that could overlap with those typically attributed to contaminant exposure [12], such as damage to the seminal vesicles, degeneration of sperm morulae [11,13] resulting in reduced sperm availability, lowered cocoon production, delayed maturation, and increased overall fitness costs [13,14].

*Monocystis * sp. infections are widely documented and extensively characterized in Lumbricus terrestris [11,13]; however, several species of Monocystis and related monocystid gregarines have also been reported in E. fetida. Historical parasitological records include M. lanceata in E. fetida [15] and the presence of Monocystis agilis in E. fetida and Eisenia andrei [16,17]. Nevertheless, studies about pathological effects in E. fetida remain extremely limited, especially when compared to the great amount of literature available for L. terrestris [11,13,14,18]. This gap in knowledge highlights the possibility that parasitic infections may be under-reported in E. fetida and may introduce an “unknown” into reproductive endpoints. Consequently, the parasite-induced changes may not be distinguished from those caused by contaminants, compromising the reliability of E. fetida as a soil-quality bioindicator.

The aims of our study are: (i) to document the presence and effects of natural *Monocystis * sp. infections within the seminal vesicles; (ii)to discuss how such infections may potentially bias ecotoxicological and ecopathological interpretations when E. fetida is used as a model species.

2. Materials and Methods

The present study was carried out at the laboratory of Comparative and Ecopathology of the Department of Biology, University of Naples Federico II.

2.1. Animals

Earthworms (E. fetida S.) used in this study were purchased from an earthworm breeding farm in Northern Italy and maintained under laboratory-controlled conditions following OECD guidelines [19]. Temperature was kept at 20 ± 2 °C, and 16 h of light and 8 h of darkness were applied as a light-day cycle using illumination levels between 400 and 800 lux, while soil moisture was kept within the range of 40–60% of water holding capacity.

After acclimatization, one hundred viable adult individuals with well-developed clitella were collected from the enclosure, rinsed with distilled water, and dried with absorbent paper. To guarantee the homogeneity of the earthworm population, each individual was weighed to ensure wheight between 300 mg and 500 mg. Individuals did not show any macroscopic alterations suggestive of an underlying pathological condition. The earthworms were anesthetized with 5% ethanol, as already described by Cooper and Roch, 1986 [20]. Animals were then analyzed by histological techniques.

2.2. Histology

Each sample was fixed in Bouin’s solution for 24 h. Samples were subsequently washed in distilled water to remove Bouin’s residues, dehydrated in increasing alcohol gradations, and finally immersed in xylene for 3 h. Samples were transversely sectioned after the clitellum in an anterior and posterior segment, and the anterior segment containing the clitellum was embedded in paraffin. At least five longitudinal sections measuring 3 μm were cut from paraffin-embedded blocks with a semiautomatic microtome (Thermo Scientific Microm HM 300, Carlsbad, CA, USA), stained with hematoxylin and eosin (H&E), and examined under light microscopy (AXIO SCOPE.A1, Carl Zeiss S.p.A., Oberkochen, Germany). The slides were photographed by a microphotography-system digital camera (Axiocam 105 color, Carl Zeiss S.p.A., Oberkochen, Germany). The male reproductive apparatus of all samples was analyzed to detect natural *Monocystis * sp. infection in the seminal vesicles and possible associated lesions. To provide a quantitative estimate of parasite-associated tissue alteration, a morphometric analysis of infected seminal vesicles was performed using ImageJ software (version 1.54r). For each infected individual, the total area of the seminal vesicle and the area showing tissue disruption associated/substition with *Monocystis * sp. infection were manually delineated (40× magnification). The amount of lesioned/substituted area was expressed as a percentage according to the formula: lesion% = (lesioned or substituted area/total vesicle area) × 100. Descriptive statistics (mean ± standard deviation) were calculated across infected individuals.

