Abstract
Daphnia (Ctenodaphnia) magna, a species widely used for ecotoxicological tests, was recorded for the first time in waterbodies of the Mexican protected area and RAMSAR site Ciénegas del Lerma. The identity of the species was confirmed using morphological traits and cytochrome oxidase I as a molecular marker. Haplotypes were 100% identical to cultures used in laboratory bioassays in México and Canada. Individuals analyzed are related to strains from Europe, and their sequences differed from natural populations reported from the United States of America and Canada. This Mexican wetland supports a rich community of migratory waterfowl; therefore, D. magna could quickly spread to other waterbodies and cause potential adverse effects on local Daphnia species. The utilization of non-native species in ecotoxicological tests must be undertaken with great care, to ensure escape to natural waters is prevented.
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Daphnia (Ctenodaphnia) magna Strauss, 1820, is an anomopod cladoceran naturally distributed in the Holarctic and African zones, with a broad presence in Europe and Asia (Benzie 2005). Two large groups of haplotypes (European–West Siberian and Siberian–Beringian) are recognized; however, haplotypes from the European–West Siberian group have a high divergence and a true North American origin (De Gelas and De Meester 2005; Bekker et al. 2018). This species was introduced to Mexican laboratories in 1988 after a training course offered by the U.S. Environmental Protection Agency (Alcocer et al. 2022) and from Canadian laboratories around the year 2000 (Mendoza et al. 2013). In Mexico and some other Central and South American countries, D. magna has been widely used as a model organism for toxicity tests of diverse chemical substances, effluents, and receiving waters (US EPA 2002; Castillo-Morales 2004). Although some authors (Benzie 2005; Karabanov et al. 2021) have recognized Mexico as part of the natural distribution range for D. magna, there are no official records of this species in Mexican aquatic environments or checklists (Elías-Gutiérrez et al. 2008a). As such, we consider D. magna to be a non-native species that may threaten local cladoceran species. The use of this species as a test organism in tropical regions has been criticized (Martínez-Jerónimo et al. 2008), and it has been proposed to replace D. magna with native species in ecotoxicological bioassays (Sarma and Nandini 2006; Alcocer et al. 2022).
There are no molecular data to date on D. magna in Mexico other than those obtained from laboratory strains of the Mexican Institute of Water Technology (IMTA) and National School of Biological Sciences (IPN) (Elías-Gutiérrez et al. 2008b; Prosser et al. 2013; Bekker et al. 2018). We registered for the first time the presence of Daphnia magna living in a natural water body, the RAMSAR site and natural protected area Ciénegas del Lerma, particularly in the portion named Chimaliapan wetland, at the geographical coordinates 19° 45′ 04″ N and 99° 30′ 14″ W at 2580 m above sea level. The morphology of Daphnia found in Chimaliapan coincided with that typical of D. magna (Fig. 1a, 1b): the top of the head shield with ridges on both sides of the mid-line (Fig. 1c), the dorsal margin of the postabdomen deeply embayed (Fig. 1d), the dorso-cephalic suture in “W” and relatively large size from 2 to 6 mm (Benzie 2005). Morphological determination of non-native species is essential; nevertheless, genetic information is important for corroborating its geographic origin and proposing better management strategies (Kotov et al. 2022).
We confirmed the identity of the Ciénegas del Lerma Daphnia using nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene for three specimens independently isolated from the Chimaliapan wetland. First, DNA was extracted from a live organism using the standard HotSHOT method (Montero-Pau et al. 2008). Following the extraction, 3 μL of the DNA was added to a PCR mix (12.5 μL of polymerase Taq and 7.5 μL ultrapure water). The mitochondrial COI sequence (about 600 base pairs length) was amplified using the widely used Folmer primers F-1490 and R-2198 (Folmer et al. 1994), with the sequences LCO1490: 5′-ggtcaacaaatcataaagatattgg-3′ and HC02198: 5′-taaacttcagggtgaccaaaaaatca-3′. The resulting PCR products were visualized on a 1.5% agarose gel using UV light. DNA was sequenced by Sanger methods at the Institute of Biology, UNAM (Mexico). Then, the informatical work was carried using Geneious 2023.0.1; the DNA sequencing chromatograms were reviewed, edited by cutting the beginning and the end nucleotides because of low quality and no clear differentiation due to the nature of the Sanger method, and aligned using the MAFFT algorithm.
