Mini pam

Photosynthetic parameters were measured during the experiment by in vivo chlorophyll a fluorescence using a Mini-PAM (Walz, Effeltrich, Germany). The effective quantum yield (Y(II)) and maximum quantum yield (F_v/F_m) of photosystem II were assessed as described by Figueroa et al., 2014 [55 (link)] on days 1, 5, 8 and 12, in four different periods throughout the day: 8:20 (F_v/F_m), 9:45, 15:00 and 18:20 h.
Y(II) data were obtained three times during the day and they were used to calculate in situ electron transport rates. Rapid light curves (RLC) were performed on the 1st, 7th and 14th days during the period from 9:00 to 14:00 h, using the Mini-PAM fluorometer connected to the WinControl-3 software (Walz, Effeltrich, Germany). During this interval, radiation treatments remained constant to avoid interfering with the test due to the variation in irradiances. Algal samples from each treatment were acclimated during 15 min in darkness and subsequently exposed to the following increasing irradiances: 0, 25, 45, 66, 90, 125, 190, 285, 420, 625, 845, 1150 and 1500 μmol photons m⁻². s⁻¹ of red actinic light. After the incubation of each irradiance, the samples received a saturation pulse. Electron transport rates (ETR) were calculated according to the following equation:
where Y(II) corresponds to the effective quantum yield of PSII, E corresponds to the irradiance to which the algae was exposed, and 0.15 is equivalent to the factor for adjusting the irradiance captured and used by photosystem II (PSII) by red macroalgae. A refers to the absorptance calculated by the following equation according to Grzymski et al., 1997 [56 (link)] and Figueroa et al., 2003 [57 (link)]: $$\mathrm{A}=1-{\displaystyle \frac{E_F}{E_T}},$$
where EF corresponds to the irradiance transmitted throughout the algae thalli and ET is the total irradiance, measured with the Li-189 radiometer (LICOR Ltd., Lincoln, NE, USA).

A portable pulse amplitude fluorometer (Mini-PAM) (Walz GmbH, Effeltrich, Germany) combined with WinControl software was used to evaluate the effect of EOs on photosynthetic activity. To ensure maximum potential quantum yield, samples were dark adapted for 30 min prior to each measurement. All analyses were performed in a dark room, and the probe was equipped with a holder that held the fibre 6 mm from the biofilm (Rugnini et al. 2020 (link)). The yield value was calculated as follows: $$\text{Yield}=\frac{Fm-Fo}{\mathit{Fm}}$$ where F₀ is the minimum and F_m is the maximum fluorescence of the dark-adapted sample (Bilger et al. 1995 (link); Schreiber and Bilger 1993 ). The efficacy of EOs at concentrations of 5% and 0.5% against photosynthetic microorganisms growing on agar plates was quantified by determining the maximum quantum yield of photosynthesis on each triplicate sample before each treatment (t₀) and after 24 h (t_24h), 1 week (t_1w), 1 month (t_1m) and 2 months (t_2m).

Elysia timida was collected on Giglio, Italy (42°22′ N, 10°52′ E and 42°21′ N, 10°52′ E) between 3 and 6 m depth. Elysia cornigera was collected at the Florida Keys (24°38′ N, 81°18′ W) at about 1 m depth. Both species were kept at a 12 L : 12 D rhythm at 25 µmol quanta m⁻² s⁻¹ in 3.7% salt water (Tropic Marine) and at 21°C. Acetabularia acetabulum DI1 (isolate of Professor Diedrik Menzel, Bonn University, Germany) was grown at the same conditions as the animals, but in algal f/2 medium. For starvation experiments, animals were moved to Petri dishes (∅︀200 mm) and 50% of the water was changed every other day. Monolinuron (JBL GmbH) was added from a stock solution of 4000 mg l⁻¹ to a final concentration of 2 µg ml⁻¹. Bleaching was performed by illuminating the slugs with 1000 µmol quanta m⁻² s⁻¹ for 1 h every day at the same time. For each pigment extraction, pooled slugs were transferred to 90% acetone for each time point, homogenized and kept at −20°C for 1 day. Extractions for every time point were carried out as biological triplicates, each replicate consisting of at least three E. cornigera and two E. timida. Crude extracts were filtered through a 200 nM polytetrafluoroethylene membrane and then analysed by reversed-phases high pressure liquid chromatography with ultraviolet/visible spectroscopy detection (Hitachi/Merk) as described earlier [32 (link)]. Pigment concentrations were determined using external pigment standards isolated from spinach thylakoids. Dry weight was determined on dried slug homogenate after extraction.
