The invasive ctenophore Mnemiopsis leidyi A. Agassiz, 1865 along the English Channel and the North Sea French coasts: another introduction pathway in northern European waters?

The presence of Mnemiopsis leidyi A. Agassiz, 1865 along the French coasts of the Eastern English Channel and the North Sea (EEC-NS) was established via morphological observation and molecular evidence. The earliest records were from surveys carried out in autumn 2005 in Le Havre harbour (Bay of Seine, EEC) and coincided with the historical introduction of the species in other Northern European waters. Since 2009, the species has also been frequently observed along the French coast of the North Sea. Results indicate M. leidyi has established a selfsustaining population in the Bay of Seine, which may act as a source population for northern European harbours via commercial shipping.


Introduction
The voracious zooplanktivore Mnemiopsis leidyi A. Agassiz, 1865 is a lobate ctenophore native to the Atlantic coasts of North and South America (Gesamp 1997). This eurythermic and euryhaline species can tolerate a wide range of environmental conditions (Kremer 1994;Purcell et al. 2001). These features, together with its regeneration (Henry and Martindale 2000) and reproductive ability (self-fertile hermaphrodite with high fecundity and rapid growth rates), explain the success of M. leidyi as an invasive species (Baker and Reeve 1974;Costello et al. 2006;Jaspers et al. 2011). The establishment of M. leidyi had severe ecological and economic impacts in the Black Sea in the 1980's (Shiganova 1998) and in the Caspian Sea in the late 1990's (Roohi et al. 2010). Consequenly, recent (since 2005) discoveries of this species in Norwegian fjords (Oliveira 2007;, the Baltic Sea (Hansson 2006;Javidpour et al. 2006;Kube et al. 2007), Danish territorial waters (Tendal et al. 2007), the German Bight (Boersma et al. 2007), the Netherlands (Faasse and Bayha 2006) and in Belgian coastal waters (Dumoulin 2007;Van Ginderdeuren et al. 2012), has alarmed the scientific community, especially as these northern European waters are amongst the world's most important fishing grounds. However, work by Hamer et al. (2011) concludes that the densities of M. leidyi currently observed in northern waters do not represent a threat to eggs and fish larvae, although they might compete with larval fishes for small plankton prey and it is unknown whether the species represents an immediate ecological threat. In this context, the aim of this article is to document the first records of M. leidyi along the French coasts of the Eastern English Channel-North Sea ecosystem (EEC-NS), and discuss possible invasion routes from its native range.

Methods
Sample sites were located along the French coast between the western part of the Bay of Seine (BoS) and the North Sea ( Figure 1A-C). A variety of sampling devices and observations were used depending on the sampling location (Table 1S). Scuba diving observations by amateurs were the source of most M. leidyi records in the BoS (mainly in Le Havre Harbour). Photographs taken by divers (Figure 2A-C) permitted morphological identification of M. leidyi based on the position of the oral lobes extending to the apical statocyst (sense organ) over nearly the entire body length (Faasse and Bayha 2006;Oliveira and Migotto 2006).
Within research monitoring surveys, two types of plankton nets were used along the French coast of the North sea for quantitative estimate of M. leidyi, a WP2 net (opening area 0.25m² and 200 µm mesh size; Fraser 1968a) and a WP3 net (opening area 1.0 m² and 1.0 mm mesh size; Fraser 1968b). The M. leidyi record from the Seine estuary represents specimens collected during a bottom trawl survey (280  40cm aperture, 20 mm mesh size).
When available, abundance and approximate sizes are provided in Table 1S of supplementary material. When actual numbers per m 3 were not available, abundance was expressed as less than ten (+), sparse (++), or common (+++). Freshly caught individuals from all sites were observed and identified alive, and some of them were preserved in 70-100 % ethanol and stored at 4°C for further genetic analyses (following Van Ginderdeuren et al. 2012; see superscript in Table 1S).

