New invasive copepod species from the Levantine Sea, north-eastern Mediterranean: Oithona davisae (Ferrari and Orsi, 1984) (Cyclopoida: Oithonidae)

İskenderun Bay is a hotspot for bioinvasions in the Mediterranean Sea, where alien species are frequently introduced, easily settle, and therefore, can often be encountered. The small-sized invasive copepod Oithona davisae (Ferrari and Orsi, 1984) has rapidly spread in several ecosystems worldwide, including the Black Sea, Wadden Sea, Marmara Sea, Adriatic Sea, and Aegean Sea, becoming an integral part of the pelagic communities there. Here, we report O. davisae in İskenderun Bay, in the north-eastern p art of the Mediterranean Sea, and provide information on its spatiotemporal variation and assess its potential impact on the seasonal abundance and biomass of the mesozooplankton community, at present and in the near future. Zooplankton abundance and biomass were at their lowest levels in winter (1066 ± 313 ind. m -3 , 1.9 ± 0.8 mg m -3 ) and highest in spring (7738 ± 1583 ind. m -3 , 18.5 ± 5.8 mg m -3 ). However, the relative abundance of oithonid copepods was highest during the winter (35.7 ± 10%). The proportion of copepodites in the oithonid assemblage was high in spring (72.5 ± 9.3%), summer (72.7 ± 13.3%) and winter (68.5 ± 3.6%) but the relative abundance of male oithonids remained low across all seasons. Oithona davisae was observed only in the autumn, in abundances of 8.4 to 12.6 ind. m -3 . These low abundances could be attributed to the new arrival of this species and the relatively large mesh size used in this study (200 µm). Nevertheless, the successful introduction and assimilation into other mesozooplan kton communities elsewhere, rapid adaptability and wide tolerance to environmental conditions of O. davisae, suggest that this species may become a dominant part of the zooplankton community across the Levantine Basin in the very near future.


Introduction
Zooplanktonic organisms hold great importance in pelagic ecosystems by maintaining the cycles of matter and energy between lower trophic levels and upper predators in the food web (Lenz 2000).Among zooplankters, the small-bodied oithonid copepods are of particular significance.They link protozoan and metazoan food webs as the prey of fish larvae while predating on small flagellates and ciliates (Nakamura and Turner 1997).Oithona davisae is a small-sized (female: 0.48-0.62µm; male: 0.47-0.54µm, Razouls et al. 2005Razouls et al. -2021) ) neritic oithonid that generally prefers coastal and estuarine habitats.This species displays marked adaptability and can easily settle shortly after being introduced to a novel ecosystem, becoming an integral and dominant part of the recipient community (Altukhov et al. 2014;Doğan and İşinibilir 2016;Zagami et al. 2018;Terbıyık Kurt and Beşiktepe 2019).Oithona davisae can tolerate a wide range of environmental parameters (Zagami et al. 2018), including temperature (−1.8 to 29 °C) and salinity (12 to 35 psu) (Ferrari and Orsi 1984;Uye and Sano 1998;Temnykh and Nishida 2012;Mihneva and Stefanova 2013;Cornils and Wend-Heckman 2015;Doğan and İşinibilir 2016;İşinibilir et al. 2016a;Zagami et al. 2018;Terbıyık Kurt and Beşiktepe 2019).Nonetheless, it seems that this species generally thrives in warmer conditions (Zagami et al. 2018).Oithona davisae was first noticed in the inshore waters of San Francisco Bay (Sacramento-San Joaquin Estuary) in 1963 (Ferrari and Orsi 1984;Cordell et al. 2008), but was not identified as a new species until 1984 (Ferrari and Orsi 1984).This species, endemic to the Eastern Asian coast with a native range limited to the Japanese and Chinese seas (Nishida 1985;Razouls et al. 2005Razouls et al. -2021)), has increased its worldwide distribution utilizing various introduction vectors (e.g.ballast water, mariculture, and currents).Oithona davisae has established viable populations in several ecosystems, such as the Black Sea (Altukhov et al. 2014), Mediterranean Sea (Saiz et al. 2003), Bilbao estuary (Uriarte et al. 2015) and Wadden Sea (Cornils and Wend-Heckmann 2015).Along the coast of Turkey, O. davisae was first observed from the Sinop coast in 2009 (Üstün and Terbıyık Kurt 2016), then in 2014 from the Marmara Sea (Doğan and İşinibilir 2016), and finally, in 2015 from the Aegean Sea (Terbıyık Kurt and Beşiktepe 2019).
Due to its unique hydrological conditions, İskenderun Bay is one of the most invaded regions in the Mediterranean Sea and has experienced a high rate of bioinvasions (Çevik et al. 2006).Human-induced stressors, such as thermal discharge, shipping, coastal development and industrial activities, affect the ecosystem of the bay (Doygun and Alphan 2006).In addition to the plethora of alien Indo-Pacific species of fish, cnidarians, molluscs, and polychaetes, which are frequently observed in the eastern Mediterranean (Çınar 2009;Bilecenoğlu 2010;Özcan et al. 2010;Bakır et al. 2012;Çevik et al. 2006), established populations of alien zooplanktonic species such as Ferrosagitta galerita (Terbıyık et al. 2007), Pleopis schmackeri (Terbıyık Kurt and Polat 2017) and Dioithona oculata (Terbıyık Kurt 2018) have been reported in İskenderun Bay.While the goals of our original project were to characterize the mesozooplankton community in İskenderun Bay, we report here the first record of O. davisae as part of the mesozooplankton community in the Levantine Basin.

