Presence of the introduced ctenophore Mnemiopsis leidyi A. Agassiz, 1865 in a lagoon system within the River Rhône delta (southeast France)

The introduced ctenophore Mnemiopsis leidyi A. Agassiz, 1865 was observed in the Vaccarès Lagoon (River Rhône delta, southeast France) for the first time in 2016. Large blooms had been observed during the summer in 2016, with monthly zooplankton community monitoring carried out from November 2016 to October 2017. This was done to better understand the dynamics of this ctenophore within this brackish Lagoon. In parallel, we report the first record of Mnemiopsis leidyi in another Mediterranean lagoon near Camargue delta (Estomac Lagoon) in 2019.


Introduction
Non-indigenous species introduction in recipient ecosystems constitute a major source of biological pollution (Boudouresque and Verlaque 2002;Elliott 2003). As some of these species become invasive, they often have strong ecological and economic impacts: modifying the diversity, the ecosystem functioning (e.g. competition, predation) and impacting human activities (e.g. fisheries, industrial complex, and tourism) (Gallardo et al. 2016).
The ctenophore Mnemiopsis leidyi has been ranked within the top 100 worst marine bio-invaders by the International Union for Conservation of Nature (IUCN; Lowe et al. 2000). Due to high reproductive rates (Jaspers et al. 2015) and high predation rates on zooplankton communities (Purcell and Decker 2005;Condon and Steinberg 2008), this ctenophore can strongly impact ecosystems both at ecological and economical levels (Purcell et al. 2001a;Gallardo et al. 2016).
Mnemiopsis leidyi originates from estuaries and coastal waters along Atlantic coastlines from the Chesapeake Bay, USA, to Argentina (GESAMP 1997). Through ballast waters release, the species has spread the past 30 years over Eurasia (Costello et al. 2012;Jaspers et al. 2018a). M. leidyi was  In this article, we report on the presence of Mnemiopsis leidyi in the Vaccarès Lagoon and on its variability in relation to environmental parameters. In order to better understand the ecology of M. leidyi within the Vaccarès Lagoon and to assess its environmental requirements, monthly monitoring was conducted from November 2016 to October 2017.

Materials and methods
The Vaccarès Lagoon, 6 500 ha, 2 m maximum depth, is in the center part of the Rhône River delta (South of France). It is includes a protected area the National Natural Reserve of Camargue, which is managed by the National Society of Nature Protection (SNPN). Nonetheless, it is contaminated by agricultural runoff, especially during spring and summer seasons. Connections with the sea are distant (more than 5 km) and limited to narrow passages equipped with sluices.
Given the difficulty to access to the banks of the Vaccarès Lagoon (restricted access, siltation, quicksand, bird nesting areas) and the prohibition of motorboats, only the Capelière station was studied (Figure 2A; 43.5353°N; 4.6370°E).
From November 2016 to October 2017, once a month (except in July 2017), sampling of the zooplankton component was conducted (Table 2). Due to the shallowness of the site and to the prohibition of motorboating, sampling was conducted by feet. Zooplankton and ctenophore were sampled using a conic net (80 μm mesh, 30 cm opening area, 1.2 m long).
The operator was placed on the left side of the net to limit the influence of his movements. A round trip along a 50 m linear transect parallel to the banks, delimited by two sticks, was performed ( Figure 2B, C). This operation was replicated (14.1 m 3 sampled). A total of 20 samples were analysed. Ctenophores were isolated and washed with filtered lagoon water. The total number of M. leidyi were counted, the whole sample was then preserved in buffered sea water-formalin solution (4% final concentration) for further zooplankton taxonomic identification. Just before each zooplankton transect, temperature was measured with a digital thermometer and salinity with a refractometer. To measure the chlorophyll a concentration, water samples were collected at the surface with 2 L plastic containers and kept in the dark. Volumes of 25 or 50 mL of water were filtered separately on GF/F (0.7 μm porosity). Filters were kept frozen (−20 °C until further analysis). Chlorophyll a was extracted using acetone (90%) and held in the dark at −20 °C overnight. The acidification method was used to measure chlorophyll a concentration according to Lorenzen (1967).
The Capelière station has long-term monitoring data for temperature, salinity, and chlorophyll a using the same method as previously mentioned. Monthly measures were provided by the SNPN from 2008-2016 for temperature/salinity and from 2008-2015 for chlorophyll a.
To identify which parameters influenced the ctenophores presence/absence, a principal component analysis (PCA) was performed using R 3.5.0, on monthly data for the sampling period 2016-2017. Groups of data were obtained by a hierarchical agglomerative clustering analysis Additionally, in 2019, after a report by a naturalist (T. Mosca pers. comm.), ctenophore identifications were conducted in June 2019 by Guillaume Marchessaux in the Estomac Lagoon (southeast France). Individuals were sampled using a landing net, temperature and salinity were measured as explained above.

