Trophic-symbiotic interactions among bacteria, meiobenthos, and macrobenthos in the Antarctic environment

Although the ecology of macrofauna, meiofauna and bacteria has been extensively studied, there is still little information on the three benthic groups in the Ross Sea, Antarctica, and the trophic-symbiotic interactions among them. Therefore, the project aimed to investigate the interactions between bacteria and benthic fauna (meio- and macrofauna). The project was structured over a 2-year time frame, in order to obtain significant results regarding the population dynamics of the benthic groups under study (which is known to be significantly slower than that of planktonic communities), and to allow verification of the mechanisms of cellular communication and production of microbial-derived biomolecules.

The objective of the project was to study the ecology of the macrofauna, meiofauna and bacteria present in Antarctica with special reference to the area included in the Ross Sea. Therefore, the aim of the project was the investigation of the bacterial community and benthic fauna (meio- and macrofauna) present in water and sediment samples collected during the XXXIII and XXXIV campaigns in Antarctica. In fact, the study was structured over a period of two years in view of the fact that the dynamics of marine benthos is markedly slower than that of planktonic communities. As for the benthic fauna, the objectives included estimation of abundance, biomass, and spatial distribution along transects; these investigations were considered appropriate to be coupled with assessment of environmental factors, such as Eh and organic matter in the sediment and salinity, temperature, and pH in the water column. With regard to the microbial community, the aim of the project included the study of the biodiversity of the Antarctic microbial community, which is of considerable interest both from the evolutionary aspect and in relation to potential biotechnological applications. Main objectives in this regard included the taxonomic identification of existing species and possible new extremophilic microorganisms using classical techniques for taxonomic assignment; as well as the evaluation of the production of both low molecular weight molecules such as enzymes and/or molecules potentially involved in "quorum sensing" (QS) processes.

The studies conducted under this project involved the analysis of the bacterial community and benthic fauna (meio- and macrofauna) present in the area included in the Ross Sea. The sampling activities necessary to collect samples for study were carried out as part of the XXXIII and XXXIV Italian Expeditions to Antarctica.

Regarding the XXXIII campaign, sampling activities started on December 10, 2017 and were extended until February 3, 2018: during this period, 3 sampling modes were used according to the pack agility and its presence in the areas in front of the "Mario Zucchelli" Base (MZS) (Figure 1). In addition to shoreline sediment sampling for benthic community analysis, water and sediment samples were also taken for subsequent chemical and physical analysis in order to characterize the environment in which the communities were located at the time of sampling. Sampling from Pack was done using the holes opened for previous projects, resorting to the aid of the 5-liter Van Veen bucket and the core of divers. The points sampled included: Mareograph and Amorphus Glaciar, both at a depth of about 20 meters; and, thanks to the support of Navy divers and Italian Army incursors, the Tethys Bay Spiaggetta, the point with coordinates 74°40.545' S, 164°04.095' E and within Adelie Cove, in protected area ASPA 161. Sampling from shoreline, requested and authorized during the mission by the Polar Research Committee, was possible at sites named Edmons Point and Cape Halletet. Sampling from the sea was done by dinghy, and allowed sampling at predetermined areas within ASPA 161, such as Adelie Cove, Faraglione Field, and an area in between them at the 20- and 50-meter bathymetries. All samples collected for macrobenthos analysis, were sieved in the aquarium by seawater pump, removing all the inorganic and organic part smaller than 1 mm in size, while a part of the sieved samples was sorted in the laboratory by stereomicroscope.