Study area
İskenderun Bay, characterized by its rectangular shape, is located in the north-eastern corner of the eastern Mediterranean and was formed by recessing into Anatolia.The bay is ~ 65 km long, 35 km wide and has an area of 2275 km 2 .The average depth of the bay is 70 m, and it is one of the areas with the largest continental shelf (after the Nile Delta) in the eastern Mediterranean Sea (Avşar 1999).The Yumurtalık district and the thermal power plant, Botaş oil filling facilities, BTC pipeline and the Toros fertilizer factory are located on the north-western shores of İskenderun Bay.On the south-eastern shores are the districts of İskenderun and Dörtyol, the iron and steel factory and İskenderun port.There are also summer cottages and camping areas on these shores, so the human population residing around the bay increases exponentially in the summer months.For these reasons, the anthropogenic impacts in İskenderun Bay are assumed to be high.However, eutrophication has not been observed due to the dynamic nature of water flow into and out of the bay (Yılmaz et al. 1992).

Sampling and laboratory studies
Sampling was carried out seasonally in the second week of April, July, October, and December 2018 at five stations in İskenderun Bay during the daytime (Figure 1).The stations were located close to the shore and the deepest depth was 15 m.Samples were collected vertically with a WP-2 zooplankton net (200 µm mesh) and fixed with a 4% formaldehyde seawater solution.In the laboratory, sub-samples were taken from the zooplankton samples with a Folsom splitter.Counting and identification were performed with an Olympus SZX16 stereomicroscope and Leica microscope.
Copepod abundance was calculated as number of individuals per species per cubic meter.The filtered water volume was determined based on the vertical cylinder volume, calculated using the diameter of the mouth opening of the net and the hauling depth.Temnykh and Nishida (2012) identification keys were used to identify O. davisae.
Environmental variables (e.g.temperature, salinity) were measured with a YSI 6600 at the surface, and 5 m, 10 m and 15 m depth.Seawater samples were taken by Niskin bottles at corresponding depths to determine chlorophyll-a concentrations, phytoplankton and protozooplankton abundances.For chlorophyll-a analysis, two litres of seawater were filtered through Whatman GF/F glass fibre filters, extracted with 90% acetone solution, and measured using the spectrophotometric method according to Strickland and Parsons (1972).To count the protozooplankton, 10 litres of seawater were collected from the surface then filtered through 18-µm mesh, fixed with Lugol solution (2.5%), and then 2% formaldehyde fixation several hours after.For phytoplankton identification and counting, two-liters seawater samples were fixed with a Lugol solution (2.5%).In the laboratory, the samples were placed in a settling chamber for 48 hours.The cells were counted by the Utermohl method using a CK 40 Olympus inverted microscope for each plankton group (Utermöhl 1958;Venrick 1978).

Data analysis
A two-way ANOVA tested the significance of season and site and their interaction on zooplankton abundance and biomass data, which were normally distributed.Posthoc Tukey's HSD pairwise tests tested for significant differences in abundance and biomass between seasons.R Statistics version 4.0.2 was used for the statistical analysis.Ferrari and Orsi, 1984 The total length of O. davisae (n = 5) ranged from 500 to 550 µm (Figure 2A).Oithona davisae had a curved, sharply pointed rostrum, and dorsal hair rows were absent on urosome (Figure 2B).The formula of the exopod spine on the swimming legs was 1, 1, 3 in P1-P3 (Figure 2C, D, E) and 1, 1, 2 (Figure 2F) in P4.A long distal spine was present in the first inner lobe of the maxillule, and individuals had one seta on the endopod (Figure 2G).There were four setae on the endopod of mandible (Figure 2H).