2.3. DNA Extraction

DNA was extracted from 3 paraffin-embedded specimens naturally infected with Monocystis sp. using the QIAmp DNA Tissue FFPE (QIAGEN, Düsseldorf, Germany), following the manufacturer’s protocol. The lysates were incubated at 55 °C for 3 h to ensure complete tissue digestion. DNA was subsequently purified using the provided spin columns and eluted in 50 µL of elution buffer. DNA quality and concentration were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA), and samples were stored at –20 °C until further use.

2.4. PCR and Sequence Analysis

The specific primers used for amplifying Monocystis ITS rDNA are as follows: MITS-F: 5′-GAGAATG-GTCAAGTCGTAAC and MITS-R: 5′-GTTCA-ACGGGTATACTTGTTCAATTTCAGG [21]. PCR reactions were performed using the OneTaq 2X Master Mix (NEB, Ipswich, MA, USA), following the manufacturer’s instructions, with reaction conditions in the MiniAmp Plus Thermal Cycler (Thermo Fisher Scientific, Carlsbad, CA, USA) as follows: initial denaturation at 94 °C for 30 s, followed by 30 cycles of 30 s at 94 °C denaturation, 1 min at 45 °C annealing temperature, 1 min at 68 °C extension, followed by a final extension of 5 min at 68 °C.

The amplified products were separated on a 1.5% agarose gel and visualized using the UV transilluminator E-Gel Power Snap (Thermo Fisher Scientific, Carlsbad, CA, USA). PCR products were then purified using the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Purified amplicons were subjected to Sanger sequencing by Eurofins Genomics (Ebesberg, Germany). The resulting sequences were analyzed using the BLASTn (version BLAST+ 2.17.0) tool available at the National Center for Biotechnology Information (NCBI).

3. Results

3.1. Male Reproductive System of Uninfected Individuals

37/100 earthworms analyzed exhibited a normal morphology of the male reproductive apparatus in line with previous descriptions [22,23]. Briefly, the testis was observed in the 10–11 segment attached by a short stalk to the anterior dissepiment of the segment. Within the testes, the germinal epithelium showed the full sequence of early spermatogenic stages: from protogonia to primary spermatocytes (SP-I). Primary spermatogonia (SP) occurred singularly or in small clusters throughout the testis and appeared as large cells presenting thick filaments of chromatin. Secondary spermatogonia were pear-shaped, with spherical nuclei bearing a centrally located nucleolus and only a thin rim of cytoplasm. The first maturation division produced SP-I, smaller than spermatogonia and relatively abundant. The SP-I morulae then migrated into the seminal vesicles, where they continued their synchronous proliferation (Figure 1a). Within the vesicles, successive stages of differentiation were observed: secondary spermatocytes undergoing further maturation divisions and spermatids arranged in large aggregates around a central cytophore. In the more advanced regions of the seminal vesicles, elongated spermatozoa were evident, indicating a complete differentiation of spermatid (Figure 1b). In segments 10 and 11, the ciliated sperm funnels of the vasa deferentia were clearly identifiable and formed a highly folded sperm reservoir. The reservoir was lined by a ciliated epithelium resting on a thin extracellular matrix and externally covered by peritoneal cells. The lumen contained eosinophilic material secreted by the epithelial cells, with mature spermatozoa interspersed among the cilia (Figure 1c). The spermatheca was lined internally by a simple columnar epithelium characterized by basal nuclei and a well-developed apical brush border. In the apical region of the cell, numerous eosinophilic granules were visible, corresponding to mucin-rich secretions produced by the epithelial cells. Occasional incorporation of sperm tails into the epithelial cytoplasm was also observed, indicating the interactions between the spermatozoa and the epithelial surface and a structural retention by the brush border (Figure 1d).