Photosystem II maximum quantum yield (F_v/F_m) was determined using a WALZ MINI-PAM as described previously [33 (link)], on the same group of individuals that were used for ¹⁴CO₂ incorporation measurements. For every time point for which ¹⁴CO₂ incorporation was determined, an individual group of slugs (at least 12 E. cornigera or nine E. timida) was kept in isolated dishes. For each of these groups, separate F_v/F_m measurements were performed, and each individual slug of a group was measured at least three times. For each data point, the mean of all individual triplicate measurements was calculated. The light-driven incorporation of ¹⁴CO₂ was determined as described earlier [5 (link)]. Briefly, for each triplicate measurement four individuals of E. cornigera and three of E. timida were used. Slugs were incubated in 1.2 ml artificial seawater supplemented with 0.40 mM [¹⁴C]-NaHCO₂ (25 µCi per incubation, NEN-radiochemicals, MA, USA) for 2 h at room temperature and 72 µmol quanta m⁻² s⁻¹ illumination. After rinsing, homogenization and acidification of the material with 150 µl 1 M HCl, all the substrate was expelled from the homogenate. Incorporation of ¹⁴C by the slugs was determined in a scintillation counter after the addition of 12 ml LUMA-Gel scintillation cocktail (LUMAC, The Netherlands).
For ROS-imaging, slugs were incubated for 45 min with 100 µM DHE (DHE; excitation/emission HeNe 543/610 nm; Sigma) or 100 µM DCF (2′,7′-dichlorofluorescin; excitation/emission Argon 488/529 nm) plus 2 µM MitoTracker Red CMXRos (excitation/emission HeNe 543/599 nm; LifeTechnologies) in artificial seawater. Slugs were rinsed twice with seawater and decapitated before mounting. Confocal laser scanning microscopy was carried out using a Zeiss LSM 710. Imaging was always performed with the same settings and at a similar position along the parapodial rim at the same depth relative to the epidermis. Images were processed using Fiji/ImageJ 1.48f [34 (link)] and statistics were performed using R [35 ]. Normality was tested via a Shapiro–Wilk test [36 (link)] and significance using a Mann–Whitney U-test [37 (link)].
Total RNA was extracted three times from seven E. cornigera (a total of 21) and three times from five E. timida (a total of 15) feeding on A. acetabulum (t₀, feeding control group), 28 individuals of E. cornigera that were starved for 4 days, of which nine individuals had only starved (S), nine had starved and were treated with monolinuron (S + M), and 10 of which had starved and were treated with 1000 µmol quanta m⁻² s⁻¹ for 1 h each day (S + B). The RNA of 20 E. timida's (7 S, 7 S + M, 6 S + B) was isolated after four days of starvation (t₁), of 30 E. cornigera (10 S, 9 S + M, 11 S + B) and of 20 E. timida (6 S, 8 S + M, 6 S + B) after 7 days of starvation (t₂), and finally of 32 E. timida (11 S, 10 S + M, 11 S + B) after 30 days of starvation (t₃). RNA was isolated using TRIzol (Life Technologies) according to the manufacturer's protocol and an additional DNAse treatment (Thermo Fisher Scientific). Poly(A) mRNA enrichment, library preparation using the TruSeq kit (Illumina) and 100 bp paired end sequencing using the Illumina HiSeq2000 system was performed by the Beijing Genome Institute (Hong Kong). Reads with remaining adapter sequences, more than 5% of unknown nucleotides or more than 20% of nucleotides with quality scores less than 10 (Illumina 1.5) were removed as part of the raw data extraction.
A total of 1 186 405 486 reads (a minimum of 52 million reads/library) were obtained (electronic supplementary material, table S2). Reads were inspected by FastQC v. 0.10.1 [38 ] and filtered and trimmed using Trimmomatic 0.32 [39 (link)] (parameters: ILLUMINACLIP:TruSeq3-PE.fa:2 : 30 : 10; HEADCROP:10; TRAILING:3; SLIDINGWINDOW:4 : 20; MINLEN:36). Reads of all samples were assembled using Trinity r20131110 [40 (link)], which yielded 249 855 and 150 314 transcripts for E. cornigera and E. timida, respectively. Expression values for transcript clusters (or unigenes) were extracted and analysed using the Trinity pipeline [41 (link)] via RSEM 1.2.11 [42 (link)], Bowtie 1.0.1 [43 (link)] and edgeR 3.4.2 [44 (link)]. Only unigenes with raw read counts of greater than or equal to 100 over at least two samples were analysed, resulting in 36 380 and 32 897 unigenes, respectively. Differential expression was determined by log₂fc of at least ±1 compared to the control set of unigenes (with raw read counts of greater than or equal to 100). Expression change significance was assessed based upon the dispersion of available replicate information in edgeR. The longest isoform of each cluster was defined as the representative sequence for a unigene.
