Philippa Kaur

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Isolation and cultivationIn total, 39 mixotrophic flagellates were investigated (Table 1). Thirty-three isolates were enriched and isolated from euphotic zone samples at Station ALOHA (22° 45′ N, 158° 00′ W) in February and May 2019. To select for mixotrophic grazers of Prochlorococcus, whole seawater was amended with K medium [28] (1/20 final concentration), and live Procholorococcus (MIT9301) was added as prey (~5 × 106 cells mL−1 final concentration). Enriched seawater samples were incubated under ~70 µmol photons m−2 s−1 irradiance on a 12 h:12 h light:dark cycle and monitored by microscopy daily up to five days. Each day, samples were serially diluted to extinction (9 dilution steps, 12 replicates per dilution) in 96-well plates in nutrient-reduced K medium (1/20 concentration) with a constant background of Prochlorococcus cells. Wells at the highest dilution showing growth of putative grazers were subjected to 3–6 further rounds of dilution to extinction. Four additional mixotrophs were isolated in full K medium using water from earlier cruises, and two (dictyochophyte strains UHM3021 described in [24] and UHM3050) were enriched in K minus nitrogen medium (K-N) without Prochlorococcus enrichment. All isolates were rendered unialgal, but not axenic, and maintained at 24 °C in K-N medium (~0.2 µM N) amended with Prochlorococcus prey, under the same light conditions as above. Dense Prochlorococcus cells grown in Pro99 medium [29], were harvested and concentrated through gentle centrifugation at 2000 RCF for 5 minutes and resuspended in fresh K-N medium to minimize nutrient carryover. To ensure their long-term accessibility, the isolates used in this study are being transferred to the National Center for Marine Algae and Microbiota at the Bigelow Laboratory for Ocean Science, East Boothbay, ME, USA (ncma.bigelow.org).Table 1 Mixotrophic flagellates investigated in this study.Full size table18S rRNA gene sequencing and phylogenetic analysisCells were harvested by centrifuging 25–50 mL dense cultures at 3000 RCF for 10 min at 4 °C. Genomic DNA was extracted from the pellets using the ZymoBIOMICS DNA Kit (Zymo Research, Irvine, CA, USA). A near-full-length section of the eukaryotic small-subunit ribosomal RNA (18S rRNA) gene was amplified by PCR with the Roche Expand High Fidelity PCR System (Sigma-Aldrich, St. Louis, MO, USA) using either forward primer 5′-ACCTGGTTGATCCTGCCAG-3′ and reverse primer 5′-TGATCCTTCYGCAGGTTCAC-3′ [30], or Euk63F 5′-ACGCTTGTCTCAAAGATTA-3 and Euk1818R 5′-ACGGAAACCTTGTTACGA-3′ [31]. Amplicons were purified using spin columns (DNA Clean & Concentrator-25; Zymo Research, Irvine, CA, USA) and sequenced (Sanger) using the same PCR amplification primers and an additional reverse primer 1174R, 5′-CCCGTGTTGAGTCAAA-3′ [32], when necessary to connect two ends. For phylogenetic analyses, similar sequences were retrieved from the PR2 database [33] based on BLAST similarity, and two environmental homologs (GenBank Acc. FJ537342 and FJ537336) were retrieved from NCBI GenBank for the undescribed haptophyte taxon, which was not affiliated with any reference sequence from the PR2 database. Sequence alignments including 39 isolates, 29 reference and 2 outgroup taxa were created with MAFFT v7.450 using the G-INS-i algorithm [34] in Geneious R11.1.5 (http://www.geneious.com) [35]. Terminal sites that lacked data for any of the sequences were trimmed and any sites with greater than 25% gaps were removed from the alignment, which generated a total sequence length of 1617 bases. Phylogenetic analysis was performed using MrBayes v3.2.6 in Geneious R11.1.5 [36] with two runs of four chains for 1,000,000 generations, subsampling every 200 generations with burn-in length 100,000, under the GTR substitution model. The Bayesian majority consensus tree was further edited within iTOL v5 [37]. All 18 S rRNA gene sequences were deposited in GenBank with accession numbers MZ611704–MZ611740; MN615710–MN615711.Microscopic observationThe average diameter of flagellates in the exponential growth phase (n = 20 cells per strain) was measured by transmitted light microscopy using image analysis software (NIS-Elements AR, Nikon, Minato City, Tokyo, Japan) calibrated with a stage micrometer. Equivalent spherical diameter (ESD) and biovolumes were calculated assuming spherical cells. Chloroplasts were visualized by autofluorescence under