Migratory behavior of free-living marine nematodes surrounded by sediments experimentally contaminated by mixtures of polycyclic aromatic hydrocarbons

1 Introduction

Free-living marine nematodes represent an excellent group for experimental and ecotoxicological studies because of their small size (1–5 mm), high abundance (up to 23 million per m²), and short life cycles (Mahmoudi et al., 2005; Moreno et al. 2011; Semprucci and Balsamo, 2012). To date, their migratory behavior has been studied in artificially defaunated sediments with different grain size to clarify the granulometric preference of these organisms (Schratzberger et al., 2004) and to determine if differences in the availability of organic matter drive their colonization patterns (Zhou, 2001). Focusing on this latter aspect, Gallucci et al. (2008) showed that deep-sea nematodes tended to preferentially colonize sediments with higher amounts of organic matter than those not similarly enriched.

Bazzicalupo et al. (1994) showed that nematodes detected and produced chemical signals for feeding, reproduction, and communication. In this context, contaminants, such as polycyclic aromatic hydrocarbons (PAHs), heavy metals, or pesticides, may act as chemical signals that induce reactions, such as the escape from harmful environmental conditions.

Various studies (Carman et al., 2000; Hägerbäumer et al., 2015; Mahmoudi et al., 2005) in closed microcosms have shown the toxic effect of PAHs on marine meiofauna, inducing a loss of abundance and nematode diversity. Thus, the disappearance of sensitive species has been observed in favor of more tolerant species, implying a major change in community structure (Muyssen et al., 2006). However, an approach based on closed enclosures does not simulate in situ conditions because it does not allow the progressive escape of nematodes from hotspots of harmful chemicals, thereby hindering the determination of the actual tolerance of a target species to a specific contaminant. In contrast to closed systems, open experimental enclosures allow the organisms to escape, if desired, making this approach closer to approximating in situ conditions.

In the present study, the migratory behavior of nematodes was investigated in defaunated sediment with different mixtures of PAHs. The use of open experimental enclosures allowed for the observation of spontaneous migration patterns that reflected the ability of nematodes to withstand contaminants and, in turn, their upper limit of tolerance.

2 Material and methods

3 Results

Overall, 28 species belonging to 28 genera were observed during the experiment (Table 1). Each compartment was characterized by a specific sub-community of nematodes. Although Rhabditis sp. presented a relatively high relative abundance in all treatments, this ubiquitous taxon was variably associated with other nematode species according to the compartment: Calamicrolaimus honestus in N (16.60 ± 4.38%), R (18.46 ± 2.78%), and CF (20.88 ± 3.08%), Cyartonema germanicum (14.14 ± 7.94%) in RR, and Oncholaimus campylocercoides (15.5 ± 3.94%) in RCP. Furthermore, Rhabditis sp. was the dominant taxon in almost all compartments; in particular, in RCF, it accounted for 98.41 ± 2.74% of the entire assemblage. The only two exceptions were C and CP, where O. campylocercoides and, to a lesser extent, Calamicrolaimus honestus had a higher relative abundance than that of Rhabditis sp.

Nematode abundance varied significantly among compartments and values ranged between 2.6 ± 0.5 individuals throughout the RC compartments and 69.3 ± 5.8 individuals throughout the initial field community (I) used to start the experiment (Fig. 2). At the end of the experiment, the compartments presented the following descending numerical gradient in abundance: I-R-CF-E-RCF-RR-CP-RCP-C-RC. Post-hoc significant differences were determined by the Tukey HSD test between central samples (i.e., I and E) and those contaminated with PAHs (Fig. 2).

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Fig. 1

Microcosm design (A) and horizontal distribution of the different sedimentary compartments into the microcosm. Natural sediment collected to start the experiment (I); natural sediment at the end of the experiment (E); defaunated sediments contaminated with chrysene (C); defaunated sediments contaminated with a mixture of chrysene and fluoranthene (CF); defaunated sediments contaminated with a mixture of chrysene and phenanthrene (CP); (RC, RCF, RCP, R, and RR) defaunated and uncontaminated sediments.