Before constructing the phylogenetic tree, we assessed the evolutionary model of substitution for our sequences using jModeltest 2.1.10 (Darriba et al. 2012), finding that it was HKY 85 + gamma. In order to estimate phylogenies using maximum likelihood (PhyML), we selected the following standard analysis options within the software Seaview 5: the best tree of nearest neighbor interchange (NNI), as well as subtree pruning and redrafting (SPR) simultaneously; support values were calculated selecting approximate likelihood ratios aLRTs, SH-like at the same time (Gouy et al. 2010). The sequences obtained were compared with those found in GenBank (Table 1) based on the survey of molecular analysis by Bekker et al. (2018), considering the origin of sequenced organisms, including some invaders and those commonly used in laboratories. According to the maximum likelihood tree (Fig. 1e), there are two deeply divergent clades for the COI gene of Daphnia magna, one from the United States and Canada and the other including Europe, the Mediterranean region, Caucasus, and Karelia. The organisms collected in the wetland in Mexico were identical (100% of the identity of 471 base pairs) to those reported for toxicology laboratory cultures (Elías-Gutiérrez et al. 2008b; Prosser et al. 2013) and as possible invaders in Mexico and Canada (Bekker et al. 2018).
In Mexico, the genus Daphnia has the highest species richness among the cladocerans, with at least 19 species (Silva-Briano et al. 2010), mostly belonging to the subgenus Daphnia, which are mainly distributed in the Nearctic biogeographic zone (Elías-Gutiérrez et al. 2008a). On the other hand, the subgenus Ctenodaphnia is barely represented in Mexico by the presence of D. exilis (Elías-Gutiérrez et al. 2008a), the exotic D. lumholtzi (Silva-Briano et al. 2010), and recently the presence of D. similis, which has been identified in Tepeyahualco lake in Central Mexico (unpublished data). Large Daphnia are mostly represented by six species in the Daphnia pulex complex (D. cheraphila, D. obtusa, D. pileata, D. pulex s. str., D. pulicaria, and D. prolata), mainly inhabiting fishless or clay-water environments (Hebert and Finston 1996; Elías-Gutiérrez et al. 2008a) where turbidity reduces the effect of fish predation.
Aquaculture, commercial shipping, the aquarium trade, and recreational boating are the main anthropogenic activities that have been observed to disperse zooplankton species across large geographic distances (Dexter and Bollens 2020); however, there is no record of the establishment of non-native species from ecotoxicological tests as a vector. It has been suggested that the spread of Daphnia magna can be attributed to human activity; a possible contributing factor to its distribution in North America may be aquaculture, with laboratory strains originating from Aquatic BioSystems Inc. Based on a comprehensive analysis of the genetic diversity of Daphnia magna, Bekker et al. (2018) grouped a clade (A) containing organisms found in ballast water, Europe, the Mediterranean region, Middle East, Turkey, Caucasus, and Western Siberia. The clade also contains Genbank sequences from Mexico, the United States, and Canada, but they argued that these sequences could have originated from European laboratory cultures. In addition, Mendoza et al. (2013) also noted that D. magna used in some Mexican laboratories had a Canadian origin. If these cultures were taken to North America and later to Mexico, it is possible to explain the high degree of genetic similarity observed between the organisms found in the field in this study and the sequences obtained from European organisms. On the other hand, bird transportation may not be discarded as a natural vector for cladoceran dispersal (Green and Figuerola 2005) because in Chimaliapan, high migratory bird species richness has been recorded (CONANP 2020); moreover, waterfowl transport significantly explains the gene flow of cladocerans in North America (Green et al. 2023).