As for the XXXIV campaign, sampling activities were carried out during the period between January 8, 2019 and February 1, 2019. During this period, 12 sites were sampled (Figure 2), 6 of which were in the immediate vicinity of the Italian Mario Zucchelli Base (MZS) and 6 within the ASPA 161 Protected Area. All sampling was carried out from the sea, performing not only sediment sampling for the study of benthic communities at different scales (micro-, meio- and macro-fauna), but also the collection of water and sediment samples for subsequent chemical-physical analysis in order to characterize the environment in which the communities (microbial, meio- and macrofauna) were located at the time of sampling. The effectiveness of the structure mounted on the dinghy allowed sampling at sites resulting from the sampling carried out during the XXXIII expedition, both within ASPA 161, such as Adelie Cove, Campo Faraglione and an area intermediate to them (named ASPA1), and in areas adjacent to the Italian base (named Rod Bay, Mareograph and Tethys Bay beach) at bathymetries of 20 and 50 meters.

Regarding the study of meio- and macrofauna, samples taken at stations located around the Antarctic Base Mario Zucchelli were frozen at -20°C there and transported to Italy for subsequent laboratory analysis.

Specifically:

- following the Castelli et al. (2003) protocols, the sediment samples were sorted under the stereomicroscope in order to take the macrozoobenthic organisms from the inorganic fraction, which were primarily classified by large taxa and fixed in 70% ethanol. Identification of organisms belonging to each taxon proceeded at the lowest possible taxonomic level, and each species was assigned a trophic category for a functional diversity analysis of the macrobenthic community. The binomial nomenclature of each species was ultimately checked in the World Register of Marine Species WoRMS (http://www.marinespecies.org).
 

- Sediments related to Meiobenthos samples were decanted and sieved repeatedly through a 30 µm mesh sieve. When the sediments consisted of mud, the fraction remaining within the sieve was resuspended and centrifuged in the presence of Ludox HS40 colloidal silica gel (Heip et al., 1985). All meiobenthic animal organisms were counted under the stereomicroscope and classified by major taxa. As for the phylum Nematoda, about 100 individuals were taken for each sample, fixed in anhydrous glycerin according to the Formaldehyde-Ethanol-Glycerin protocol (De Grisse, 1969) and mounted on kerosene ring slides for identification by compound light microscopy. The nematodes, then, were identified to the genus level and classified according to the specialist literature and in accordance with the taxonomic descriptions available on the NeMys database (http://www.nemys.ugent.be). The biomass of nematodes was calculated from their biovolume (Andrassy, 1967) according to the formula: V = L x W2 x 0.063 x 10-5, where L is the length of the organism and W its width. Ultimately, nematodes were categorized into trophic guilds on the basis of mouth cavity morphology (Wieser, 1953).
 

- Interstitial water samples were filtered using cellulose filters with porosity of 0.20µm, then treated with hydrogen peroxide (100µl in 100 ml of sample) to digest the organic content. The samples were then fractionated into two aliquots for determination of ions and metals. One of the two aliquots, before being used for ion chromatography readings, was used for reading chemical and physical parameters such as: pH, dissolved oxygen, conductivity, dissolved salts using a multiparameter probe (VWR pHenomenal/ pH1100L). As for ion concentration analysis, interstitial water samples were sent to the ICS1100 chromatographic system equipped with a dual column for simultaneous determination of anions and cations. Anions were detected with an AS22 column working with a volume of 100 µl sample and a 3.5mM sodium carbonate/sodium bicarbonate buffer solution as the eluent; cations were determined using a CS12A column working with a volume of 25 µl sample and a 20 mM methanesulfonic acid solution as the eluent. For both anions and cations, the calibration curve was worked out using certified multistandard solutions. This allowed the detection of Cl-, F-, Br-, NO2-, NO3-, PO43-, SO42-, Li+, Na+, K+, NH4+, Ca2+ and Mg2+ (as inorganic species) and HCOO-, CH3COO-, C2O42- (as organic species). Heavy metals in solution (Pb2+, Cd2+, Cu2+ and Zn2+) were determined using a polarographic method working with a mercury electrode for measurements and an Ag/AgCl electrode as a control, thus allowing simultaneous determination of zinc, cadmium, lead and copper.
 