Seasonal distribution
Total zooplankton abundance and biomass were significantly different across seasons (two-way ANOVA, p < 0.01), but neither site nor the interaction of site and season affected total zooplankton abundance and biomass (p > 0.1 and p > 0.05, respectively).The abundance and biomass in spring were significantly higher than all other seasons (Tukey's HSD, p < 0.05).The lowest zooplankton abundance and biomass values were observed in winter (1066 ± 313 ind.m -3 , 1.9 ± 0.8 mg m -3 , respectively), and the highest values were observed in spring (7738 ± 1583 ind.m -3 , 18.5 ± 5.8 mg m -3 ) (Figure 5).The relative ratio of copepods in the mesozooplankton  was low in spring and summer (32 ± 2.5%, 20 ± 4.4%) and highest in the winter (61.7 ± 1.8%) (Figure 6).Similarly, the relative abundance of oithonids (the group to which O. davisae belongs) in the copepod community was low in spring (3.6% to 6.4%).The contribution ratio of oithonids to the Copepoda community was the highest in the winter and ranged from 23.3% to 52.9% in this season, depending on the site (Figure 6).The proportion of copepodites among oithonids was high, reaching 87% (Z4) in July.In autumn, the proportion of oithonid copepodites remained comparatively low and fluctuated between 30.8% and 49.5%.The proportion of oithonid males was very low and did not exceed 4.7% on average (Figure 7).Our first observations of O. davisae in the study area were in autumn (October).The abundance of this species was relatively low as compared to other Oithona species and varied from 8.4 to 12.6 ind.m -3 , with the highest densities observed at the station closest to the coast.Oithona nana, which is a similar species of a similar size, was observed in all seasons.Its average abundance was highest in spring (20 ind.m -3 ), and ~ 6 ind.m -3 were found in the other seasons.