epifluorescence microscopy. An average ESD of 0.64 µm was used for Prochlorococcus prey [24].Visual evidence of phagocytosis was obtained by adding fluorescent beads (0.5 μm YG Fluoresbrite Microspheres; Polysciences) to each culture. Samples post incubation (~2 h) were fixed with an equal volume of 4% ice-cold glutaraldehyde, and subsamples (20 µL) were mounted on a glass slide under a coverslip. Paired images captured using epifluorescence and transmitted light microscopy (Olympus BX51 with Leica DFC 7000 T color digital camera) were overlain to identify cells with ingested beads.Grazing experimentsLong-term grazing experiments were conducted for all 39 grazers, and 31 were used to quantify grazing rates based on rates of disappearance of Prochlorococcus cells, which persist but do not readily grow in K-N medium [24]. Rates were not calculated for eight isolates (seven Florenciella and one DictyX) because they were sampled at a lower frequency. Fifteen isolates representing all genera (or approximately genus-level clades) were examined in more detail by replicating grazing experiments two times (marked in bold in Supplementary Table S1), while the remaining sixteen isolates were tested once to survey within- and across- genus variation. Prior to the experiments, all grazer cultures were maintained/acclimated in the experimental medium (K-N with prey). Experiments were initialized by inoculating late-exponential-phase grazers into fresh K-N medium at a final concentration of ~103 flagellates mL−1, and adding live, unstained prey at a final concentration of 2–3 × 106 Prochlorococcus mL−1. Grazers were incubated for 3–8 days in total, depending on how fast prey were ingested. To minimize carryover of dissolved nutrients and prey growth in the grazing experiments, Prochlorococcus were grown to stationary phase in Pro99 medium, then pelleted (2000 RCF for 3–5 min) and resuspended in fresh K-N medium prior to addition to control and experimental cultures. Control cultures of grazer without added prey and prey without grazers were included during each grazing experiment to confirm that grazer growth and prey removal were attributable to grazing. Cell concentrations of prey and grazers were measured every 12–24 h by flow cytometry of glutaraldehyde-fixed samples at final concentration of 0.5% (Attune NxT; Thermo Fisher Scientific, Waltham, MA, USA, and CytoFLEX; Beckman Coulter Life Sciences, Indianapolis, IN, USA). Populations were distinguished based on light scatter and pigment autofluorescence and occasionally confirmed with DNA fluorescence (stained post-sampling with DNA stain SYBR Green I). Ambient bacteria concentrations, monitored using DNA stains, were ≤10% of the added Prochlorococcus at the start and during most periods of all grazing experiments.Grazing rates and biovolume conversion efficiencyWe calculated ingestion rates, I (prey grazer−1 h−1) for each grazer as:$$I = frac{{P_t – P_{t + 1}}}{{G_{{{{{rm{avg}}}}}}(T_{t + 1} – T_t)}}$$
(1)
where (P_t) and (P_{t + 1}) are the prey abundance at sampling interval t and t + 1 (cells mL−1), Gavg is the arithmetic mean grazer abundance (cells mL−1) over the time interval, and ((T_{t + 1} – T_t)) is the time (h) between two sampling intervals. Clearance rates (C, nL grazer−1 h−1) were calculated by dividing the ingestion rate by average prey concentration over the same interval, and specific clearance rate (body volume grazer−1 h−1) was calculated by dividing the clearance rate by cellular biovolume (µm3) of each grazer. Equation (1) uses a linear approximation of prey and grazer trajectories over the sampling interval, which was appropriate for our data where change could be relatively linear, concave-up, or concave-down. Other commonly used ingestion rate calculations assume exponential prey decline [38] and/or exponential grazer growth [39].Clearance rates over time in each experiment were assessed visually to obtain a representative series of rates that minimized potential influence of modest prey growth/decline observed in control cultures (Supplementary Fig. S1), as well as potential slowing of ingestion as the grazer neared carrying capacity or depleted prey to a low concentration. For each experiment a contiguous set of relatively constant rates were used to calculate a mean clearance rate. This assessment sometimes excluded the first 12–24 h, but not when removal rates were particularly fast. Intervals when the grazer neared carrying