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Fig. 2

Abundance of free-living marine nematodes in the different sedimentary compartments (see Fig. 1 for the meaning of the abbreviations coding for the compartments).

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The species numbers were significantly lower in both contaminated and uncontaminated compartments than at the beginning of the experiment (I), as indicated by one-way ANOVA results and post-hoc Tukey HSD comparisons (Fig. 3). In particular, overall nematode assemblages in the reference compartments (R, RR, RC, RCF, and RCP) were less diverse than in that in I (Fig. 3).

The values for the Shannon diversity index were lower at the end of the experiment than at the beginning (I). Paired Tukey HSD test outputs showed significant differences between the mean Shannon values in different compartments, especially for I vs. RR, RC, CF, and RCF and E vs. RC, RCF and C vs. RC and CF vs. RCF (Fig. 3). Comparable results were observed for Margalef’s species richness (d).

In the case of evenness (J’), no discernible differences from the initial community were recorded. However, multiple comparisons with the Tukey HSD test highlighted significant differences for I vs. RCF, E vs. RCF, and CF vs. RCF.

The nMDS results (stress = 0.18) (Fig. 4) indicated a very weak impact of the PAHs on the specific distribution. The most important effect was noted for contaminated compartments C and RC. Additionally, the ANOSIM analysis did not indicate significant differences in the composition of nematodes among the compartments, most likely because of the low numbers of replicates (n = 3). Despite this, a dissimilarity exceeding 60% was noted between the initial community (I) and communities from all compartments after 30 days of exposure (Table 2). Furthermore, the average dissimilarities for C vs. E and C vs. RC exceeded 60% (Table 2).

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Fig. 3

Values of univariate indices at each sedimentary compartment (see Fig. 1 for the meaning of the abbreviations coding for the compartments). Species number (S); Shannon diversity index (H'); Margalef’s species richness (d); Pielou’s evenness (J’). The stars above bars indicate significant differences when compared with the starting nematofauna ‘I’.

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Fig. 4

nMDS ordination based on the abundances of nematode species (see Fig. 1 for the meaning of the abbreviations coding for the compartments).

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As shown in Table 2, SIMPER results confirmed that Rhabditis sp. was the main taxon that contributed to dissimilarity. Two other species, O. campylocercoides and Calamicrolaimus honestus, were also discernibly responsible for the dissimilarity between compartments. In particular, the average dissimilarity between I and all the communities at the end of the experiment was mainly caused by both a decrease in the abundance of O. campylocercoïdes, Calamicrolaimus honestus, and P. proteus and an increase in that of Rhabditis sp.

4 Discussion

This work was conducted to test the effects of PAHs that are primarily released from the oil industry in the Bizerte area, Tunisia (i.e., chrysene, phenanthrene, and fluoranthene) on the migratory behavior of a local free-living nematode community. An original microcosm design was then created using PVC tubes and compartments with different sediment qualities were directly connected.

Our results are in accordance with those of previous studies (Bazzicalupo et al., 1994; Troemel et al., 1995), which showed that the nematodes can detect relatively distant contamination hotspots and react by migrating from them at a distance dependent upon the taxon. After 30 days, Rhabditis sp. represented more than 50% of all nematodes in the natural sediments, although this taxon accounted for <1% at the beginning of the experiment. Because this taxon exhibited substantial abundance in the compartments treated with PAHs, Rhabditis sp. may be considered an opportunist and able to colonize contaminated sediments that would not be inhabited by more sensitive nematode species. Notwithstanding, the dominance of Rhabditis sp. in all compartments could also be ascribed to its small size and pointed and elongated tail. Indeed, according to Boufahja et al. (2016), the locomotion of a given species inevitably depends on its morphology, physiology, and lifestyle.

Two other species, Calamicrolaimus honestus and O. campylocercoides were widespread among all compartments. However, compared to Rhabditis sp., these two species were more represented in the initial natural sediment (I), as well as in the C and CP compartments. It is therefore likely that Calamicrolaimus honestus and O. campylocercoides are resistant to low levels of stress and compete with Rhabditis sp. in these compartments. However, these species failed to dominate Rhabditis sp. in the compartments with higher stress, namely a mixture of PAHs with four cycles, such as CF.