To plot the overall taxonomic distribution of the assembled transcripts, unigenes were subjected to a BLASTx-based [45 (link)] search (e-value cut-off 10⁻¹⁰) against a database consisting of protein sequences of RefSeq version 64 [46 (link)] plus those of the genomes of Crassostrea gigas [47 (link)] and Lottia gigantean [48 (link)] (electronic supplementary material, figure S2). For all downstream analyses, only those genes were included for which top hits to the mentioned organisms or other metazoans were retrieved. Excluded were those with best blast hits to plants, protists, prokaryotes and viruses. Protein annotations for the 14 848 E. cornigera and 13 875 E. timida unigenes were extracted from mentioned BLAST hits and from a second BLASTx search to the UniProtKB/Swiss-Prot database [49 (link)]. KOG/NOG (EuKaryotic/Non-supervised Orthologous Groups [50 (link)–52 (link)]) categories were determined based on the best BLASTx hit to protein sequences of eggNOG v. 4 [53 (link)]. KEGG [54 (link)] accessions were obtained using the KAAS 1.6a webservice [55 (link)] against metazoa.
For qRT-PCR, 200 ng RNA was reverse transcribed using random hexamers and the iScript Select cDNA Synthesis Kit (Bio-Rad). Two-step qRT-PCR was performed on biological triplicates for each time point and treatment (each containing pooled RNA from greater than or equal to five slugs) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions on a StepOne Plus (Applied Biosystems) real-time PCR system. Primers were designed using Primer-BLAST [56 (link)] (for primer sequences, see the electronic supplementary material, table S4) and data were analysed according to Pfaffl [57 (link)], using the EcRPL38 (Eco000149), EtRPL19 (Eti000121) and EtSETMAR (Eti000317) as reference genes.

The freezing resistance of leaves of R. glacialis was assessed by the simulation of artificial frost nights with different severities in the temperature-controlled commercial chest freezers. The experimental protocol is described precisely but briefly: Leaves were laid out on wet paper towels inside of a sealable plastic bag. To avoid the artificial supercooling of the samples, ice nucleation was triggered by the application of a few droplets of an ice nucleation active (INA) bacteria suspension (Pseudomonas syringae van HALL 1902) to the wet paper towels. For each treatment temperature, 10 randomly selected leaves were chosen. Treatment temperatures were set in a sequence with at maximum 3 °C difference. After controlled cooling (−3 °C h⁻¹) to the target temperature, the leaves were exposed to the target temperature for 4 h, followed by controlled thawing (+3 °C h⁻¹). After the treatment, the samples were stored at +20 °C under moderate light conditions until the frost damage became visible. Damage was assessed by the chlorophyll fluorescence. The chlorophyll fluorescence (Fv/Fm) was measured either with the MINI PAM (Walz, Effeltrich, Germany) or the IMAGING-PAM M-Series (MAXI Version, Walz).
$L{T}_{50}$ is the temperature at which 50% of the tested leaves are considered damaged. For the calculation of $L{T}_{50},$ a logistic function (Boltzmann function) was fitted to the data: $Fv/Fm=\frac{{\left(\frac{Fv}{Fm}\right)}_{min}-{\left(\frac{Fv}{Fm}\right)}_{max}}{1+e^{\frac{T-T_{LT50}}{dx}}}+{\left(\frac{Fv}{Fm}\right)}_{max}$ , where $T$ is the temperature, $\frac{Fv}{Fm}$ is photosynthetic yield or viability in %, ${\left(\frac{Fv}{Fm}\right)}_{min}$ and ${\left(\frac{Fv}{Fm}\right)}_{max}$ are the asymptotic lower and upper limits of the curve (minimum yield, maximum yield), $T_{LT50}$ is the temperature at the inflection point, and $dx$ is a slope factor. After randomly selecting 4 yield values from each target temperature, a fit analysis was performed. This procedure was repeated 250 times. The mean LT₅₀ values and standard deviation are presented.