capacity were also often excluded, if grazing rates slowed down. To assess whether clearance rates increased as prey were depleted we plotted clearance rate and ingestion rate as a function of prey concentration (Supplementary Figs. S2 and S3). In general, there was no relationship between clearance rate and prey concentration, and ingestion increased linearly with prey concentration. These patterns imply that the prey concentrations in this experiment did not saturate the ingestion rates of these grazers. Under non-saturating prey concentrations the average clearance rate over an experiment should be a good estimate of the maximum clearance rate (Cmax). Consistent with this interpretation, functional responses fit to these experiments yielded Cmax estimates that were similar to the reported average clearance rates. Because these experiments were not designed with a sufficient range of prey density to esimate functional responses, we do not report the Cmax estimates, but we note here that our reported average clearance rates may be useful as approximate Cmax numbers in future work.Six grazers representing three classes (three dictyochophytes, two haptophytes, and one chrysophyte) were further investigated to determine functional grazing responses using a wide range of initial prey densities (105–107 cells mL−1). Functional responses were modeled using the Holling type II curve, (I = frac{{I_{{{{{rm{max}}}}}}P}}{{P + frac{{I_{{{{{rm{max}}}}}}}}{{C_{{{{{rm{max}}}}}}}}}}), where I is the ingestion rate over a sampling interval (Eq. 1), Imax is the maximum ingestion rate, Cmax is the maximum clearance rate and P is the arithmetic mean prey density between two sampling points. This curve was fit to ingestion rate data using maximum likelihood with R package bbmle [40].For 31 isolates we calculated the amount of grazer biovolume created per prey biovolume consumed, using data from the same grazing experiments used to calculate grazing rates. It was calculated based on the following formula:$$E = frac{{(F_{{{{{rm{f}}}}}} – F_{{{{{rm{i}}}}}})}}{{(P_{{{{{rm{i}}}}}} – P_{{{{{rm{f}}}}}})}}frac{{B_{{{{{rm{F}}}}}}}}{{B_{{{{{rm{p}}}}}}}}$$
(2)
where Ff and Fi are the final and initial flagellate concentrations, Pi and Pf are initial and final prey concentrations in each culture, and BF and BP are the cellular biovolume of prey and grazer. We refer to the quantity E as the biovolume conversion efficiency, and we use it as an indicator of physiological differences among diverse mixotrophs. Note that biovolume conversion efficiency can be greater than 1, if prey have greater nutrient:biovolume than the grazer.Quantitative PCRReal-time, quantitative PCR (qPCR) was performed to quantify the 18S rRNA gene abundances of representative mixotroph groups discriminated at approximately the genus level, including Florenciella, Rhizochromulina and another undescribed clade within the class Dictyochophyceae; Chrysochromulina and another undescribed clade within the division Haptophyta; clade H in the class Chrysophyceae; and Triparma eleuthera and Triparma mediterranea in the class Bolidophyceae. Primers (Supplementary Table S2) were designed to target a short region (95–176 bases) of the 18S rRNA gene and meet basic criteria (≤2 °C difference in melting temperature between members of a pair, %G + C content between 45 and 65%, ≤1 degenerate position per primer, no predicted primer dimers). Sequences considered targets for a given primer set had ≤1 mismatch across both primers, which included all or most known members within the corresponding targeted clade. Members in the nearest non-targeted clade had ≥3 mismatches distributed across both primers. Efficiency and specificity of the synthesized primers (IDT Inc., Coralville, IA, USA) was tested by ensuring there was specific amplification (qPCR followed by melting curve analysis and gel electrophoresis) when using DNA from cultures within the targeted group and no amplification when using DNA from cultures close to, but outside of the targeted group (Supplementary Table S3). Empirical observations of amplification success using control cultures were used to infer whether species known only by environmental sequences were likely to amplify with a given primer set (Supplementary Fig. S4).In situ gene abundances were quantified in water samples collected from Station ALOHA at 5, 25, 45, 75, 100, 125, 150, and 175 m, during HOT cruise numbers 259 (Jan), 262 (Apr), 264 (Jul), and 266 (Oct) of 2014. Seawater (ca. 2 L) was filtered through 0.02 μm pore-size, aluminum oxide filters (Whatman Anotop, Sigma-Aldrich, Saint Louis, MO, USA) and stored at −80 °C. Genomic DNA of both grazer cultures and environmental samples was extracted (MasterPure Complete DNA and RNA Purification Kit; Epicentre) as described elsewhere [41]. Four replicated PCR reactions (10 μL) were carried out for each sample except for Triparma (duplicates) and consisted of 5 μl of 2× PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific, USA), 10 ng environmental DNA, 500 nM of each primer, and nuclease-free water. Reactions were run on an Eppendorf Mastercycler epgradient S realplex2 real-time PCR instrument. Each run contained fresh serial dilutions (1–6 log gene copies) of target-specific, 750-bp synthetic standards (gBlocks, IDT) prepared in triplicate. The cycling program included an initial denaturation step of 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 55 °C for 30 s. Specificity of amplification was checked with a melting curve run immediately after the PCR program and occasionally, by gel electrophoresis. Amplification efficiencies ranged from 95% to 106% for all the primers.To convert gene copies to cell numbers, 18S rRNA gene copy number per cell−1 was determined for representative isolates in the seven targeted genera/clades. Known quantities of cultured cells (106–107 cells) from each isolate with 2–8 replicates were pelleted at 4000 RCF for 15 min at 4 °C. DNA was extracted from the pelleted cells (MasterPure Complete DNA and RNA Purification Kit, Lucigen), quantified by fluorometry (Qubit, Invitrogen) and the extract volume adjusted to achieve a DNA concentration of 10 ng µL−1. The expected number of eukaryotic cells µL−1 of extract was calculated as the difference between the total cells in the sample prior to centrifugation and in the supernatant afterward (as determined by flow cytometry) divided by the final extract volume. Copy number of the 18S rRNA genes µL−1 of extract was determined by qPCR with the appropriate group-specific primers. The resulting value of gene copies µL−1 was divided by the equivalent number of eukaryotic cells µL−1 in the extract (assuming 100% extraction efficiency) to derive minimum estimates of gene copies cell−1. An average value for representatives within each genus/clade (1–5 isolates) was used to calculate in situ cell concentrations for the genus. These derived in situ abundances were compared to flow cytometric counts of total photosynthetic picoeukaryotes at Station ALOHA obtained from the Hawai’i Ocean Time-series Data Organization and Graphical System (https://hahana.soest.hawaii.edu/hot/hot-dogs/).Global distribution revealed through Tara Oceans 18S rRNA metabarcodesTo estimate the relative abundance of the OTUs closely related to our diverse isolates on a broader geographic scale, we searched the 18S rRNA-V9 sequence data from the 0.8–5 µm fraction of surface water sampled at 40 stations by the Tara Oceans project (http://taraoceans.sb-roscoff.fr/EukDiv/). Reads for ‘Tara lineages’ with highest similarity (E-value < 10−15) to each of our targeted clades (Supplementary Table S1) were expressed as a fraction of total reads excluding dinoflagellates but included all other Tara Oceans phytoplankton ‘taxogroups’: Bacillariophyta, Bolidophyceae, Chlorarachnea, Chlorophyceae, Chrysophyceae/Synurophyceae, Cryptophyta, Dictyochophyceae, Euglenida, Glaucocystophyta, Haptophyta, Mamiellophyceae, Other Archaeplastida, Other Chlorophyta, Pelagophyceae, Phaeophyceae, Pinguiophyceae, Prasino-Clade-7, Pyramimonadales, Raphidophyceae, Rhodophyta and Trebouxiophyceae. Dinoflagellates were excluded because of the difficulty in assigning phototrophic vs. heterotrophic status to all taxa, and because nearly all dinoflagellate reads were from a single, poorly annotated OTU that was also highly abundant in larger size fractions. More

