Layout 1 INTRODUCTION Protists are important components of plankton and in- clude many categories of obligate and facultative het- erotrophs, which are known, respectively, as protozoo-plankton (from protozoan plankton, including, e.g., oligotrich and choreotrich ciliates) and mixoplankton (from mixotrophic plankton, including, e.g., dinoflagellates and some oligotrich ciliates) (Flynn et al., 2019). Mixotrophs, being capable of both phagocytosis and photosynthesis within a single cell (Stoecker et al., 2009; Flynn et al., 2013, 2019), encompass different functional types (Mitra et al., 2016), namely: constitutive mixotrophs, bearing innate plastids (e.g., dinoflagellates; Stoecker et al., 2017); non-constitutive mixotrophs, acquiring plastids from photosynthetic preys (i.e., a strategy known as kleptoplasty, which is present in oligotrich ciliates, predating mainly cryptophytic microalgae; Johnson, 2011; Hughes et al., 2021); moreover, some non-constitutive mixotrophs can transmit plastids to the progeny (e.g., Mesodinium rubrum; Johnson et al., 2007; Smith and Hansen, 2007), while some other must acquire new plastids after any cell replication (i.e., most oligotrich ciliates). Such a behavioural diversity along with a high trophic plasticity allow mixoplankton to cope with intermittent resources, by easily shifting between autotrophy and het- erotrophy (e.g., Haraguchi et al., 2018). This ecological property is of much higher relevance in the circulation of matter and energy throughout the plankton food web, in which protists play as connecting nodes between primary producers and metazoan consumers (D’Alelio et al., 2016a; Flynn et al., 2019; Leles et al., 2021). In this brief communication we report on the diver- sity of heterotrophic and mixotrophic protists occurring in the Gulf of Naples (Tyrrhenian Sea, Italy) during late summer/early autumn; this period sets in the middle be- tween the spring/summer and mid-autumn phytoplank- ton blooms and it is characterized by similar amounts of hetero- and mixotrophic protists, based on information collected at the Long-Term Ecological Research station MareChiara (LTER-MC; Ribera d’Alcalà et al., 2004; Zingone et al., 2019). As the planktonic system gener- ally shifts from autotrophy to heterotrophy during the late summer/early autumn phase, the relevance of mixotrophy is expected as highest in the period under investigation. By analysing water samples collected from two oceano- graphic stations in the Gulf of Naples, we provide abun- dance data for planktonic protists (mainly focusing on dinoflagellates, oligotrich and choreotrich ciliates), identify their trophic modes (either mixo- or heterotrophic) based on information available in the literature, report on compo- sitional differences at spatial scale and discuss these latter by considering the standing environmental and trophic con- ditions. Though preliminary, our survey provides useful biodiversity data about mixo- and protozooplankton at local scale, as taxonomical information about these organisms have not been updated in the Gulf of Naples in the last two decades (Modigh, 2001; Modigh and Castaldo, 2002; Rib- era d’Alcalà et al., 2004). ARTICLE An autumn biodiversity survey on heterotrophic and mixotrophic protists along a coast-to-offshore transect in the Gulf of Naples (Italy) Gabriele Del Gaizo1,*, Luca Russo1,2, Maria Abagnale3, Angela Buondonno3, Marta Furia3, Simona Saviano3, Mauro Vargiu3, Fabio Conversano3, Francesca Margiotta3, Maria Saggiomo3, Isabella Percopo4, Domenico D’Alelio1,* 1Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, Italy; 2PhD Program in Evolutionary Biology and Ecology, Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy; 3Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn, Naples, Italy; 4Marine Organism Taxonomy Core Facility – MOTax, Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn, Naples, Italy ABSTRACT Plankton communities include both unicellular and multicellular organisms. An important unicellular component is represented by those protists (i.e., unicellular eukaryotes) that