Relative water content (RWC) was determined according to Escandón et al. [18 (link)], using three needle fragments (1 cm long) in each six replicates per sampling point. Samples fresh weight (FW) was registered and needles maintained with de-ionized water for 24 h in dark at 4 °C, after which turgid weight (TW) was recorded. Then, needles were dried at 80 °C for 72 h, and dry weight (DW) was registered. RWC was calculated by using the following equation: RWC (%) = (FW-DW)/(TW-DW) × 100.
Proline was quantified in needles according to Bates et al. [81 (link)] with modifications, in six replicates per treatment. About 100 mg of frozen needles were homogenized in 3% sulfosalicylic acid (5 μL/mg FW), and centrifuged at 13,000 rpm for 5 min. A mixture with 100 μL of 3% sulfosalicylic acid, 200 μL of glacial acetic acid, and 200 μL of acidic ninhydrin was added to 100 μL of the supernatant of the extract, and the resulting mixture was vortexed and incubated at 96 °C for 1 h. After that, the reaction was finished on ice for 10 min. Samples were extracted in 1 mL of toluene and vortexed for 20 s, and the formation of two phases was observed. The absorbance of the chromophore-containing toluene phase was read at 520 nm (Eppendorf BioSpectrometer^® basic) using toluene as blank reagent, and proline concentration was determined from a Sigma-Aldrich^® L-proline standard curve with 6 points (0–150 μg/mL).
To estimate photosynthetic activity in maritime pine plants growing at the greenhouse, (ϕPSII) was analyzed in needles using a pulse-amplitude modulation fluorimeter (MINI PAM; Walz, Effeltrich, Alemania), according to Nebauer et al. [82 (link)]. Two measurements of five needles in the mid part of the plant were measured up to a total of ten replicates for each plant group, treatment and sampling period. Needles were pre-adapted in the dark for 20 min and then exposed to a light flash, taking one measure in darkness and another in light, both at a wave length of 350–400 nm. Estimates of ϕPSII were obtained by measuring variable fluorescence (Fv), and calculating the difference (Fv = Fm-F0) between the maximum fluorescence (Fm), after the light flash, and the minimum fluorescence (F0), in the absence of light. The estimated ϕPSII represents the proportion of the energy absorbed by the chlorophyll of PSII that is being used to drive the photochemical process, therefore it is a measure of the efficiency of linear electron transport.
Chlorophyll and carotenoids were extracted and analyzed in needles of maritime pine plants according to Lichtenthaler [83 (link)] with some modifications, in nine replicates per treatment. A total of 300 mg of needles were grinded in metal containers with a metal sphere (50 mm ø) using the MM 400-Retsch mixer for 30 s. After grinding, three aliquots of 100 mg each were prepared and pigments were extracted in 10 mL of 100% (v/v) acetone and centrifugated (10,000 rpm, 10 min, 4 °C). After that, absorbances of the supernatants were read at 470, 645 and 662 nm (Eppendorf BioSpectrometer^® basic), and the concentration of each pigment was determined.
Total soluble sugars (TSS) and starch contents were determined in needles of maritime pine plants as described by Rodríguez et al. [84 ], in six replicates per treatment. Extracts were obtained from 50 mg of frozen needles grinded and homogenized in 10 mL of 80 % (v/v) ethanol, by using a MM 400-Retsch mixer for 40 s. The mixture was incubated at 80 °C for 1 h and centrifuged (6000 rpm, 20 min, 4 °C). After that, 2.5 mL of a solution of Sigma-Aldrich^® anthrone (0.25 g of anthrone in 100 mL of 95% sulfuric acid) were added to 1 mL of the supernatant, and the mixture was vortexed and incubated at 100 °C for 15 min. After cooling down, absorbance was read at 620 nm. Starch content was determined from the pellet resulting from the centrifugation of the initial extract, that was incubated in 10 mL of 30% (v/v) perchloric acid for 16 h at room temperature. After centrifugation, 1 mL of the supernatant was mixed with 2.5 mL of anthrone, vortexed and incubated at 100 °C for 15 min. The mixture was cooled down and the absorbance was read at 620 nm. Both TSS and starch contents were calculated against a Sigma-Aldrich^® D-glucose standard curve (0–800 μM), using anthrone as a blank.