Biological invasions pose a significant threat to aquatic ecosystems, and the spread of uncontrolled non-indigenous species can have detrimental effects on biodiversity, ecosystem processes, and economic activities. The Chesapeake blue crab, Callinectes sapidus, native to the western Atlantic Ocean, is non-indigenous in the Black Sea region. This study presents novel findings on its presence and breeding in the Black Sea, particularly in North-Western part within the territorial confines of Ukraine. The study provides evidence of successful reproduction by a female blue crab with eggs in this non-native habitat, i.e. off the coast of Ukraine. Furthermore, molecular DNA barcoding analysis revealed the Ukrainian crab to be of a haplotype found in Italy and the United States, indicating potential connectivity between the Black Sea population and other established populations. Additionally, other haplotypes were detected in the Black Sea region, suggesting the possibility of multiple introductions and admixture between non-indigenous populations of different origins. The successful establishment and spread of C. sapidus in the Black Sea region may be attributed to its adaptability to a wide range of environmental conditions and the lack of natural predators or competitors in the invaded regions. This crab is commercialized in the USA, which may have implications for fisheries and aquaculture activities in Ukraine.

Callinectes sapidus, cox1, DNA-barcoding, egg-bearing female, non-indigenous decapod, North-Western Black Sea

One of the main modern challenges for aquatic ecosystems is biological invasion: the spread of non-indigenous or cryptogenic species, i.e. those of uncertain or unknown origin, to areas where they did not previously occur (Ruiz et al. 2000; Olenin et al. 2017). Such invasion is accompanied by threats to the ecosystem, as the species can act as vectors for new parasites and pathogens, alter ecosystem processes, reduce biodiversity, and cause major economic losses (Vitousek et al. 1996; Mack et al. 2000). In marine ecosystems, successful invasions are more common in the deltaic and estuarine zones with salinity levels lower than 20‰, because the brackish waters are poor in species and empty niches can be exploited by immigrants (Wolff 1999). As the Black Sea is a brackish water marginal part of the Mediterranean Sea, it is hence characterised by low biological diversity and high range of biological invasions (Gomoiu et al. 2002; Băncilă et al. 2022).

The Chesapeake blue crab Callinectes sapidus Rathbun, 1896, is a widely-distributed species, native to the western Atlantic Ocean, ranging from Nova Scotia, Canada, to northern Argentina (Onyekachi 2014), which has gained significant attention due to its ecological and economic importance (Kamita 1963). Despite its wide distribution, its taxonomic status remains undecided. Recent studies have suggested that the species complex within the genus Callinectes may require a more detailed examination, as there are subtle morphological differences that may indicate the presence of cryptic species (Spitznagel et al. 2019).

The Chesapeake blue crab has been documented as an invasive species in many Mediterranean European coasts, as well as in the Levantine Sea and the Black Sea basin (Ribeiro and Veríssimo 2014; Mancinelli et al. 2017; Falsone et al. 2020; Öztürk et al. 2020; Pipitone et al. 2020; Kara and Chaoui 2021; Castriota et al. 2022; Năstase et al. 2023). The species is believed to have entered the region through various pathways, such as ballast water discharge, aquaculture activities, and natural dispersal.

The initial documentation of C. sapidus in the Black Sea dates back to 1967, where it was first observed in the Gulf of Varna, Bulgaria (Bulgurkov 1968). Subsequently, the species has exhibited a long history of expansion throughout the Black Sea, initially characterized by sluggish dispersion and limited recorded instances. However, in the 1970s, multiple sightings were documented in the eastern region of the basin, specifically in the Kerch Strait (Zaitsev 1998), alongside the coasts of Russia and Georgia (Shaverdashvili and Ninua 1975; Monin 1984). By the early 2000s, the species had been sighted in Romania, Crimea, and various locations in the Sea of Azov (Bashtannyy et al. 2002; Diripasko et al. 2009; Khvorov 2010), and in 2013, its presence was confirmed in Türkiye (Yağlıoğlu et al. 2014). Notably, in specific sectors of the Black Sea, the expansion of this species has escalated significantly, as evidenced in Bulgaria (Stefanov 2021) and Türkiye (Aydın 2017; Ceylan 2020, 2024; Aydın et al. 2024); this escalation in the North-West Black Sea within the territorial confines of Ukraine forms the topic of this paper.

Two hypothesised routs of Chesapeake blue crab invasion into the Black Sea basin were discussed by Stefanov (2021). In the first stage (approximately till the end of the 20^th century), the species was initially introduced in the area with ballast waters, but in the second stage, most of the blue crab founds in the area due to natural dispersal from the Mediterranean Sea trough the Bosporus Strait. A trend towards rapid increase in cases of natural spread across the Bosporus Strait to the Black Sea since the beginning of the 21^st century has also been noted for marine fish and turtles (Kvach and Kutsokon 2017; Zinenko et al. 2021). This is explained by climate changes, which supports the hypothesis. While before the significant warming, the Black Sea may have been unfavourable (too cold) for most of the warm-water species, but the current climate changes dramatically improve the situation. For example, ecological niche modelling, which was applied for C. sapidus, turned out that all shallow coastal areas of the Black Sea can be suitable habitats for this species (Ceylan et al. 2024).