Discussion
This study presents the first observation of O. davisae in the Levantine coastal waters of Turkey.Previously, the species had been commonly and abundantly observed in coastal areas of Turkey in the Black Sea (Üstün and Terbıyık Kurt 2016), Marmara Sea (Doğan and İşinibilir 2016) and Aegean Sea (Terbıyık Kurt and Beşiktepe 2019).In the Mediterranean Sea, the spread of the species has been restricted and only reported from Barcelona harbour (Saiz et al. 2003), Lake Faro (Zagami et al. 2018), Lake Ganziri (Zagami et al. 2018) and the port of Venice (Vidjak et al. 2019).Therefore, it appears that O. davisae has expanded its distribution eastwards towards the Turkish seas and the Levantine Basin in the Eastern Mediterranean Sea.
The Mediterranean Sea is a hotspot for marine bioinvasions.The opening of the Suez Canal in 1869 connected two different biogeographic provinces, becoming a major pathway in the passage of alien species from the Red Sea to the Mediterranean Sea in a process termed the Lessepsian migration (Por 1978).In addition to passage via the Canal, species are introduced into the Mediterranean Sea by other pathways, including shipping, aquaculture and aquarium trade (Ruiz et al. 1997;Galil and Zenetos 2002;Zenetos et al. 2010).Of these, shipping has been assumed by many researchers to be the most important vector transporting O. davisae to novel environments (Uriarte et al. 2015;Altukhov et al. 2014;Cornils and Wendhackman 2016;Üstün and Terbıyık Kurt 2016;Terbıyık Kurt and Beşiktepe 2019).A local zooplankton distribution monitoring program, conducted since 2004 in the study area (Terbıyık Kurt and Polat 2013, 2014, 2015, 2017;Terbıyık Kurt 2018, 2019), has not observed O. davisae nor has the species been reported along the coasts of other countries in the Levantine Sea prior to 2019.It is likely that O. davisae has been transported to İskenderun Bay through ballast waters.Indeed, İskenderun Bay plays an important role in maritime transport, and it is the second-most important marine transportation and trading centre that serves the Middle East in the Mediterranean region of Turkey (Doygun and Alphan 2006).
Oithonids are an important contributor to zooplankton communities in İskenderun Bay (Terbıyık Kurt and Polat 2013).According to published records, six species belonging to the family Oithonidae (Oithona plumifera, O. nana, O. similis, O. tenuis, O. setigera and D. oculata) are found in İskenderun Bay (Toklu and Sarıhan 2003;Terbıyık Kurt and Polat 2013;Toklu-Alıçlı and Sarıhan 2016;Terbıyık Kurt 2018).Previous sampling in 2012 at our study sites found that O. plumifera was the dominant species during the summer and autumn (Terbıyık Kurt and Polat 2013).However, the abundance of the small oithonid O. nana, which is similar in size to O. davisae, was relatively low and peaked in the spring.The relative abundance of male oithonids was low.However, previous studies also found higher abundances of oithonid copepodites in autumn and winter in İskenderun Bay (Terbıyık Kurt and Polat 2013), as was also observed in the present study.
The abundance of O. davisae seems low at present in İskenderun Bay, similar to the reported abundances from the Venice Lagoon (Adriatic Sea) (Vidjak et al. 2019).In addition, the net mesh size (200 µm), which is regularly used for sampling, is too large to efficiently collect this smallsized species.Therefore, these abundances are likely an underestimation.Alternatively, low population numbers could be indicative of a newly arrived population of O. davisae to this region.Although the reported abundances were low, O. davisae has a high tolerance to variable temperatures (Svetlichny et al. 2021), and the conditions in İskenderun Bay are ideal for it to settle and thrive.In Sevastopol Bay (Black Sea), the highest abundance of O. davisae was observed from September to November, when the water temperature was 15-23 °C, and abundances dropped sharply at temperatures below 8 °C (Svetlichny et al. 2016).Only adult females were observed between 8 and 17.5 °C (from the end of February to the middle of May), and male individuals were observed when the temperature reached 22 °C (at the end of May).At 17.5 °C, copepodites started to appear in the community (Svetlichny et al. 2016).In Fukuyama harbour (inland Sea of Japan), the highest abundance of O. davisae was observed during the summer, when the water temperature was relatively high (20-28 °C in June, July and September) (Uye and Sano 1995).In the coastal waters of İskenderun Bay, the water temperature shows a clear seasonal cycle that fluctuates between 15.6 and 29.6 °C (Terbıyık Kurt and Polat 2013, 2014, 2015, 2017;Terbıyık Kurt 2018), but in the present study, the temperature did not fall below 18 °C.Further work directly monitoring O. davisae in İskenderun Bay is needed to understand the seasonal cycling of this species in the non-native waters and to uncover if it has already based an established population.
In addition to having the correct thermal regime, the regions in which high abundances of O. davisae have been reported are generally productive areas (İşinibilir et al. 2016b;Üstün and Terbıyık Kurt 2016;Zagami et al. 2018;Terbıyık Kurt and Beşiktepe 2019).In Lakes Faro and Ganziri, the highest abundance of O. davisae was observed during late spring and early summer, which is when the density of small-sized flagellates is the highest (Zagami et al. 2018).Although Iskenderun Bay has 2−4 times higher chlorophyll-a concentrations compared to surrounding regions, it is generally considered oligotrophic (Yılmaz et al. 1992).In the study sites, chlorophyll-a concentrations and the abundance of dinoflagellates and protozooplankton (mainly ciliates), which could serve as food to O. davisae, were at low levels in autumn (which is the only season in which we found O. davisae).These low satiating food concentrations are sufficient for feeding and egg production in the adult females of O. davisae (Almeda et al. 2010;Zamora-Terol and Saiz 2013), suggesting an adaptation to oligotrophic environments in which O. davisae can survive, reproduce and outcompete native species.This may explain the ecological success of this species where food is a limiting factor for other copepods (Almeda et al. 2010), enabling its invasiveness as it settles easily in novel environments and becomes dominant in a short period of time (Zagami et al. 2018;Altukhov et al. 2014;Doğan and İşinibilir 2016;Terbıyık Kurt and Beşiktepe 2019).
Although no harmful effects have been documented in ecosystems where O. davisae has settled, its presence in the community could signal an ecosystem change (Ivanova et al. 2017).Therefore, to determine the status and impact of O. davisae in the northeast Levantine Basin, this species should be monitored using suitable sampling nets with a smaller mesh size (< 200 µm).This will provide reliable estimations of its population structure and standing stocks in the productive areas of İskenderun Bay, such as the coast of Toros fertilizer factory, the mouth of the Ceyhan River and the lagoon systems.

Figure 1 .
Figure 1.The sampling stations in the study area of İskenderun Bay, along the coast of Turkey.

Figure 3 .
Figure 3. Seasonal changes of temperature and salinity at sampling stations in İskenderun Bay during 2018.

Figure 4 .
Figure 4.The abundance and distribution of some biotic elements (Total phytoplankton, dinoflagellate, Protozooplankton and chlorophyll-a) at sampling stations of İskenderun Bay in autumn 2018.

Figure 6 .
Figure 6.Proportional distribution of oithonids and Copepoda in zooplankton at sampling stations in İskenderun Bay across the four seasons of 2018.

Figure 7 .
Figure 7. Seasonal distribution of Oithona davisae and O. nana abundance, and ratio of oithonid females, males and copepodites at sampling stations in İskenderun Bay in 2018.