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Chemicals and reagentsEOs of basil (Ocimum basilicum L.), bergamot (Citrus bergamia Risso & Poit), camphor [Cinnamomum camphora (L.) J. Presl.], cinnamon (Cinnamomum zeylanicum Blume), citronella [Cymbopogon nardus (L.) Rendle], clove (Eugenia caryophyllus Wight), eucalyptus (Eucalyptus globulus Labill.), jasmine (Jasminum officinale L.), lavender (Lavandula angustifolia Mill.), lemon grass [Cymbopogan citratus (DC.) Stapf], mentha (Mentha piperita L.), rosemary (Rosmarinus officinalis L.), patchouli (Pogostemon patchouli Benth), and wild turmeric (Curcuma aromatica Salisb.) were procured from Talent Technologies (Talent Technologies, Kanpur, India). Acetylcholinesterase (AChE) activity assay kit, Anti-OBP2A antibody, ELISA kits, 1,1-diphenyl-2-picrylhydrazyl (DPPH), radioimmunoprecipitation (RIPA) buffer and phosphate buffer saline (PBS) were purchased from Sigma Aldrich (Sigma Aldrich Chemical Co., St. Luis, USA). TRPV1 antibody was purchased from Santa Cruz (Santa Cruz, California, USA). 1-chloro-2,4-dinitrobenzene (CDNB) was purchased from Cayman (Cayman Chemical Company, Michigan, USA). Human normal lung cell line (L-132) was obtained from the National Centre for Cell Sciences (NCCS), Pune, India. High performance liquid chromatography (HPLC) grade acetone was purchased from Merck (Merck Pvt. Ltd., Mumbai, India). All other chemicals used were of the highest analytical grade available.Test insects5–7 days old adult female Ae. albopictus mosquitoes were housed at the laboratory insectary, Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur, Assam, India. Mosquitoes were reared by maintaining temperature at 27 ± 2 °C, relative humidity: 75 ± 5% RH and 14L:10D h of light–dark alternative cycles in standard-sized wooden cages (75 cm × 60 cm × 60 cm) with a sleeve opening on one side as described previously63. 10% sucrose solution ad libitum were provided for nourishment. Before testing, the mosquitoes were starved for 24 h.Screening of EOsDose response study was performed to evaluate the best oils among the fourteen EOs. This study was approved (approval number: 032/2021TMCH, 28/08/2018) by the Institutional Human Ethical Committee (IHEC), of the Tezpur Medical College & Hospital (TMCH), Tezpur, Assam, India, and all experiments were performed in accordance with relevant guidelines and regulations. Five volunteers are chosen, not allergic to mosquito bite and all volunteers provided written informed consent. A volunteer’s thigh was marked according to the door opening hole of the K&D module as described by Klun and Debboun64. It is made of Plexiglas and the base of the rectangular cage (26 cm × 5 cm × 5 cm) has six holes, each with rectangular 3 × 4 cm holes that are opened and closed by a sliding door (Supplementary Fig. S8: Provide the photograph of K&D module). The flexor region of the forearms of a human volunteer was outlined with four rectangular (3 cm × 4 cm) test areas. A volume of 25 µL of each concentration of the EOs in soybean oil (40, 4 and 0.4 µg/cm2) and 25 µL of the soybean oil (diluent) as control was applied to the marked areas. After air drying for 5 min, a K&D module with matching cut outs in its floor was placed over the treated areas, containing five nulliparous 5–7 days old female mosquitoes in each hole. The doors of the cells were opened and the number of mosquitoes biting in each cell was recorded within a 2 min exposure, after which the doors were closed. After completion of each observation, mosquitoes were freed by opening cells of the K&D module in a sleeved screened cage. For each test, fresh sets of mosquitoes are used. Five replications for each test were carried out. The efficacy of EOs were determined by the percentage repellency against mosquitoes, using the formula or Eq. (2) described by WHO46.$$% ;{text{repellency}} = frac{C – T}{C} times 100$$
(2)
where, C is the number of mosquitoes landing, or biting at the control area; T is the number of mosquitoes landing or biting at the treated area.Fourier transform-infra red spectroscopy (FT-IR)Study of chemical compatibility for each formulation ingredients are necessary. All formulation ingredients possess specific value of vibrational frequency and have varied functional groups in their chemical structures. For compatibility study, each EOs, excipients to be used in cream formulation, and their physical mixture was placed one by one over the sample plate of the FT-IR instrument (Bruker, ALPHA, Billerica, MA, USA). The covering probe was placed over the sample and IR spectra was obtained over a wavelength of 2.5–25 μm at room temperature. Functional groups possessed by each individual ingredient should be identical in their physical mixture which confirms their compatibility37.Thermogravimetric analysis (TGA)The thermal behaviour of citronella oil, clove oil, lemon grass oil, their mixture and EO-MRC were evaluated using a thermal analyser (TG 209 F1 