Results
The water temperature varied similar to established Mediterranean seasonal pattern with low temperatures in winter (3 °C) and high temperatures in summer (30 °C, Figure 3A, Table S1). During 2017, the minimum of 3 °C was recorded in January and the maximum of 29 °C in August ( Figure 3A, Table 2).
The salinity in the Vaccarès Lagoon strongly varies over the years, depending highly on freshwater inflow (precipitations and especially agricultural pumping during the spring and summer seasons) and water management. Between 2008 and 2016, salinity have varied from 2.8 psu (November 2011) to 28 psu (March 2008; Figure 3B). During 2016-2017, the salinity remained between 12 psu (September 2017) and 25 psu (October 2017) but is generally stable between November 2016 and August 2017 (19 ± 1 psu) ( Figure 3B, Table 2), decreased in September (S = 12 psu) and an increase of 25 psu in October 2017.
Chlorophyll a concentration has remained low, especially during winter (4.5 ± 2.1 μg Chla L -1 ), except during some annual blooms such as in 2012   Thermocyclops sp. (Cyclopoid), 3 species/genus of rotifers (Asplanchna sp., Hexarthra fennica, Notommata sp.) and other reported taxa such as bivalves larvae, gastropods larvae, annelids larvae, eggs and fish larvae. Rotifers were mostly abundant during the winter, while copepods represented between 80.0% and 99.6% of the zooplankton community during the spring, summer and fall seasons.
M. leidyi has been observed between December 2016 and February 2017 (Table S2)  Lagoon, the abundance of copepods decreased significantly primarily with holoplankton (i.e. rotifers) during winter periods. However, no correlation was observed with salinity and chlorophyll a concentration.

