Occurrence of the eastern mosquitofish Gambusia holbrooki Girard, 1859 (Poeciliidae, Cyprinodontiformes) in the Madeira Archipelago (NE Atlantic)

BioInvasions Records (2025) Volume 14, Issue 3: 641–651 Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 641 CORRECTED PROOF Rapid Communication Occurrence of the eastern mosquitofish Gambusia holbrooki Girard, 1859 (Poeciliidae, Cyprinodontiformes) in the Madeira Archipelago (NE Atlantic) Inês Órfão1,2, Luís P. da Silva3,4, Dinis Girão3,4,5, Pedro Raposeiro6,7, José Jesus2, Manuel Biscoito1,8,9, João Canning-Clode1,2,10, Filipe Ribeiro11 and Sónia Ferreira3,4 1MARE (Marine and Environmental Sciences Centre) / ARNET (Aquatic Research Network), ARDITI (Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação), Funchal, Madeira, Portugal 2Faculty of Life Sciences, University of Madeira, Funchal, Madeira, Portugal 3CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão, Universidade do Porto, 4485-661 Vairão, Vila do Conde, Portugal 4BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, 4485-661 Vairão, Vila do Conde, Portugal 5Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4099-002 Porto, Portugal 6CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Pólo dos Açores, UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands, Universidade dos Açores, Ponta Delgada, Portugal 7Faculdade de Ciências e Tecnologia, Universidade dos Açores, Ponta Delgada, Portugal 8Museu de História Natural do Funchal, Estação de Biologia Marinha do Funchal, Funchal, Madeira, Portugal 9OOM – Oceanic Observatory of Madeira, Funchal, Portugal 10Smithsonian Environmental Research Center, Edgewater, USA 11MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Faculty of Sciences of the University of Lisbon, Lisbon, Portugal Corresponding author: Inês Órfão ([email protected]) Abstract Gambusia holbrooki is one of the most problematic invasive species globally, but its presence has yet to be confirmed in some regions. This is the case of the particularly conservation-sensitive freshwater insular ecosystems of Madeira Archipelago in Macaronesia. Freshwater ecosystems on the two main islands were inspected based on previous unconfirmed observations of mosquitofish. Specimens were found in three artificial ponds in Madeira Island and in one stream of Porto Santo Island. The morphology and mitochondrial DNA of the collected specimens confirmed their identification as G. holbrooki, confirming its presence on this archipelago for the first time. However, previous records suggest that the species may have inhabited Madeira Island for decades. Also, the high number of individuals detected in Porto Santo suggests an established population on this island. The apparent population size in Porto Santo may make an eradication action extremely challenging. Nonetheless, this may still be achievable on Madeira Island, which is extensively covered by inland waters. Proactive measures are essential to prevent further spread and mitigate potential biodiversity loss in freshwater ecosystems of this archipelago. Measures must be adapted to account for the current mosquitofish distribution and the reduced carrying capacity of Madeira’s freshwater ecosystems. It is also important to consider that these ecosystems serve as habitat for an endangered native fish species and several endemic macroinvertebrates. Ultimately, it is essential to develop a monitoring plan to avoid introductions into adjacent natural areas and towards other insular ecosystems. Key words: DNA barcoding, first records, freshwater, insular ecosystems, Macaronesia, mosquitofish Citation: Órfão I, da Silva LP, Girão D, Raposeiro P, Jesus J, Biscoito M, CanningClode J, Ribeiro F, Ferreira S (2025) Occurrence of the eastern mosquitofish Gambusia holbrooki Girard, 1859 (Poeciliidae, Cyprinodontiformes) in the Madeira Archipelago (NE Atlantic) . BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 Received: 6 March 2025 Accepted: 19 June 2025 Published: 8 September 2025 Handling editor: Enrique Gonzalez Ortegon Thematic editor: Kenneth A. Hayes Copyright: © Órfão et al. This is an open access article distributed under terms of the Creative Commons Attribution License ( Attribution 4.0 International - CC BY 4.0). OPEN ACCESS. Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 642 Introduction The eastern mosquitofish Gambusia holbrooki Girard, 1859, is considered one of the most invasive freshwater fish (Lowe et al. 2000; ISSG 2013). Native to East North America, the poecilids G. holbrooki and G. affinis (Baird & Girard, 1853) were intentionally introduced at the beginning of the twentieth century to control mosquito and mosquito-borne diseases (Seale 1917; Pyke 2005). Currently, mosquitofish G. holbrooki represents one of the most widespread freshwater fish with populations established on all continents except Antarctica (Krumholz 1948; Pyke 2008). In Europe, the eastern mosquitofish was first introduced in Spain in 1921, having expanded and established populations throughout the mainland and in some islands (Pyke 2008; Vidal et al. 2010; Florencio and Lamelas-López 2016; Anastácio et al. 2019; Costa et al. 2021). Clear evidence of the severe and drastic negative impacts caused by G. holbrooki led to its inclusion in the European Union legal framework as an invasive alien species of Union Concern (Nr. 1143/2014). Despite such concerns, its presence and distribution remain undocumented and unstudied in certain regions, particularly in oceanic islands. Invasive species can particularly threaten these isolated islands characterized by high endemism (Leclerc et al. 2018; Simberloff 1995). The invasive success of eastern mosquitofish relies on its phenotypic plasticity, life-history traits and aggressive behaviour (Pyke 2005). Mosquitofish phenotypic plasticity allows them to tolerate distinct environmental conditions and survive in different habitats. It can survive and succeed in highly modified and polluted habitats, in divergent water salinities and temperatures (Pyke 2008; Ruiz-Navarro et al. 2011: Kurtul et al. 2024). For instance, they can develop resistance to DDT (dichlorodiphenyl-trichloroethane), survive and prosper in water temperatures from 0° to 45 °C, and salinity from 0 to 40 ppt (Pyke 2008). The species exhibits an opportunistic and omnivorous diet, consuming insects, crustaceans, fish, amphibians, algae, zooplankton and diatoms (Pyke 2005). Life-history traits are correlated with fast development and a high reproductive rate that allows them to establish in an ecosystem quickly (Ribeiro et al. 2008). For instance, it presents several breeding seasons. Females can have multiple broods during each season and their fecundity is adaptable to environmental conditions (Santi et al. 2020). At last, its voracious and aggressive behaviour led G. holbrooki to dominate fish assemblages (e.g. Moreno-Valcárcel et al. 2013). Consequently, this species tends to reduce diversity and abundance of native and endemic fish, macroinvertebrates and zooplankton (e.g. Margaritora et al. 2001; Nicol et al. 2015; Florencio and Lamelas-López 2016; Haiahem et al. 2017). This ecological displacement can disrupt trophic dynamics and deteriorate water quality (Akhurst et al. 2012; but see Angeler et al. 2002). Further range expansion is expected in response to global climate change and habitat Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 643 Figure 1. Sampling sites in Madeira Archipelago surveyed in the present study. White dots indicate sites surveyed and stars indicate sites with confirmed presence of Gambusia holbrooki. This species was found in artificial urban ponds in Madeira Island and in a stream in Porto Santo Island. alterations given its preference for warm and disturbed waters (Murphy et al. 2015; Jourdan et al. 2021; Nekrasova et al. 2021). Considering the significant negative consequences that G. holbrooki can have on ecosystems, it is essential to determine its presence, identify invasive routes and create adjusted control actions throughout its non-native distribution range. Madeira Archipelago is one such area lacking comprehensive knowledge (Ribeiro et al. 2009). There are sporadic unconfirmed observations of the genus in artificial urban ponds for the main island – Madeira (M. Biscoito, J. Jesus personal data; Observation.org 2024; iNaturalist 2024; respectively). In this context, we investigated the presence of G. holbrooki in urban ponds and natural habitats across the Madeira and Porto Santo islands. Collection and examination of suspected specimens for detailed morphological and genetic analyses allowed to confirm the presence of G. holbrooki in the studied region. Ultimately, this first official record contributes to a more comprehensive understanding of this species’ invasive distribution in the world. Materials and methods Freshwater aquatic invertebrate sampling was conducted on Madeira and Porto Santo islands using kick-nets in 36 sites in 2024 (Figure 1, Supplementary material Table S1). A hand net with a 250 µm mesh size was used to perform 2 to 3 sampling procedures at each site. Each sampling lasted 1–3 minutes and covered a wide variety of habitats such as riffles and pools to represent the distinct microhabitats present at each sampling location. Most sites were natural habitats (i.e. streams or ponds), but four artificial ponds in Madeira were visited to validate previous unconfirmed records of mosquitofish. Photographic records of the habitat with mosquitofish were made. Then, some specimens were collected and preserved in 96% ethanol for morphological and genetic analyses in the laboratory. Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 644 To confirm the genus identification a combination of the following morphological characters was considered: small size (less than 6 cm), large eyes and a flattened head, mouth superior, with lower jaw protruding further than upper jaw, dorsal fin origin well behind anal fin origin, visible scales, rounded tail fins with black dots, silver colouration lighter in the ventral area, with scattered grey colour and black markings. Each specimen was analysed on a stereoscopic microscope (Leica MZ12, 8x to 100x; Olympus SZX16, 7x to 115x). Additionally, photographic records made in 2022 in Porto Santo were analysed. The dorsal and anal fin ray counts were used for species identification. Gambusia holbrooki has 8 dorsal and 11 anal fin rays, while the similar G. affinis has 7 dorsal and 10 anal fin rays (Walters and Freeman 2000). Additionally, collected fish were sexed based on the presence (males) or absence (females) of a gonopodium, and of a black spot in the genital region close to the ventral fin (females). Total genomic DNA was isolated from the muscle tissue of 3 selected specimens using EZ-10 Spin Column Animal Genomic DNA Miniprep Kit (BIO BASICS). Partial sequences of mtDNA COI gene were amplified using the LCO1490 and HC02198 primers (Folmer et al. 1994). Both PCR reactions had 10 μL of final volume, containing 5 μL of Multiplex PCR Master Mix (QIAGEN), 0.4 μM of each primer, and 1–2 μL of DNA. PCR amplification was carried out on a T100 Thermal Cycler (BioRad) using the following conditions: initial denaturation at 95 °C for 15 min; 5 cycles at 95 °C for 30 s, 47 °C for 45 s, 72 °C for 45 s; then 40 cycles at 95 °C for 30 s, 51 °C for 45 s, 72 °C for 45 s; and a final elongation step at 60 °C for 10 min. Amplified DNA fragments were purified and sequenced using the same primers as in the PCR, following ABI PRISM BigDye Terminator protocols. Sequences were visualized on a 310 Applied Biosystem DNA Sequencing Apparatus. Sequences were curated using Geneious 9.1.8. (https://www. geneious.com), compared and submitted to BOLD and GenBank databases. Results In Madeira Island, no specimens were found in any of the natural sites visited. Species of mosquitofish were found in three of the five artificial ponds visited (Figure 2A, B). Two of the four artificial ponds where citizens previously reported seeing mosquitofish no longer exist (Table S1). Only a few specimens were observed at each occupied pond in Madeira Island (fewer than 10). The two specimens collected were a female and a juvenile. Specimens photographed in 2022 in Porto Santo Island at Ribeiro Salgado were identified as Gambusia sp. (Figure 2C). Hundreds of similar fish were again observed at two sampling points in the same stream in 2024 (Figure 2D, E, Table 1). The stream had dense vegetation, warm still water and shallow margins in the area where we found the specimens. From the five specimens collected, two were adult males, two were adult females, and one Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 645 Figure 2. Representative sampling sites in Madeira Archipelago where Gambusia holbrooki was detected: A and B – Madeira Botanical Garden artificial ponds; C – mosquitofish observed in Porto Santo in 2022; D and E – standing water sections of the interrupted stream of Ribeiro Salgado in Porto Santo in 2022 (D) and in 2024 (E). Photographs by Inês Órfão (A, B, C and D) and Luís P. da Silva (E). Table 1. Sampling sites where the presence of Gambusia holbrooki was confirmed in the Madeira Archipelago. Specimens were found in artificial (*) and natural waterbodies (º). Date Island Region Site Latitude Longitude Voucher number 2024.12.17 Madeira Santa Cruz Água de Pena* 32.7055 −16.7651 MMF51452 2024.12.18 Madeira Funchal Funchal* 32.6510 −16.9145 MMF51453 2025.01.02 Madeira Funchal Madeira Botanical Garden* 32.6624 −16.8947 na 2022.09.05 Porto Santo Porto Santo Lapeirasº 33.0525 −16.3646 na 2024.09.07 Porto Santo Porto Santo Lapeirasº 33.0523 −16.3600 MMF51327 2024.09.07 Porto Santo Porto Santo Lapeirasº 33.0525 −16.3646 MMF51451 was a juvenile of undetermined sex. Morphologic identification of the seven collected specimens resulted in the validation of the presence of Gambusia holbrooki in both islands. The collected specimens were deposited at the regional natural history museum, Museu de História Natural do Funchal (voucher numbers MMF51327, MMF51451, MMF51452, MMF51453). We obtained 658 bp COI DNA barcoding sequences from 3 selected specimens. The DNA barcodes generated enabled the unequivocal identification of the specimens as G. holbrooki. DNA barcodes matched others deposited in BOLDSYSTEMS database at 100%. Database DNA barcodes were from specimens collected in Europe (France, Greece, Spain), Asia (India, Turkey, Uzbekistan), Australia and United States of America. DNA sequences were deposited in BOLD (IBDIP001-24, IBDIP002-24 and IBDIP003-24; http://dx.doi.org/10.5883/DS-IBIGH) and GenBank public databases (Accession numbers: PQ629089, PQ629090, PQ629091). Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 646 Discussion Morphological traits and COI DNA barcoding sequences of fish from Porto Santo and Madeira islands confirmed the presence of the eastern mosquitofish Gambusia holbrooki in the Madeira Archipelago. In Porto Santo, hundreds of mosquitofish were found in a stream with some favourable conditions for this species, i.e., at lower altitudes, in a reach with dense vegetation, warm still water and shallow margins (Cherry et al. 1976; Casterlin and Reynolds 1977; Mesquita et al. 2006; Pyke 2008; Murphy et al. 2015). Still, the arid landscape, high water salinity and calcareous composition of freshwater found on this island support the remarkable plasticity of mosquitofish (e.g. Santi et al. 2020). In Madeira, the species was found in artificial ponds in urban areas but not in natural habitats, suggesting a distribution restricted to highly anthropized areas. Our findings add the Madeira Archipelago as part of the national distribution range of the eastern mosquitofish G. holbrooki (see Ribeiro et al. 2009). This underscores the potential risks posed by further introductions and expansion to neighbouring water bodies and oceanic islands. The specimens from the Madeira Archipelago are identical to the widespread haplotype present in Europe, Asia, Australia and America. No information is available that allows us to trace its origin. Despite no official record of mosquitofish releases in the Madeira Archipelago, the mosquitofish was known to be present in irrigation ponds and urban lakes in Madeira Island several decades ago (M. Biscoito and J. Jesus personal data). Specimens have been officially introduced in other regions of Portugal since 1941 for mosquito control (Lourenço 2004; Ribeiro et al. 2009; Anastácio et al. 2019). Later, studies suggest that mosquito larvae generally represent a small percentage of the stomach content of G. holbrooki (Pyke 2005). Still, mosquitofish is occasionally and informally introduced in mainland Portugal (F. Ribeiro personal observation). Regarding Porto Santo, the introduction of mosquitofish is likely more recent than in Madeira. Mosquitofish could have arrived in Porto Santo for ornamental purposes, and the population’s founders could have escaped private aquaria. This seems to have been the case of the introduction in the Azores Archipelago (Costa et al. 2021), and potentially of the New Zealand mud snail Potamopyrgus antipodarum in the Madeira Archipelago (Órfão et al. 2024a). The high density of G. holbrooki at sites in Porto Santo suggests a wellestablished population. The impacts remain unknown but may already be difficult to reverse. Given its presence in a natural habitat, eradication would be logistically challenging, costly, and likely unsuccessful (Kalogianni et al. 2025). However, its confinement to isolated water bodies could facilitate localized control measures, particularly if population density and structure are considered, and long-term physical removal is consistently implemented (Ruiz-Navarro et al. 2013). The likelihood of Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 647 mosquitofish spreading is reduced within Porto Santo Island due to few potential habitats and almost no connected watercourses. In contrast, translocations are easier to occur from artificial ponds in urban areas to streams in Madeira Island. Ponds could serve as stepping stones to the more than 200 streams covering the island. Such expansion to Madeira Island’s natural freshwater ecosystems would be highly risky for biodiversity and the ecosystem. The introduction of G. holbrooki was correlated with a reduction in native and endemic macroinvertebrate populations on another Macaronesia island (Florencio and Lamelas-López 2016; Raposeiro et al. 2017). These findings are aligned with a large body of evidence proving that mosquitofish reduce the diversity and abundance of native species (Pyke 2005; Beatty et al. 2022). Nevertheless, it is unknown whether the mosquitofish could compete for food resources with the critically endangered European eel Anguilla anguilla during its early life stages (Pike et al. 2020; Órfão et al. 2024b). The combined effect of the eastern