consume bacteria, other protists, and even small animals. These organisms are an important link between primary producers and metazoans, and they can also be ‘mixotrophic’, coupling phagotrophic and pho- toautotrophic behaviours. In this note, we report on the diversity of heterotrophic protists, or protozoo-plankton (from ‘protozoan plankton’), and mixotrophic protists, or mixo-plankton, which were sampled at two sites (coastal and offshore stations), at two depths (0 and 10 m), in the Gulf of Naples during the early autumn of 2020. Our survey identified dinoflagellates and oligotrich ciliates as the most abundant groups, while tintinnids (choreotrich ciliates) were less quantitatively relevant. The taxonomic com- position of samples investigated herein remarked that reported by previous studies, except for the tintinnid Ascampbeliella armilla, which was never reported in the area of study. A coast-to-offshore differentiation in the taxonomical composition of heterotrophic and mixotrophic protists was also observed, with some species more abundant within coastal waters and other better thriving in offshore ones. These differences were associated with distinct environmental and trophic conditions. Non -co mmerc ial us e o nly An autumn biodiversity survey on protists in the Gulf of Naples 75 MATERIALS AND METHODS The Gulf of Naples (Southern Italy) is a coastal em- bayment open to the Tyrrhenian Sea (Figure 1a). Water sampling was carried out at two hydrographic stations named FE_11 (coordinates: 40°47.285, 14°20.883; dis- tance from the coast: 0.3 NM; maximum depth: 26 m) and FE_12 (coordinates: 40°46.706, 14°19.858; distance from the coast: 1.3 NM; maximum depth: 100 m). These sta- tions were integrated in a basin-scale oceanographic Figure 1. Diversity of protistan grazers in the Gulf of Naples. (a) map of the area and localization of sampling sites. (b) relative abundance of the main taxonomical groups at the two samplings sites and depths. (c) heatmap showing the community composition in different samples. Heatmap’s colour scale is indicated on the right. Unit variance scaling is applied, i.e., a difference of +1 or -1 means that the local abundance value for a taxon is one standard deviation away (+ or -) from the average abundance of the taxon considering the whole row. Non -co mmerc ial us e o nly G. Del Gaizo et al.76 cruise run on 17 September 2020 and focusing on the characterization of the costal system in the eastern sector of the Gulf of Naples (project FEAMP-ISSPA, funded by the government of Regione Campania, Italy). As these sta- tions were located at different distances from the coast, for more simplicity, we herein refer to FE_11 and FE_12 as coastal station and offshore station (CS and OS), re- spectively. Both CS and OS were ~5 NM away from LTER-MC (Ribera d’Alcalà et al., 2004; Zingone et al., 2019), which was not part of the cruise mentioned above. Water column characterization and sampling.At both CS and OS stations, the structure of the water column was described by the acquisition of CTD data, which were ob- tained with a SeaBird 911 Plus multi-parametric probe with sensors for temperature, conductivity, and other aux- iliary proxies, such as dissolved oxygen sensor, fluores- cence, water turbidity, and photosynthetically active radiation (PAR). The multi-parametric probe was mounted on a Rosette bearing 12 Niskin bottles with a volume of 10 L. Water samples analysed in the present study were collected from Niskin bottles closed at two depths, 0 and 10 m, in both CS and OS. These depths cor- responded to the top and the bottom of the seasonal sur- face mixed layer, i.e., the water column layer in which a higher concentration of unicellular organisms is expected based on previous research in the study area (e.g., D’Ale- lio et al., 2015). Chemical analyses. Samples for dissolved inorganic nu- trients were collected directly from the Niskin bottles and immediately stored in 20 ml HDPE vials at -20°C. Chem- ical analyses were carried out with a continuous flow auto- analyzer (Flow-Sys System), based on an updated version