The reasons behind the successful invasion of C. sapidus are not fully understood, but it is likely that its adaptability to a wide range of environmental conditions, as well as the lack of natural predators or competitors in the invaded regions, have contributed to its establishment and spread (Epifanio 2013).

DNA barcoding is an important tool for species identification and conservation management in the region. It uses a short, standardized gene region as a molecular marker for species identification (Hebert et al. 2004; Vernooy et al. 2010). This method rapidly and accurately identifies known and new species (Hebert et al. 2009; Vernooy et al. 2010; Tahir and Akhtar 2015). It can be used to establish the source of initial invasion (Son et al. 2020) and provide information on whether the bottleneck effect took place, or whether there were multiple introductions and admixture between invasive populations of different origins (Kolbe et al. 2007; Roman and Darling 2007; Estoup et al. 2016). Additionally, it is important for food authentication and disease vector identification (Vernooy et al. 2010).

This study presents novel findings on the presence and breeding of the non-indigenous Chesapeake blue crab, Callinectes sapidus, in the Black Sea region. The aim of the study was to provide the DNA barcoding of the specimen found, provide its detailed morphometric description and a clutch characteristics, also to analyse the perspectives of the further spread of the species and its commercial use.

The species was sampled on 20.06.2024 using 45 × 50 cm dipnet, 8.5-mm mesh, near the City of Chornomorsk (southwestern Ukraine) from 0.5 m depth, in the marina harbour (46.288722°N, 30.663694°E; Fig. 1). The sampled individual was transported alive into the laboratory of the Institute of Marine Biology of the National Academy of Sciences of Ukraine, Odesa, and was kept alive until detailed study. The specimen was identified to the genus or species level based on Makarov (2004), as well as descriptions from the previous findings (Niță and Nenciu 2021).

Figure 1. 

Map of findings of Callinectes sapidus in the Black Sea and the Sea of Azov. Grey circle – this study; red circles – data from the used sources: 1–8 (data from the Table 1), 9 (Zaitsev 1998), 10 (Diripasko et al. 2009); 11 (Monin 1984; Pashkov et al. 2012); 12 (Shaverdashvili and Ninua 1975); 13 (Yağlıoğlu et al. 2014; Ak et al. 2015; Öztürk et al. 2020; Aydın et al. 2024; Ceylan 2020, 2024); 14 (Khvorov 2010); 15 (Stefanov 2021); 16 (Bashtannyy et al. 2002); 17 (Snigirev et al. 2020) (for details see Suppl. material 1: table S1).

In addition, comprehensive information about the distribution of this species in Ukraine was gathered from amateur contributors, datasets, and media sources. Our approach provided a more comprehensive overview compared to what is available in peer-reviewed publications.

The living crab was sexed and weighed, and the following measurements were determined (in mm): carapax length (CL), carapax width (including the longest spines; CW), length of each pereiopod (P1L, P2L, P3L, P4L, P5L) both right and left, abdomen length (AL) and abdomen width (AW). The total weight of the wet egg clutch was measured. The egg number was calculated in the isolated 1-g part of the living wet clutch using a Konus Crystal 7×–45× STEREO stereomicroscope. Following this, the number of eggs in 1 g was used to calculate the total egg count of the clutch. The egg length was measured (in µm) for 25 specimens. The mean parameters and standard deviation (sd) were calculated for all measures, as well as minimum and maximum values.

Small parts of uropods were dissected and preserved in 96% ethanol for molecular study. The specimen was then preserved in 10% formalin and deposited in the zoological collection of the National Museum of Natural History at the National Academy of Sciences of Ukraine (voucher # IKNDFZ-It-648).

Genomic DNA was isolated at the Department of Ecology and Vertebrate Zoology at the University of Lodz (Poland) using the buffer method (Chelex) (see Casquet et al. 2012 for details). The mitochondrial cox1 gene was amplified with standard cox1 primers HCO2198 and LCO1490 (Folmer et al. 1994). The following PCR protocol was used: 3 min – 94 °C; (30 s – 94 °C, 1:30 min – 45 °C and 1 min – 72 °C) × 5; (30 s – 94 °C, 1:30 min – 51 °C and 1 min – 72 °C) × 35; 5 min – 72 °C (Hou et al. 2007). The resulting amplicons were purified with exonuclease I (20 U/μl; EURx) and Fast Polar-BAP alkaline phosphatase (1 U/μl, EURx).