mosquitofish and other introduced species is also unknown. For instance, it would be worth investigating the impact that both G. holbrooki and the snail P. antipodarum may have on water quality (Akhurst et al. 2012; Sandvik et al. 2022). Currently, G. holbrooki is included in the List of Species of Union Concern (EU Regulation 1143/2014). Therefore, member states must implement a management plan that will lead to local adjusted measures. An effective strategy must prioritize the protection of the native and, especially, the endemic biodiversity. Furthermore, urgent monitoring of Madeira’s freshwater ecosystems is required to prevent further biological invasions and to assess the status of existing invasive species, such as rainbow trout Oncorhynchus mykiss (Ribeiro et al. 2009). Priority should be given to creating a robust monitoring plan involving the local community and several stakeholders to increase the effectiveness of prevention and control measures. For instance, citizens’ involvement can contribute to study mosquitofish current distribution covering a larger area, in a shorter period and at a lower cost (MacPhail and Colla 2020; Maasri et al. 2022). Also, raising awareness of the threats imposed by invasive species helps reduce the likelihood of new introductions. Other important stakeholders, such as aquarium enthusiasts, should also be included in awareness programs. At the research level, such monitoring of invasive species’ presence will benefit from combining traditional and new methods (e.g. transects and eDNA – Radinger et al. 2019). Ultimately, a comprehensive approach to monitoring and controlling biological invasions is crucial and should align with the UN Water Action Agenda 2023 and the EU Nature Restoration Law. Special attention should be given to susceptible ecosystems, such as insular freshwater with reduced and unique biodiversity (Whittaker et al. 2017; Fernández-Palacios et al. 2021). This is the case of the Macaronesia archipelagos with a high rate of Gambusia holbrooki in Madeira Archipelago Órfão et al. (2025), BioInvasions Records 14(3): 641–651, https://doi.org/10.3391/bir.2025.14.3.13 648 endemic macroinvertebrates (Stauder 1995; Hughes et al. 1998; Hughes 2003; Raposeiro et al. 2012; Pešić et al. 2025). Authors’ contribution Research conceptualisation: IO, LPS and SF; sample design and methodology: IO, LPS, PR and SF; investigation and data collection: IO, LPS, DG, PR and SF; data analysis and interpretation: IO, LPS, DG and SF; funding provision: JCC; writing: IO, LPS, DG and SF (original draft), PR, JJ, MB, JCC, and FR (review and edit). Authors’ ORCIDs Inês Órfão: 0000-0001-9261-324X; Luís P. da Silva: 0000-0003-2358-1277; Dinis Girão: 00090008-4842-0721; Pedro Raposeiro: 0000-0002-7461-0851; José Jesus: 0000-0001-8509-2757; Manuel Biscoito: 0000-0002-9347-0823; João Canning-Clode: 0000-0003-2143-6535; Filipe Ribeiro: 0000-0003-3531-5072; Sónia Ferreira: 0000-0002-6884-3966. Acknowledgements The authors would like to acknowledge the support provided by the Direção Regional de Pescas and Direção Regional de Agricultura e Desenvolvimento Rural, in particular to José Luís da Silva Ferreira and Marco Alexandre Caldeira da Costa, in the search for evidence of the species in the archipelago.We also thank the two anonymous reviewers for their valuable comments and suggestions. Funding declaration FR, LPS and SF were funded by the FCT—Fundação para a Ciência e a Tecnologia, I.P., through the program “Stimulus of Scientific Employment, Individual Support” (CEEC/0482/2020.CEECIND/CP1595/CT0001, CEECIND/02064/2017/CP1423/CP1645/CT0009, 2020.03526.CEECIND/CP1601/CP1649/CT0007, respectively) (DOI identifiers: https://doi.org/ 10.54499/2020.00482.CEECIND/CP1595/CT0001, https://doi.org/10.54499/CEECIND/02064/ 2017/CP1423/CP1645/CT0009 and https://doi.org/10.54499/2020.03526.CEECIND/CP1601/ CP1649/CT0007). JCC was also funded by national funds through FCT under the Scientific Employment Stimulus Institutional Call (CEECINST/00098/2018). The project MEGAPREDATOR: A giant on the water: from predation pressure to population control of the European catfish (Silurus glanis) (https://doi.org/10.54499/PTDC/ASP-PES/4181/2021), supports currently FR. DG benefited from a grant from the project Biodiversity Genomics Europe (Grant no.101059492) funded by Horizon Europe under the Biodiversity, Circular Economy and Environment call (REA.B.3); co-funded by the Swiss State Secretariat for Education, Research and Innovation (SERI) under contract numbers 22.00173 and 24.00054; and by the UK Research and Innovation (UKRI) under the Department for Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme. 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