Results
All observed specimens collected during this study were lobate individuals, and no cyddipid larvae were recorded. The earliest records of M. leidyi that we could verify with photographs provided by divers ( Figure 2A) were from Le Havre Harbour (Figure 1B and Table 1S, N° 8, 12 and 13) in September and October 2005. Since then, there have been frequent observations of individuals (ranging from 3 to 8 cm in length) reported during diving sessions in the same area ( Figure 1B and Table 1S N° 4 to 16). Some individuals collected in 2011 ( Figure 1B and Table  1S, N° 9 and 10) were genetically identified and confirmed our initial morphological identification (E. Antajan, unpublished data). In Le Havre Harbour, the greatest numbers of M. leidyi were recorded during July to October; however, a few individuals were still observed in winter ( Figure  1B and Table 1S, N° 13).
The first records of M. leidyi in the North Sea were made in September 2009 (Antajan et al. 2010, Figure 1C; N° 24 to 27). Specimens of M. leidyi were also collected during 2010, 2012 and 2013 in the two nearby harbours of Calais (N° 19 to 23) and Dunkirk (N° 28 to 32). M. leidyi was recorded from these sites from September to December at temperature ranging from 5.5 to 20.2°C and salinity between 29.7 and 34.9.
In January 2011, the launch of the European MEMO project (InterReg IVa-2 seas, Mnemiopsis Ecology and Modelling: Observation of an invasive comb jelly in the North Sea) was accompanied by a major public communication effort and has led many reports by citizens of the presence of the species in the BoS ( Figure 1A-B and Table  1S, N° 1 to 3 and 18). Following the collection of M. leidyi in the Seine estuary during a bottom trawl survey ( Figure 1B and Table 1S, N°17), the Somme, Canche and Authie estuaries (Figure 3), and a long-term monitoring station (SOMLIT stations) were also surveyed to assess whether M. leidyi had spread northward. However, no M. leidyi were detected at any of these four sites.