3.2. Male Reproductive System of Infected Individuals

Natural *Monocystis * sp. infection was detected in 63 out of 100 individuals, corresponding to a prevalence of 63% (95% CI: 53–72%). *Monocystis * sp. The seminal vesicles contained numerous rounded oocysts distributed irregularly throughout the vesicular tissue; they varied in diameter and displayed a well-defined wall. Several oocysts contained clearly distinguishable sporocysts with multiple elongate sporozoites. Isolated sporocysts were also present outside the oocyst wall, dispersed within the surrounding tissue. An inflammatory encapsulation reaction was observed surrounding the oocysts. Nodular aggregates of celomocytes and free sporocysts were also observed inside the capsule. Brown-stained granular deposits occurred in some regions of the encapsulation, suggesting possible melanization.

The combination of oocysts and encapsulation resulted in a heterogeneous appearance of the seminal vesicle parenchyma, with noticeable local degeneration or compression of the surrounding tissue (Figure 2a,b).

Within the seminal vesicle, among the developing morulae, a rounded parasitic stage was observed. This structure appeared as a spherical eosinophilic body surrounded by the flagella of consumed spermatozoa or late spermatids. The tails attached to the parasitic body were arranged irregularly, creating a “ciliated-like” appearance. The absence of an oocyst wall or internal compartmentalization suggests a trophozoite developmental stage. The adjacent germ cell clusters exhibited normal organization, with morulae composed of tightly packed germinal cells (Figure 3). Morphometric analysis revealed that *Monocystis * sp. infection affected a substantial portion of the seminal vesicle tissue. The mean percentage of lesioned/substituted area was 36.59 ± 15.58% across the 63 infected individuals analyzed, indicating marked inter-individual variability in the extent of tissue alteration/substitution. Details about morphometric analysis on lesioned/substituted area compared to the total area of infected seminal vesicles are reported in Table S1.

The general architecture of the spermathecal wall appeared preserved. Compared with uninfected spermathecae, the lumen seemed to contained less spermatozoa, and these were located free in the lumen rather than adhered to the epithelial surface: no spermatozoa were observed attached to the brush border or aligned along the apical microvilli (Figure 4).

3.3. PCR

Finally, PCR analysis using the specific primers MITS-F and MITS-R successfully amplified the ITS rDNA region of Monocystis sp. A single clear band of approximately 500 base pairs was observed in all analyzed samples (Figure S1). Sequencing of the purified amplicons followed by BLASTn analysis confirmed that the obtained sequences matched the Monocystis ITS rDNA sequence deposited in GenBank under accession number OQ955756.1, with 100% query coverage and 100% average sequence identity.

4. Discussion

This histological study offers a new point of view into the interaction between *Monocystis * sp. and E. fetida, highlighting how parasitism can affect key reproductive and immunological processes in a species widely used in soil ecotoxicology. Despite the importance of E. fetida as a model organism, the potential influence of health status and of biotic agents (i.e., natural parasitic infections) on ecotoxicological endpoints has received little attention. It is well known that in L. terrestris infection by *Monocystis * sp. and other gregarin parasitic species occurs frequently, and host–parasite dynamics are fairly understood [11,13]. On the contrary, comparable data for E. fetida remain scarce [15,16,17]. The infection prevalence observed in our population (~60%) was notable. Compared to prevalence rates previously detected in wild individuals, our results underline that Monocystis may be more widespread in laboratory strains than previously assumed [24,25], representing a threat to the health of managed individuals as well as for the wild ones.

Infection was associated with reproductive alterations such as diminished sperm content within spermathecae, degeneration of seminal vesicle tissue, and inflammation. These alterations indicate that *Monocystis * sp. can directly impair reproduction by reducing the number of mature spermatozoa and thus the fertility potential. This is particularly relevant given that common ecotoxicological endpoints to assess the impact of soil contaminants and other pollutants include cocoon production and hatching, sperm quality, and integrity of the reproductive tissue [4,26,27]. Therefore, our study strongly suggests that the reproductive effects associated with *Monocystis * sp. infection closely overlap with endpoints routinely used to assess chemical toxicity in E. fetida. Thus, the negative results of reproductive endpoints could come from either the exposure to chemical stressors [7,9,27] or the presence of Monocystis spp. alone.