Libra®, NETZSCH-Gerätebau GmbH, 95100 Selb, Germany). Approximately about 10 mg sample weight was placed in the crucible each time. Nitrogen was used as a shielding gas. Heating program was fixed as 30–600 °C at a rate of 10 °C/min.Formulation development and optimizationFor optimization, a 17-run, 3-factor, 3-level Box-Behnken design (BBD) was utilized. A second order polynomial model was constructed by quadratic response surface methodology (RSM) using Design-Expert software (Version 6.0.8, Stat-Ease Inc., USA). Total seventeen formulations were obtained using EO concentrations as dependent variables against complete protection time (CPT) as independent variable or response variable. Analysis of variance (ANOVA) was performed using the same software to obtain the most effective formulation.Preparation of creamPhase inversion temperature method was applied for the preparation of EO-based mosquito repellent cream (EO-MRC). About 50 g cream sample was prepared in order to get enough for performing the various qualitative and quantitative assay. The oil phase (phase B) was prepared by dissolving the oil soluble excipients, except phase A (mosquito repellent active ingredients) under mild heating at 200 rpm in a hot magnetic plate stirrer (Magnetic Stirrer IKA RCT basic) and heated to 65 °C. The aqueous phase was prepared by mixing various aqueous soluble ingredients (phase C) under gentle heating and stirring. Temperature of the aqueous phase was raised to 65 °C. Phase A was gently added to the oil phase at a stirring speed of 200 rpm and 55 ± 2 °C. The mixture was then emulsified by adding phase C slowly and kept for 1 h at a stirring rate of 800 rpm and 60 ± 2 °C. The formulated EO-MRC was then kept for natural cooling.Efficacy assessmentCPT of the developed cream (EO-MRC) formulation was carried out by arm in cage bioassay. 1 mL EO-MRC was applied to ≈ 600 cm2 area of the forearm skin between the wrist and elbow and 1 mL of the 12% N, N-di ethyl benzamide (DEBA) based marketed cream (DBMC) was compared on the other arm. Two mosquito cages (size: 40 × 40 × 40 cm) each containing 200–250 non-blood-fed female Ae. Albopictus were used. One cage is designated for testing the EO-MRC and the other for the positive control (DBMC). During testing, hands were protected by surgical gloves for which the mosquitoes cannot bite while the volunteer avoids movement of the arm. EO-MRC and DBMC treated arms were exposed for 3 min at 30 min intervals to determine landing and/or probing activity. A single landing or probing of mosquito within a 3 min test interval concludes the test. CPT was calculated as the time (min) required for the first mosquito landing or probing after repellent application to the treated area. The median CPT and confidence intervals were estimated from the Kaplan–Meier Survival Function46.Efficacy was correlated with DEBA based marketed cream (DBMC). The inclusion of the specific commercial product DBMC is for comparison and does not constitute any recommendations.CharacterizationGas chromatography-mass spectroscopy (GC–MS)Qualitative studyDifferent chemical components in fourteen EOs and the selected blend were identified by a GC–MS system of Agilent Technologies (5301 Stevens Creek Blvd. Santa Clara, CA 95051, United States). Test sample concentration of 500 μg/mL was prepared in GC grade acetone. A sample volume of 1 μL was introduced into the injector held at 250 °C. Oven temperature of 40–300 °C was programmed at 20 °C/min. Helium was used as carrier gas at flow rate 1 mL/min. The injector and detector temperature were set at 250 °C and 230 °C (quad) and 150 °C (core) respectively37. Standard C7–C30 saturated alkanes were purchased from Sigma Aldrich Chemicals Co., St. Louis, USA. Retention indices (RI) of the identified components were determined for identification of the detected components.