The sequences of the amplicons were determined by Macrogen Inc. under Sanger procedure and then analysed in the online GenBank repositories using BLAST (blast.ncbi.nlm.nih.gov) and BOLD to find similarities. The data were registered in BOLD, where they were assigned BINs (Barcode Index Number) based on the genetic distance to other similar sequences in the database (Ratnasingham and Hebert 2013). Every sequence in the BOLD repository undergoes this taxonomic revision. The algorithms used to assign BINs are described in detail in Ratnasingham and Hebert (2013). For invertebrates, the standard genetic distance is more than 2% for animals to have different Barcode Index Numbers (BINs 2024).

A phylogenetic analysis was performed using two of our sequences and 51 sequences available in the public repository (Morlais and Severson 2002; Keskin and Atar 2012, 2013; Windsor et al. 2019; Öztürk et al. 2020; Venera-Pontón et al. 2020; Vecchioni et al. 2022) (Suppl. material 1: appendix S1). The analysis was performed by aligning our sequences with those downloaded from the BOLD database and GeneBank using MAFFT 7.452 (Katoh et al. 2017). The phylogeny relationship was the reconstructed using maximum likelihood in IQ-TREE v1.5.5 (Nguyen et al. 2015), with branch support estimated using 1000 replicates of both the SH-like approximate likelihood-ratio test (Guindon et al. 2010) and the ultrafast bootstrapping algorithm (Minh et al. 2013). The model was assigned automatically. Haplotypes were determined using DnaSP v. 6.12.03 (Rozas et al. 2017), with a Median-Joining approach inferred in PopART (algorithm described by Clement et al. 2002).

Export and import data from foreign countries for crab goods (HS code: 030614) are sourced from the UN Comtrade database (UN Comtrade 2024). All the data present in the database all available years was analysed by date.

Our data confirms the second finding of an ovigerous (egg-bearing) female of the Chesapeake blue crab (C. sapidus) in the Black Sea; the previous specimen was identified in the port of Fatsa, Ordu, Türkiye in 2020 (Gül et al. 2021). Also, this is the first observation of a female blue crab with eggs off the coast of Ukraine, providing evidence that the species is successfully reproducing in this non-native habitat. Most of the findings of C. sapidus in the Black Sea have been mature individuals, with no ovigerous females among them (Öztürk et al. 2020). This suggests that the population in the Black Sea may be sustained through continuous introductions, rather than successful local reproduction.

The size of previous gravid female recorded near the Turkish coast was approximately the same as the present crab, and similar to other findings in the Black Sea: the gravid female in Ordu had a carapax (CL/CW) measuring 81.33/200 mm, while ours measures 77.64/204 mm (Gül et al. 2021). The carapax width (largest parameter) of the crabs from the Turkish coasts varied between 157–203 mm (Aydin et al. 2024), compared to 205 mm for a sample from Romanian waters (Niță and Nenciu 2021). In the Ukrainian waters, the carapax width varied between 140–150 mm for a crab in the Sea of Azov (Diripasko et al. 2009) to 200 mm for one near the Crimean coasts (Monin 1984).

The egg sizes in the gravid female registered near the Turkish coasts varied between 261.7–309.5 µm (281 ± 18.26) (Gül et al. 2021), which is close to our data. The wet clutch weight of the newly-recorded female (48 g) was larger than the Turkish one, with 33.84 g (Gül et al. 2021); the new specimen also had a higher egg count (n = 1,410,816), than the Turkish one (n = 1,166,879).

The size of the gravid female recorded in the study could support the hypothesis of successful reproduction in the North-Western Black Sea, because the transportation with the ballast waters of the 200-mm-size crab individual looks sceptic. The continuous findings of the adult individuals in this region in last decades (Vishnevskaya 2013; Snigirev et al. 2020; Voloshkevich 2021; Chaus and Markautsan 2023; Didenko 2023) confirm that the reproduction is possible. Potential route could involve ports along the coastlines, focusing on the transfer of goods over land for segments of the journey. For instance, logistical connections from the Black Sea ports to major hubs in Eastern Europe, followed by transport through Greece or other Mediterranean ports, could facilitate cargo movement.

Molecular DNA-barcoding analysis indicates that the haplotype found in Ukraine is also present in Italy and the United States, suggesting connectivity between the Black Sea population and other established populations. Additionally, two other haplotypes were detected in the Turkish Black Sea, form another clade that also includes haplotypes known in the invasion region only from the Levantine Sea, indicating the genetic diversity within the region. In general, the haplotypes had a mixed distribution pattern within each cluster. This genetic diversity in the region is quite consistent with the hypothesis of two waves of the blue crab invasion in the Black Sea (Stefanov 2021). In this case, the potential route for the Ukrainian haplotype could be connected with traditional long-distance shipping routes between the Black Sea ports and ports of the Western Mediterranean/Atlantic region. For Turkish haplotypes, the invasion is likely due to natural dispersion from the Eastern Mediterranean (Levantine Sea) through the Bosporus Strait and related seas. In the other hand, the Eastern Mediterranean is significantly different from the Western Mediterranean due to the leading role of species invasions from the Indo-Pacific region through the Suez Canal (Galil et al. 2018). This may fully explain the absence of these haplotypes in the Western Mediterranean and the high genetic diversity in the Black Sea.