Our data suggest that live or diapausing Daphnia magna used in toxicological tests may escape from ecotoxicology laboratories and establish in natural water bodies. However, we need to precisely determine where the first escape began. To our knowledge, ecotoxicology laboratories with alive D. magna strains in central Mexico are around 50 km from the Chimaliapan wetland and have different drainage basins; hence, direct leaking from these places seems unlikely. In any case, it is well-known that in some laboratories in Canada, Mexico, or another country that has yet to be considered, mismanagement of D. magna has occurred. Since then, dispersal has started and may continue.
The establishment of D. magna in permanent tropical environments such as lakes or reservoirs with high fish predation is not probably due to selective predation by fish. Fish predation on larger cladocerans is stronger in warm waterbodies than in temperate ones (Havens et al. 2015) because tropical fish communities in warm tropical lakes commonly have higher abundances and taxonomic diversity, less piscivorous species, smaller body size, and a predominance of omnivores with multiple or continuous reproductions throughout the year (Iglesias et al. 2017). In pools, ponds, and temporary water bodies where fish predation is low or absent, competitive interactions shape zooplankton communities. Under these conditions, body size is a crucial characteristic that determines competitive success among daphnids, with an advantage for larger species with lower threshold food concentration when food is limited (Gliwicz 1990). Therefore the establishment of large-size D. magna (2.0–6.0 mm) in these environments may threaten smaller (1.1–3.5 mm) native species. Thus, the spread of large D. magna may have consequences for diversity and ecosystem function with the potential for competition with native Daphnia.
The ecological and economic implications of using non-native species for laboratory tests are unexplored. Nevertheless, biological invasions may substantially affect the aquatic system's community structure leading to food chain disruption with potential economic effects on fisheries and drinking water quality (Kotov et al. 2022). Zooplankton invasions in the early twenty-first century are mainly represented by cladocerans from North America, where predator species such as Bythotrephes longimanus, Leptodora kindtii, and Cercopagis pengoi have the most damaging effects on ecosystem processes (Dexter and Bollens 2020; Kotov et al. 2022). For herbivores, Daphnia lumholtzi is the best example of invasiveness in America, where evidence was found for the competitive exclusion of local zooplankton populations (Dobberfuhl and Elser 2002). Therefore, it is crucial for conservation to determine the effects of the dispersion of introduced Daphnia magna on the ecosystem level and local fauna. Meanwhile, it is essential to develop ecotoxicological bioassay protocols with native species to reduce the use of exotic species with invasion potential to avoid detrimental effects on native species and ecosystem functioning.
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Acknowledgements
We thank M. en C. Alejandro Cruz Monsalvo Reyes and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) Projects IN231920 and IN229823. MAJS thanks Secretaría de Educación, Ciencia, Tecnología e Innovación de la Ciudad de Mexico.
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This study was support by PAPIIT IN231920 and IN229823 Projects, UNAM.
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Conceptualization, CAE-R and AL-V; Funding, AL-V; Data collection and field work, CAE-R, DMM-M and LR-DP; data analysis and calculations, MAJ-S, DMM-M and EP-I; writing, reviewing and editing, CAE-R, AL-V, DMM-M, MAJ-S, EP-I and LR-DP. All authors have read and approved the final version of the manuscript.
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Espinosa-Rodríguez, C.A., Jiménez-Santos, M.A., Martínez-Miranda, D.M. et al. Daphnia magna (Crustacea: Anomopoda) in central Mexico wetlands: implications of escape from ecotoxicological laboratories. Biol Invasions 26, 1–7 (2024). https://doi.org/10.1007/s10530-023-03164-7
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DOI: https://doi.org/10.1007/s10530-023-03164-7