Discussion
In this study, we report the introduction of the invasive ctenophore M. leidyi in the Vaccarès Lagoon, which has been confirmed in situ by Guillaume Marchessaux for the first time in 2016. Our data from November 2016 to October 2017 showed that Mnemiopsis presence/absence was very variable in the Vaccarès Lagoon.
At the Capelière station, a long-term fish scientific survey had been initiated in 1993, using traditional passive fishing gear, called "capechade", composed of a pound net and several fyke nets of 6 mm mesh size, that are commonly used by fishermen in lagoons. Through this survey the first observation was reported of gelatinous individuals that may correspond to M. leidyi in 1996 ( Figure 6, Table S2). Unfortunately, no sample is available to confirm this report. However, since the first report, Tour du Valat has reported monthly on the presence or absence of the supposed M. leidyi, based on scientific surveys and fishermen observations. Fishermen and scientists described the gelatinous individuals with several details (presence of lobe, cilia, rainbow colors and body transparency) which tend to confirm that the individuals may have always corresponded to Mnemiopsis leidyi. Furthermore, any other species of gelatinous zooplankton (i.e. cnidarian, etc.) has been observed in the Vaccarès Lagoon.
Since its first possible appearance in 1993, M. leidyi presence in the Vaccarès Lagoon has rather been chaotic, being totally absent during relative long periods (from 1998 to 2000, 2005 and 2010), and sometimes present all year round (in 2015, Figure 6, Table S2). Mnemiopsis has been present all of 2016 ( Figure 6) but absent in 2017 and early 2018. Last observations showed that Mnemiopsis was present between June 2018 and December 2018, and in October and November 2019 ( Figure 6, Table S2).
In our study, given the difficulties of access to the lagoon and high-water turbidity, it was difficult to quantify ctenophores abundances. We suggest to attach plankton net on sticks to sample passively zooplankton and ctenophores and longer sampling periods (1 hour for example) a biggest net (Regent net, 70 cm opening area for example) to quantify ctenophores abundances. Additionally, even if rotifers measured ~ 100 μm, the use of the 80 μm meshed net could be not appropriate to quantify these organisms. Then, we suggest use of 60 μm meshed net in future studies.
As reported in the literature, salinity and temperature are determining factors for the maintenance of ctenophores. The records of M. leidyi at temperatures ranging from 3 to 29 °C and salinity from 5 to 25 psu are consistent with the eurythermal and euryhaline tolerances of this ctenophore. M. leidyi has indeed been shown to survive in salinity from 5 to 40 psu (Kremer 1994;Purcell et al. 2001b). Even though M. leidyi has been shown to survive in extreme salinity and temperature ranges, reproduction rates under low salinity levels (< 10 psu) have been reported. Extremely low salinity and low temperature may explain its limited range expansion in Northern Europe (Jaspers et al. 2011(Jaspers et al. , 2018b. In the Vaccarès Lagoon, ctenophore presence appeared to increase when the salinity is over 20 psu ( Figure 3B).
To compare with other Mediterranean lagoons, chlorophyll a concentrations measured in our monthly study (1.5-7.4 μg L -1 ) were lower than observed in the Berre Lagoon (0.9-24.6 μg L -1 ; Delpy et al. 2016) and in the same range as Bages-Sigean Lagoon (0.3-4.7 μg L -1 ; Delpy et al. 2016). In our study, chlorophyll a concentration did not influence ctenophores presence/absence as observed by Delpy et al. (2016). Mnemiopsis leidyi was observed in the Vaccarès Lagoon for chlorophyll a concentration from 3.9 μg L -1 to 5.12 μg L -1 , whereas it was absent in our sampling for chlorophyll range from 1.5 μg L -1 to 7.1 μg L -1 . In our results, chlorophyll a concentration does not influence Mnemiopsis present/absence. More data will be needed to confirm this hypothesis especially as Mnemiopsis is an omnivorous organism, then phytoplankton could play an important role in its growth (Deason and Smayda 1982).
The presence of this gelatinous zooplankton species may have a strong impact on zooplankton community function as it has been observed in the Black Sea (Shiganova et al. 2019). The presence of this introduced species and its high predation pressure in a national protected area could have a negative impact on the biodiversity (e.g. fish, mussels, etc.) (Shiganova et al. 2019 and references within). In the Berre Lagoon and in the Black Sea meroplankton and rotifers were more predated by ctenophores than copepods (Rapoza et al. 2005;Marchessaux 2019). In the Vaccarès Lagoon it would be the reverse: low abundances of copepods were observed in the lagoon when M. leidyi was present suggesting a potential high ctenophores predation pressure on copepods.
In the Berre Lagoon, the population of M. leidyi was maintained during the 2010 to 2017 despite variable conditions: temperatures ranging from 3 to 28 °C, salinities from 10 to 30 psu, and a quantity of carbon available around 3 mgC L -1 or more (Delpy et al. 2012(Delpy et al. , 2016Marchessaux 2019). In the Vaccarès Lagoon, M. leidyi does not seem to be permanently established in the Camargue. The introduction pattern of M. leidyi by cargo ships result from trans-Atlantic (e.g. East coast of Mexico and USA) and local ship transits within the Mediterranean Sea (Bolte et al. 2013;Ghabooli et al. 2013). As no maritime traffic occurs in the Camargue area (i.e. natural reserve) its presence may probably result from regular reintroductions. Advection processes and natural transport (drift, internal circulation) are certainly a vector of secondary spread of M. leidyi from closed lagoons such as the Berre Lagoon as observed by Jaspers et al. (2018a). Currents and artificial canals between the Berre and other lagoons may be vectors of M. leidyi expansion.
Recently, January 2019 M. leidyi was observed in the Estomac Lagoon (Fos sur Mer, south France) near the Berre Lagoon but not connected to its (Thierry Mosca pers. comm.). In June 2019, ctenophores measured between 7 cm and 10 cm long (Figure 7, Table 3). The lagoon temperature was between 26.2 °C and 27.7 °C and salinity between 29-30 psu. In early June 2019, 30 individuals were observed but, 200 were identified by 26 June 2019.
In conclusion, the present study reports that the alien ctenophore M. leidyi has been present along the French lagoon ecosystems since at least 2005 (in the Berre Lagoon), and probably earlier. Long-term monitoring with population genetic investigations are needed to address source-sink dynamics of this species along the Mediterranean coastlines.