of the protocol developed by Hansen and Grasshoff (1983). For the determination of particulate organic carbon (POC), variable volumes (0,75-1 L) of seawater were filtered on Whatman GF/F pre-combusted (450 °C, 5 h) glass fibre fil- ters immediately stored at -20°C after collection. To remove the inorganic carbonate fraction, filters were exposed overnight to HCl vapours, and then analysed with a Thermo Electron CHN elemental analyser (FlashEA 1112 Series) (Hedges and Stern, 1984). Cyclohexanone-2,4- dinitro[1]phenyl hydrazone was used as a standard. For HPLC pigment analyses, 2-3 L of seawater were filtered (GF/F Whatman - 47 mm diameter) and stored in liquid ni- trogen for later analyses, performed with a Hewlett 166 Packard HPLC (1100 Series) according to Vidussi et al. (1996). Instrument calibration was carried out with external standard pigments provided by the International Agency for 14C determination-VKI Water Quality Institute. Along with chlorophyll a (Chl a), some pigments associated to cryptophyte plastids (ß-carotene and alloxanthin) and in- volved in the kleptoplasty activity by ciliates (Johnson, 2011; Hughes et al., 2021) were analysed. Biological analyses. Water samples (250 mL) for the identification and enumeration of planktonic protists were fixed with Lugol solution (final concentration 1%), stored in the dark at room temperature, and processed according to Utermöhl’s method (Utermöhl, 1958), which is de- scribed as follows: i) samples were settled in a graduated cylinder for 48 hours; ii) samples were concentrated to the volume of 100 mL by gently collecting the supernatant through free fall, using a 1 mL pipette connected to a latex tube; iii) after gentle agitation of the cylinder, the concen- trated samples were poured into dark bottles and then gen- tly shaken for 150 times; iv) finally, samples (100 mL) were settled in sedimentation chambers for 48 hours. The count was performed over the entire area of the chamber using an inverted optical microscope (Zeiss Axiovert 200) at 20x magnification following standard methods (Uter- möhl 1958). Subsamples were considered representative if the count reached 100 individuals. Optical microscopy photographs were taken with ZEISS Axiocam 305 Color. Cell size (only used to categorize broad groups of taxa, see Figure 1c, Supplementary Table 1, and to insert scale Table 1. Environmental variables of coastal station (CS) and offshore station (OS), at two depths (0 and 10 m). Environmental variables Unit Coastal station (CS) Offshore station (OS) 0 m 10 m 0 m 10 m Temperature °C 25.16 24.47 25.37 24.91 Salinity PSU 37.19 37.87 37.58 38.11 Turbidity NTU 0.37 0.22 0.33 0.22 Fluorescence RFU 2.53 1.23 2.04 0.77 Dissolved Oxygen ml l–1 4.69 4.5 4.95 4.54 PAR μE m−2 s−1 84.70 12.31 103.69 31.57 Chl a mg m–3 1.28 0.38 0.55 0.22 POC mg m–3 223.5 119.21 262.33 99.98 DIN mmol m–3 4.86 2.22 1.16 0.27 PO4 mmol m–3 0.14 0.07 0.08 0.04 Βcarotene mg m–3 0.04 0.004 0.01 0.006 Alloxanthin mg m–3 0.03 0.01 0.013 0.004 Non -co mmerc ial us e o nly An autumn biodiversity survey on protists in the Gulf of Naples 77 bars into microphotographs) was measured with an eye- piece micrometer. The trophic mode of planktonic protists was identified based on Mitra et al. (2014) and Schneider et al. (2020) plus many other studies indicated in Supple- mentary Table 1. Biodiversity analysis. Multivariate analysis on plank- tonic protists data matrices was performed using the open- source web platform ClustVis (Metsalu and Vilo, 2015). A heatmap was produced to represent differences between the taxonomic composition of planktonic protist commu- nity between samples. Both rows and columns in the heatmap were ordered using Euclidean distance and single linkage as a clustering method. RESULTS During our sampling, the water column in the Gulf of Naples was moderately stratified, with i) a very shallow freshwater layer driven by land runoff and gliding on sea- water at surface, ii) a marked mixed layer exceeding the first 10 m of the water column, and iii) a pycnocline start- ing at a depth of about 15 m at any