In the case of P. paradoxus, Encheliidae (sp.), Trichotheristus paramirabilis, and Theristus pertenuis, they seemed to avoid contaminated sediments because these organisms were observed only in uncontaminated sediments (I and E) and compartments (R). Conversely, M. pristiurus and L. longicaudatus were found only in one or more contaminated treatments, which resulted in them being almost absent from the controls. This finding may indicate that these organisms may preferentially migrate to contaminated sediments because in the uncontaminated ones the interspecific competition is excessive. Furthermore, this result suggests that they are, to some extent, tolerant to PAHs. On the other hand, some species exhibited avoidance behavior toward the contaminated compartments (C, CP, and CF). In this context, it is noteworthy that T. modicus, which avoided the stress in the CP compartment by moving forward until reaching the uncontaminated compartment, RCP. Additionally, the species C. germanicum also exhibited avoidance and migration; indeed, this species had a greater presence in the less stressful compartments (R, RR, RC, and RCP), but also was able to avoid the most stressful compartment with the heaviest PAHs, CF.

From the beginning of the experiment, the field sediments placed into the central part contained a nematode community (I), which was free to move within the microcosm in any direction. The exposure of the organisms to different mixtures of PAHs induced species-specific migratory behavior that was tightly associated with the tolerance of each taxon to contamination. Univariate analysis showed that all contaminated compartments appeared to have discernible effects on nematodes, probably in relation to the mortality of the most sensitive species (Beyrem and Aïssa, 2000; Carman et al., 2000) or their avoidance of the contaminated compartments. Overall, all peripheral control compartments (RC, RCF, and RCP) were characterized by assemblages with lower numbers of species than those observed in contaminated sediments (C, CF, and CP). Although sensitive species likely tended to remain in the natural sediments (E), the most tolerant ones seemed to migrate towards control associated areas by passing through contaminated compartments, i.e., a behavior observed for Cyartonema germanicum, T. modicus, and S. splendens. Focusing on contaminated compartments (C, CF, and CP), the lowest diversity characterized the assemblage exposed to chrysene, implying higher toxicity associated with this contaminant. Furthermore, possible antagonistic effects exerted between chrysene and the other two tested PAHs (fluoranthene and phenanthrene) may be inferred because it has been reported in previous studies (Morelis et al., 2007). Different effects on nematodes were exerted by the two tested mixtures; i.e., the assemblage exposed to phenanthrene and chrysene was more diverse than that observed in the sediments treated with fluoranthene and chrysene. This finding suggested that the antagonism between the first two contaminants was higher than that exerted by fluoranthene on chrysene.

5 Conclusion

The experimental design and protocols proposed in this work are totally original and the bioassay could be reproduced at low coast. Our experimental approach using connected compartments with different levels of stress allowed us to highlight several experimental findings. First, it was possible to highlight the “real” tolerance and sensitivity of nematode species vs. PAHs, and thus identified their natural distribution in the surrounding sediments based on their “own decisions.” Second, the observed distribution pattern of nematode species mirrored the species-specific affinity to PAHs contamination that was measurable in terms of biodiversity. The freed movement of the nematodes throughout all the compartments contaminated or not, allowed us to identify the most tolerant species, such as Rhabditis sp., and to less extent, Calamicrolaimus honestus and O. campylocercoides, that can be used in future ecotoxicological studies related to PAHs. The experiment allowed us to determine the most sensitive species, namely, S. paradoxus, Encheliidae (sp.), Trichotheristus mirabilis, and Theristus pertenuis, that existed only in uncontaminated compartments. These taxa could be classified as sentinels and their high abundance in a given water body could be indicative of pristine status.

Finally, our results suggested a possible antagonism between the PAHs used to start the experiment (chrysene, fluoranthene, and phenanthrene). Although chrysene appeared to exert a toxic effect on nematodes, its harmful effects appeared less pronounced when mixed with the other two PAHs, with more pronounced antagonism with phenanthrene than with fluoranthene.