The successful introduction of C. sapidus in the Black Sea and Levantine Sea can be attributed to its euryhaline nature, which allows the species to thrive in a wide range of salinity conditions (Öztürk et al. 2020).

The Chesapeake blue crab already has significant commercial importance, and is harvested in fisheries along the Atlantic coast of the United States (Stagg and Whilden 1997; Rhodes et al. 2001; Kennedy et al. 2007; Miller et al. 2011) and in the Gulf of Mexico (Perry et al. 1984). While the historical focus of the fishery has been the Chesapeake Bay, other regions have seen increasing prominence: Maryland, Virginia, North Carolina, and Louisiana are the primary states for commercial crab landings (Kennedy et al. 2007; DeAlteris et al. 2012; Bourgeois et al. 2014), boasting a significant number of commercial crab licence holders, and recreational crabbing (Bourgeois et al. 2014).

In Ukraine, the establishment of a viable blue crab aquaculture industry would likely face several political, social and ecological challenges. Thus, apart from getting through the policy of Ukraine (Resolution 2022), we propose some steps that should be taken to overcome the social and ecological challenges (Fig. 5).

Figure 5. 

Suggested scheme of socio-ecological steps for a blue crab commercialization into Ukrainian domestic seafood market.

It is impossible to predict the potential for the commercial use of the species based on the fertility of a single specimen. It is also necessary to understand the temporal structure of their populations and to consider their size, weight and sexual maturity. Blue crab populations usually vary considerably in their sexual maturity depending on the water body. For example, specimens from Bigulia Lagoon mature later (males: 16.16 cm; females: 16.79 cm) than individuals from Palo Lagoon (males: 14.38 cm; females: 13.86 cm) (Marchessaux et al. 2024). Seasonal size differences also occur between males and females between lagoons, and within the same lagoon. A comparison of individuals from different lagoons showed that the key influence on the growth rate, distribution and structure of populations is exerted by temperature and salinity (Marchessaux et al. 2024).

Nevertheless, care should be taken to control the spread of this crab outside farms. Thus, we offer to commercialize it under half-closed aquaculture systems to prevent its population growth in natural areas. Escaped crabs are known to cause additional costs, including the repair of damaged fishing nets and the loss of income from damaged catches (Oussellam et al. 2021).

The study demonstrates a first recorded female blue crab which carrying eggs and successfully reproduced in a non-native habitat off the coast of Ukraine. Molecular DNA barcoding analysis revealed that genetically, the Ukrainian crab is similar to a haplotype found in Italy and the United States, suggesting a potential link between the Black Sea population and other established populations. Additionally, the analysis detected other genetic variations in the Black Sea region, indicating the possibility of multiple introductions and interbreeding between non-indigenous populations from different origins. The further study of the Chesapeake blue crab spread and productivity in the Ukrainian waters is necessary to understand its commercial potential.

YK research conceptualization, species identification, funding administration; HG molecular analysis and results discussion, funding administration; AL morphology study, laboratory proceeding; MS range analysis, general discussion; SK fieldworks, sampling administration.

Yuriy Kvach https://orcid.org/0000-0002-6122-4150

Halyna Gabrielczak https://orcid.org/0000-0002-7888-477X

Anastasiia Lepekha https://orcid.org/0009-0006-8461-7025

Mikhail O. Son https://orcid.org/0000-0001-9794-4734

Sergii Khutornoi https://orcid.org/0000-0003-1351-8610

The molecular analysis was supported by the University of Lodz internal funds. Publication of this study was supported by the European Union’s Horizon Europe HORIZON-CL6-2024-BIODIV-01 project “GuardIAS - Guarding European Waters from IAS”, under grant agreement no. 101181413 (Katsanevakis et al. 2024).

Data is available in the BOLD System repository, http://www.boldsystems.org. Relevant voucher information is accessible through the public dataset DS-CSPD (DOI: http://dx.doi.org/10.5883/DS-CSPD).

Species georeferenced records are available at the European Alien Species Information Network: https://easin.jrc.ec.europa.eu/easin/RJD/Download/27fe9991-584e-4b6f-8d91-92eaa589fa6f.

We thank to Kostiantyn Redinov for providing information on the discovery of the species. We express our gratitude to a fisherman Ruslan Stavniychuk for the provided information. We also greatly thank the anonymous reviewers for their efforts and suggestions to improve the quality of our manuscript.