sampling station (Sup- plementary Figure 1). Overall, the water column at CS and OS showed different physical-chemical properties, indicating distinct abiotic and biotic features (Table 1). Specifically, while salinity was higher in offshore and deeper samples, the reverse was true for inorganic nutri- ents, Chl a and other accessory pigments, indicating a re- source richer environment towards the coastline and in surface waters. However, the total abundance of plank- tonic protists with either hetero- or mixotrophic behav- iour; complete taxonomical data are shown in Supplementary Table 1 was in the same order of magni- tude in all samples, with more similar values between samples taken from the same depth (Figure 1b). Dinoflagellates were numerically dominant in all sam- ples (relative abundance: 43-58%) except for the one taken from the OS at -10 m, where they were outnum- bered by oligotrich ciliates (51.2%). Tintinnids (choreotrich ciliates) reached the highest fraction (4.4%) in the OS at 0 m. Other taxa (e.g., Acantharia) showed minimum and maximum abundances in surface and deeper waters of the OS (0.2 vs. 2.3%), respectively. As for dinoflagellates, the genera Protoperidinium, Proro- centrum, Gyrodinium, Dinophysis and Scrippsiella were dominant. Oligotrichs were mainly represented by the genera Strombidium, Strobilidium, and Tontonia, and by the species Mesodinium rubrum. Specimens detected for taxonomical groups mentioned above are shown in Fig- ures 2-3, and their trophic habit is synthesized in Figure 1c and fully referenced in Supplementary Table 1. According to multivariate analysis performed on taxa abundance data (Figure 1c), a coast-to-offshore differen- tiation of the protist community investigated was relevant at surface, while deeper samples were very similar to each other and distinct from surface ones. Coastal samples in- cluded mostly mixotrophic taxa, while offshore samples encompassed a higher fraction of obligate heterotrophs. Overall, the ratio between potentially photosynthetic and fully heterotrophic protists in the coastal station accounted for 5.0 and 2.3, at 0 and -10 m, respectively; in the off- shore station, this ratio accounted for 2.4 and 2.8, at 0 and -10 m, respectively. Data indicated a substantial reshuffling of the plank- tonic protist community between different environmen- tal/trophic conditions. Indeed, the Chl a/POC ratio near the coast was two folds higher than offshore (Table 1), in- dicating that the microbial compartment was richer in au- totrophic cells in the first than in the second. A similar pattern was found for photosynthetic pigments associated with kleptoplasty (i.e., ß-carotene and alloxanthin from cryptophytes) and therefore mixotrophic ciliates. DISCUSSION In our preliminary survey, based on one sampling, in one date, during the late summer/early autumn season in the Gulf of Naples, the mixo-/protozoo-plankton commu- nity was quantitatively dominated by dinoflagellates and oligotrich ciliates, most of which included mixotrophic genera and species. Tintinnids, which are obligate het- erotrophs, were far less abundant, while other minor taxa, such as Radiolaria, were extremely less represented. With the sole exception of the tintinnid Ascampbeliella armilla, all taxa we detected have been already reported in the Gulf of Naples during the period of interest (Modigh, 2001; Modigh and Castaldo, 2002; Ribera d’Alcalà et al., 2004; Piredda et al., 2017). Though never reported before in the Gulf of Naples, A. armilla was already found in subtropical/temperate waters, e.g., in the Mediterranean Sea, since 1951 (Duran, 1951) and more recently from the Bay of Villefranche (France) (Dolan, 2017) and from the Bay of Mersin (Turkey) (Polat et al., 2019). Most taxa detected in our study were reported as mixotrophic, and these latter were often associated with mature aquatic ecosystems (Mitra et al., 2014; Schneider et al., 2020), in which these organisms contribute to a big part of organic nitrogen flows (Gutiérrez-Rodríguez et al., 2014). The potential access of mixotrophs to more re- sources