Discussion
Mnemiopsis leidyi has been present along the French coast since at least 2005. Earlier occurrence is possible; however, the routine monitoring surveys along the French coasts of the North Sea (e.g. Calais, Dunkirk and Gravelines) use a formalin solution to preserve zooplankton samples immediately after collection. This causes M. leidyi to disintegrate and makes species identification, counting, and measuring practically impossible (Kube et al. 2007;Engell-Sørensen et al. 2009;Gifford 2009). Purcell (1988) presented an identification method for formaldehydepreserved samples based on identification of M. leidyi tentacle bulbs. This method was tested on 2005-2009 formalin-fixed samples but given the very low number of individuals (< 2 ind.m -3 ) and the large amount of particulate matter in the samples, tentacle bulbs were impossible to detect. Since our 2009 record of M. leidyi in nonpreserved samples ( Figure 1C and Table 1S, N° 24-26), zooplankton sampling techniques were reconsidered and collected samples were first examined while specimens were alive before sample preservation. This new strategy resulted in additional records in the North Sea, and we recommend a systematic visual inspection of still living material for ctenophores and similar taxa prior to sample preservation in future zooplankton surveys.
Our first record of M. leidyi in 2005 in Le Havre Harbour coincides with the first record of M. leidyi in the northern part of the North Sea -Oslo fjord in Norway (Oliveira 2007). Although the North Atlantic Current has previously been considered as a potential introduction vector of M. leidyi in the North Sea (Oliveira 2007), it could not explain our simultaneous record in the Bay of Seine (BoS) because this current does not extend into the English Channel ). Instead, our results suggest there were multiple and simultaneous introductions of the species in the main harbours of Northern Europe, presumably by means of ballast water (Vinogradov et al. 1989;Ivanov et al. 2000;Oliveira et al. 2007;Fuentes et al. 2010 and references therein). Large ships crossing the Strait of Dover towards the North Sea regularly stop in the Calais and Dunkirk harbours and can also visit the large European harbours such as Le Havre (France), Rotterdam (Netherlands), Anvers, Zeebrugge (Belgium) and Hamburg (Germany; Figure 3). The introduction pattern of M. leidyi by cargo ships in the EEC-NS would, therefore, result from trans-Atlantic (e.g. East coast of Mexico and USA) and local ship transits as was observed along the Belgium and Dutch coasts (Wolff 2005;Faasse and Bahya 2006;Van Ginderdeuren et al. 2012) and in the Mediterranean Sea (Bolte et al. 2013;Ghabooli et al. 2013).
Advection processes and natural transport (drift, internal circulation) have previously been identified as vectors of secondary spread for M. leidyi (Lehmann and Javidpour 2010;Schaber et al. 2011;Van Ginderdeuren et al. 2012). Therefore, it is possible that M. leidyi introduced along the French coast of the North Sea originated from adjacent regions (e.g. Belgium or BoS).
The spread of M. leidyi southward from the North Sea, where established populations are observed (Faasse and Bayha 2006;Van Ginderdeuren et al. 2012), is unlikely because the residual tidal transport is oriented northward; water masses typically drift from the English Channel to the North Sea Brylinski et al. 1991; Figure 3). North-easterly winds can at times reverse the general circulation pattern and induce southward spreading of plankton organisms (Dauvin et al. 2007), but these conditions did not prevail during our sampling periods (autumn 2009(autumn -2010(autumn , 2012(autumn , 2013(autumn and January 2010. Recurring records year-round in the BoS since 2005 suggest the existence of another potential, self-maintaining, source population (sensu Costello et al. 2012). However, a northward spread from the BoS is also unlikely as no M. leidyi were recorded in the main EEC estuaries (Somme, Canche and Authie) nor at the SOMLIT stations ( Figure 3). Natural expansion from the Seine estuary would likely lead to M. leidyi spreading westward, which is consistent with the net seaward transport in the Seine estuary (Wang et al. 1995), the general water circulation pattern in the BoS (Le Hir et al. 1985;Lazure and Desmare 2011), and observations of M. leidyi in the western part of the BoS in autumn 2011 ( Figures  1A and 3; N° 1 and 2 Table 1S). Thus, simple water drifting as a vector of M. leidyi spreading from the BoS to the French coast of the North Sea is unlikely, and records suggest the existence of distinct North Sea and a BoS populations. Although the BoS population seems to be selfsustaining, the absence of M. leidyi in winter or year-round in the North Sea (in 2011) suggests a regular pattern of elimination and re-inoculation more typical of a sink population (sensu Costello 2012).
Two main invasion pathways were identified for M. leidyi introductions in Eurasia (Ghabooli et al. 2011;Reusch et al. 2010;Costello et al. 2012). While specimens from the Gulf of Mexico invaded the Black Sea and then the Caspian Sea, specimens from the Baltic Sea seem to originate from Narragansett Bay (USA). Studies of M. leidyi population genetics would permit assessment of whether EEC-NS specimens are individuals imported from their native habitats or represent mixed populations resulting from water exchanges between the EEC-NS harbours during ship transit, as was demonstrated in the Mediterranean Sea (Fuentes et al. 2010;Ghabooli et al. 2013).
In conclusion, the present study demonstrated that the alien invasive ctenophore M. leidyi has been present along the French coasts of the EEC-NS ecosystem at least since 2005. If the species has established a population in the BoS, its development and origins in North Sea French harbours (Calais and Dunkirk) are poorly understood. Long-term monitoring, along with population genetics investigations, are therefore needed to address source-sink dynamics of the species and potential establishment along the French coasts of the North Sea.

Acknowledgements
We gratefully acknowledge all divers of the association Port Vivant and F. Chevallier from Tatihou museum for reporting their observations of Mnemiopsis leidyi and for providing detailed information, photographic evidence and specimen for DNA analyses. We also acknowledge the anonymous reviewers whose constructive comments greatly improved a previous version of this paper. The Gravelines and Dunkirk data were collected within the framework of the research programmes IGA (Impact des Grands Aménagements), Vibrio-Manche and gelatinous plankton surveys conducted by IFREMER and ULCO-LOG with financial support from EDF (Electricité de France). This work was funded by the EU under the InterReg IVa-2seas (MEMO project) programme.
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