- Sediment samples were analyzed to assess their particle size composition. Samples previously dried in an oven at 60° for 48h were weighed and placed in a sieve column for determination of the percentage of gravel (fraction >2mm), sand (0.063mm<fraction<2mm) and mud (fraction < 0.063mm). The percentage of Total Organic Carbon (TOC) was also estimated following the procedure described by Schumacher (2002).

Regarding the study of meio- and macrofauna, samples taken at stations positioned around the Antarctic Base Mario Zucchelli were frozen there at -20°C and transported to Italy for subsequent laboratory analysis.

The data thus acquired are being processed using statistical and heuristic software for the preparation of scientific articles to be submitted to leading journals.

Microbial community study

Regarding the study of the microbial community, attention was initially focused on samples taken in two areas included in ASPA 161, at the bathymetry of 20 meters. These samples, labeled as ASPA1_20 and ASPA2_20 were analyzed by two different approaches namely culture-based and metagenomic, to identify the bacterial species in them. The culture-based study, aimed at isolation of cells and their taxonomic identification, was performed by performing enrichment on different culture media namely: Marine Broth (MB) (comp. for L: Peptone 5 g, Yeast extract 1 g in 0. 45 μm filtered seawater), R2A (comp. for L: Yeast extract 0.5 g, Peptone 0.5 g, Casamino acids 0.5 g, Glucose 0.5 , Na-pyruvate 0.3 g, K2HPO4 0.3 g, MgSO4-7H2O 0.05 g, pH 7.2), TH (comp. for L: peptone 8 g, yeast extract 4 g and NaCl 2g) and M162. Through this approach, 16 different cell strains could be identified and isolated for which 16S rRNA gene sequencing was performed using the EzTaxon-e server (http://www.ezbiocloud.net/eztaxon), which is an open-access database in which 16S rRNA gene sequences of bacterial species are cataloged. By comparing the gene sequences obtained with this database, it was possible to deduce that most of the microbial strains isolated belong to the genera Psychrobacter and Lyfsonia.

• Federica Semprucci, Luca Appolloni,*  Eleonora Grassi, Luigia Donnarumma, Lucia Cesaroni, Giuseppina Tirimberio, Elena Chianese, Paola Di Donato, Giovanni Fulvio Russo, Maria Balsamo, Roberto Sandulli: Antarctic Special Protected Area 161 as a Reference to Assess the Effects of Anthropogenic and Natural Impacts on Meiobenthic Assemblages, Diversity 2021, 13, 626. https://doi.org/10.3390/d13120626
• DI DONATO, Paola; Leone, Luigi; Finore, Ilaria; Abbamondi, Roberto; Poli, Annarita; Tommonaro, Giuseppina; Appolloni, Luca; Donnarumma, Luigia; Sandulli, Roberto; Russo, Giovanni, Fulvio; Nicolaus, Barbara. Extremophiles from Antarctica: the bacterial community living in association with the benthic fauna in the Ross sea, 2019, Proceedings of 60th Congress of the Italian Society of Biochemistry and Molecular Biology (SIB2019)
• Appolloni L, Di Donato P, Donnarumma L, Semprucci F, Cesaroni L, Grassi E, Balsamo M, Rendina F, Ferrigno F, Russo GF, Sandulli R: Valutazione preliminare degli impatti antropici e naturali sule comunità meiobentoniche nella Baia di Terranova (Antartide). Convegno Nazionale della Società di Biologia Marina Mediterranea (SIBM). Giugno 2020.
• Attività di divulgazione e public engagement: Seminario dal titolo "Racconto di un lungo viaggio alla scoperta della biodiversità marina bentonica in ambiente estremo" presso il Liceo Scientifico "don Lorenzo Milani" di Gragnano (NA), 9/4/2019; Seminario dal titolo "Una curiosità senza limiti: La ricerca ai confini del mondo" presso Istituto Superiore Statale "Pitagora", Pozzuoli (NA), 5/4/2019.