Our findings underscore the substantial risk of confounding effects in ecotoxicological studies when the infection status of test organisms is not assessed or controlled, thereby increasing the difficulty of correctly attributing the causes of observed reproductive impairments. Moreover, in L. terrestris, it has been shown that exposure of infected individuals to carbon-based nanomaterials exacerbates reproductive impairment by increasing the effects of *Monocystis * sp. compared to the effects of carbon- based materials on their own, thus suggesting a multiplying effect of contaminants on Monocystis spp. damage [10]. Consequently, the failure to detect and account for natural parasitic infections represents an underestimated source of biological variability in laboratory-based ecotoxicological tests and may lead to misinterpretation of toxicity thresholds, overestimation of contaminant effects, and reduced reproducibility across studies. For this reason, our findings challenge the implicit assumption that laboratory populations used in standardized assays are biologically uniform and free from confounding biological stressors. In this context, *Monocystis * sp. infection should be explicitly considered a potential biological bias in ecotoxicological assays employing E. fetida, particularly when reproductive and histopathological endpoints are used.

Moreover, we here firstly report the presence of a strong immune response made of multilayered encapsulations, celocyte nodules, and melanization occurring within the seminal vesicles of E. fetida. In L. terrestris, seminal vesicles have been described as immune-privileged sites, where no immune reaction occurs. Indeed, immune activation in L. terrestris against *Monocystis * sp. only occurs in the coelomic cavity [28,29]. Therefore, the immune aggregates observed in our specimens represent a new host–parasite interaction, suggesting a possible species-specific difference in susceptibility and immune activation between E. fetida and L. terrestris. The immunological events carry substantial energetic costs, often taking resources away from growth, somatic maintenance, and reproductive investment, which are endpoints routinely used to evaluate sublethal toxicity in soil ecotoxicology [4,14,26,27]. Therefore, the reduced growth and increased mortality often documented in previous studies could be linked to the possible reallocation of energy towards immune response against *Monocystis * sp. rather than other physiological events. Host–parasite interactions in invertebrates are increasingly recognized as dynamic processes influenced by the environment and immune status of the host, often involving trade-offs between immune function and reproductive investment [30,31,32]. In this context, our histological observations of *Monocystis * sp. infection in E. fetida show that parasitism can induce reproductive and immunological alterations that closely resemble endpoints commonly interpreted as contaminant-induced effects in ecotoxicological and ecopathological studies. Without dedicated parasitological screening, these parasite-induced effects can easily be misinterpreted as contaminant-induced toxicity events. As such, *Monocystis * sp. infection constitutes a significant potential biological variable—or bias—that may be a confounding factor in pollutant toxicity assessments using E. fetida. For this reason, we recommend implementing diagnostic screening to exclude *Monocystis * sp. infection in laboratory cultures, ensuring that test organisms used in ecotoxicological studies are free of parasitic interference. Parasitological examination should be performed on a representative sample of individuals as soon as earthworms are purchased by the breeding site, following the protocol described by Schall (2021) [32]. However, we observed that by manipulating earthworms, they release a small amount of feces, which can be suspended in water and smeared on a slide for microscopic evaluation. This technique could reduce the need for animal sacrifice; however, more detailed investigations should be carried out to validate the protocol.

5. Conclusions

*Monocystis * sp. infection significantly alters the reproductive and immunological traits of E. fetida, reducing sperm production and storage while inducing immune reactions within seminal vesicles not previously described for this species. These changes induced by the parasite resemble effects driven by chemical pollutants and, therefore, they can confound key ecotoxicological endpoints. Routine parasitological screening of laboratory cultures is recommended to ensure the reliability of soil-toxicity assessments involving E. fetida and to distinguish pollutant-related alterations from parasite-mediated impairments. Moreover, further investigations combining qualitative histopathology with quantitative parasitological results and reproductive analyses will help to better characterize the impact of *Monocystis * sp. infection in ecotoxicological contexts.