than those that are available to strictly pho- totrophic or heterotrophic protists is crucial during non-bloom phases, when the aquatic system shifts from net autotrophy to net heterotrophy (Haraguchi et al., 2018). Such conditions are typical of late summer/early autumn in the Gulf of Naples, with September being a ‘non-bloom month’ of transitions between i) the late spring/summer peaks of phytoplankton occurring in a condition of fully stratified water, and ii) the mid-autumn Non -co mmerc ial us e o nly G. Del Gaizo et al.78 and more ephemeral phytoplankton peak occurring in the surface layer of a partially mixed water column during the so-called Saint Martin’s summer (Zingone et al., 1995; Ribera d’Alcalà et al., 2004; Zingone et al., 2019). We are sure to have sampled planktonic protists in the above-mentioned non-bloom phase, based on the physi- cal-chemical data we collected as well. Firstly, the sea- sonal thermo-/pycnocline (i.e., the water layer showing the largest variation of temperature and density) started around -15 m, i.e., the same pattern as that reported at LTER-MC in the Gulf of Naples over the last four decades (Ribera d’Alcalà et al., 2004; Kokoszka et al., 2021). Sec- ondly, Chl a values (i.e., 0.22-1.28 mg m-3) in all samples were significantly below the minimum associated to phy- toplankton blooms (i.e., > ~1.7 mg m-3) in the area (Mar- giotta et al., 2013; D’Alelio et al., 2015). Based on the consideration above, the overall dominance of mixotrophs in the surface layer of the Gulf of Naples during the late summer/early autumn appears as motivated by the domi- nance of non-bloom conditions. Non-bloom conditions may favour mixotrophic di- noflagellates and ciliates, for which phototrophy and phagotrophy contribute in somewhat equal extent to the biomass production (Jeong et al., 2010; plus estimations made by D’Alelio et al., 2016b). Most mixotrophic dinofla- gellates, despite bearing innate plastids, can ingest a variety Figure 2. Dinoflagellates from the Gulf of Naples. (a) Prorocentrum gracile; (b) Prorocentrum dactylus; (c) Tripos furca; (d) Pro- toperidinium sp.; (e) Dinophysis sacculus; (f) Phalacroma oxytoxoides; (g) Karenia sp.; (h) Pselodinium vaubanii; (i) Gyrodinium sp. Scale bars: 50 µm in (c); 20 µm in (a, e, h, i); 10 µm in (b, d, f, g). Non -co mmerc ial us e o nly An autumn biodiversity survey on protists in the Gulf of Naples 79 Figure 3. Oligotrich ciliates from the Gulf of Naples. (j-m) Strombidium spp.; (n-o) Strobilidium spp.; (p-r) Leegardiella sol; (s) Pelagostrobilidium neptuni.; (t-u) Mesodinium rubrum; (v-w) Tontonia spp.; (x-y) Laboea strobila. Scale bars: 50 µm in (q); 20 µm in (j, m, r, s, v, x, y); 10 µm in (k, l, n, o, p, t, u, w). Non -co mmerc ial us e o nly G. Del Gaizo et al.80 of nanoplankton preys as well, such as prymnesiophytes, small dinoflagellates, cryptophytes and raphidophytes (Jeong et al., 2005). Concerning mixotrophic ciliates, since both photosynthesis and phagotrophy sustain their growth, they will get a benefit over completely heterotrophic ciliates in the photic zone of areas with relatively low microbial preys but sufficient amounts of inorganic nutrients (Maselli et al., 2020). Mixotrophic ciliates sequester chloroplasts from ingested phytoflagellates as they are non-constitutive mixotrophs (Dolan and Pérez, 2000; Esteban et al., 2010). Most of them are unable to induce plastid replication and must acquire new plastids after cellular division (Stoecker et al., 2009). Pico- and nano-eukaryotes (cell size = 2-12 μm), more abundant in absence of blooms of larger phyto- plankton, can be easily captured, and their photosynthetic organelles retained, by mixotrophic ciliates (Haraguchi et al., 2018; Maselli et al., 2020). In our study and based on compositional features of the community investigated, we also detected a coast-to- offshore differentiation in mixo-/protozooplankton groups (Figure 1c). In the coastal and nutrient richer station, we found a prevalence of mixotrophic dinoflagellates like those in the genera Prorocentrum (Jeong et al., 2005), Dinophysis (Caroppo, 2001), Pseliodinium (Figure 2h) (Gomez, 2018), and Torodinium (Gómez et al., 2016), and of the species Tripos furca (Figure 2c) – this latter being a photoautotroph that can also feed on ciliates if con- strained by limited nutrients conditions (Smalley et al., 2003; Hansen, 2011). On the contrary, the less nutrient- rich offshore waters showed higher abundances of strictly heterotrophic organisms, like (i) phagotrophic dinoflagel- lates, e.g., Phalacroma oxytoxoides and Protoperidinium sp. (Figure 2f and 2d; trophic modes identified by, respec- tively, Park et al., 2011 and Jeong et al., 2010), and (ii) tintinnids, e.g., Eutintinnus tubulosus and Salpingella spp. (for trophic modes, see Dolan, 2010; Dolan et al., 2019). The pattern described above can be explained by the presence, along the transect investigated, of different trophic environments (see e.g., D’Alelio et al., 2016a); this hypothesis is further supported by the spatial distribution of M. rubrum (Figure 3 t-u), which shows higher abun- dance at the coastal and nutrient richer site. This species depends heavily upon phototrophy, as up to 90% of its bio- mass production relies on photosynthesis (Stoecker et al., 2009; plus estimation made by D’Alelio et al., 2016b). Being a photoautotroph, it prefers coastal waters, charac- terized by higher nutrient concentration and lower salinity (Johnson et al., 2013). As a non-constitutive mixotroph, M. rubrummust acquire plastids from cryptophytes (Smith and Hansen, 2007), whose characteristic pigments in our study peaked at coast as well (Table 1). M. rubrum cannot grow indefinitely in the light without its preferred cryptophyte prey and it cannot grow in the dark even if supplied with copious food (Johnson et al., 2007): even though we col- lected a limited number of samples, it is remarkable that we found the highest abundance of M. rubrum at the top of the mixed layer (Figure 1b), where light is not limiting, while we found its lowest abundance at depth in the off- shore station, where both light and pigments of plastid-pro- viding cryptophytes were at the lowest. In conclusion, our study reports further evidence of the multifaceted nature of planktonic protists in temper- ate coastal areas such as the Gulf of Naples. Adding to a high taxonomical diversity, we detected signs of com- munity structuring shaped by the different abundance of distinct functional types, in relation with environmental and trophic conditions. Our study calls for further inves- tigation of the ecology of mixo-/protozoo-plankton at basin and regional scales, due to the relevance of facul- tative and obligate heterotrophic protists, including mixotrophs, in the circulation of matter and energy in the planktonic food web of coastal systems characterized by intermittent resources. Corresponding author: gabriele.delgaizo@szn.it; domenico.dalelio@szn.it Keywords: Plankton; protozoa; dinoflagellates; ciliates; tintinnids; mixotrophy. Authors’ contributions: DDA and IP supervised research. FM and FC planned oceanographic sampling. GDG counted plankton sam- ples, with the help of IP. LR collected information pertaining to trophic modes. MA, AB, MF, MV, FM, MS analysed chemical samples. SS and FC provided physical data. DDA and GDG wrote the paper, with the help of LR and IP. All the authors have read and approved the final version of the manuscript and agreed to be ac- countable for all aspects of the work. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials: All data generated or analyzed during this study are included in this published article. Acknowledgments: This study was supported by the project PO FEAMP 2014/2020 (Misura 2.51) funded by Regione Campania, Italy. The authors thank the Stazione Zoologica Unit for the Access to Marine Ecosystems and Environmental Analysis for organizing and carrying out water sampling. Two anonymous reviewers are gratefully acknowledged for their precious comments that helped to improve the quality of this paper. Received: 10 August 2021. Accepted: 22 November 2021. 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