% Assay by GC–MS studyCalibration samples of eugenol and citronellol were prepared by dissolving an appropriate amount in GC grade acetone to get concentrations of 62.5 μg/mL, 125 μg/mL, 250 μg/mL and 500 μg/mL. Test samples of EO-MRC, clove oil and citronella oil were prepared by dissolving a required amount in acetone to quantify the EO components in the final formulation. A sample volume of 1 μL was introduced into the injector as described in ‘Qualitative study’ section.Physicochemical parametersPhysical parameters of the EO-MRC and placebo formulations were determined in order to establish aesthetic compliance and consumer acceptability. To determine the viscosity, a programmable viscometer was used (Model: DV2T, Ametek Brookfield, Middleboro, MA, USA); combined with software Rheo3000, version 1.2.2019.1 [R]. Sample volume was fixed at 30 g and viscosities were determined at 10 rpm for 40 s at room temperature using a T-Bar spindle (B-92) (Helipath spindle set, Brookfield Engineering Labs. Inc). Density was determined by using a pycnometer. pH of EO-MRC was checked by using digital pH meter (Labman Scientific instruments, Tamil Nadu, India).Spread ability of EO-MRC was determined as per the method reported earlier by Sabale65. In brief, 1 g of EO-MRC was placed on 1 cm2 pre-marked circular area on the glass slide (7.5 cm × 2.5 cm). EO-MRC was compressed using another glass slide placed from edge to centre of primary slide. 200 g of commercial weight was placed on the set up and allowed the gel to spread for the period of 1 min. The spread diameter was calculated with the aid of graph paper and spread ability was evaluated using formula expressed as Eq. (3):$$mathrm{Spread, ability}=mathrm{m}times frac{mathrm{l}}{mathrm{t}}$$
(3)
where, m is the commercial weight placed on the setup; l is the length of cream spread; and t is the time.Safety assessmentCytotoxicity by MTT assayThe reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. With the help of spectrophotometric means, the resulting intracellular purple formazan can be quantified. The assay measures the cell proliferation rate and conversely, when metabolic events cause apoptosis or necrosis, the reduction in cell viability66.Cells cultured in T-25 flasks were trypsinized and aspirated into a 5 mL centrifuge tube. Cell pellet was obtained by centrifugation at 3000 rpm. The cell count was adjusted, using DMEM HG medium, such that 200 μL of suspension contained approximately 10,000 cells. To each well of the 96 well microtiter plate, 200 μL of the cell suspension was added and the plate was incubated at 37 ℃ and 5% CO2 atmosphere for 24 h. After 24 h, the spent medium was aspirated. 200 μL of different test concentrations viz. 62 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL, of EO-MRC were added to the respective wells. The plate was then incubated at 37 °C and 5% CO2 atmosphere for 24 h. The plate was removed from the incubator and the drug containing media was aspirated. 200 μL of medium containing10% MTT reagent was then added to each well to get a final concentration of 0.5 mg/mL and the plate was incubated at 37 ℃ and 5% CO2 atmosphere for 3 h. Without disturbing the crystals formed in the wells, culture medium was completely removed. 100 μL of solubilisation solution (DMSO) was added to each well and the plate was then gently shake in a rocking shaker (ROCKYMAX™, Tarsons, Kolkata, India) to solubilize the formed formazan. The absorbance was measured at a wavelength of 570 nm and also at 630 nm using a microplate reader. The percentage growth inhibition was calculated and concentration of EO-MRC needed to inhibit cell growth by 50% (IC50) was generated from the dose–response curve for the cell line.Animals and ethics statementAll experimenting protocols using animal were performed according to the “Principles of Laboratory Animal care” (NIH publication 85–23, revised 1985) and approved by the Institutional Animal Ethical Committee (IAEC) of Defence Research Laboratory (DRL), Tezpur, Assam, India (approval no. CPCSEA/DRL/Protocol no. 3, 20/06/2018). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals67. All efforts were made during the study period to minimize the suffering of animals and to reduce the number of animals used.5–8 weeks old, about 210–250 g of male healthy adult Wistar rats (Rattus norvegicus) and young and healthy New Zealand albino rabbits (Oryctolagus cuniculus) were obtained from the institutional animal housing facility and allowed to acclimatize for 7 days prior to the study. Standard food and purified water ad libitum were provided in clean and hygienic condition at 22–25 ℃, 40–70% RH with 12 h light–dark cycles.Acute dermal irritation studyAcute dermal irritation study was conducted on healthy New Zealand albino rabbits following the OECD test guidelines 40468. Approximately 24 h before the test, fur was removed from the dorsal area of the trunk. 0.5 g EO-MRC, was directly applied to the skin and after 4 h exposure period, residual EO-MRC was removed by using water without disturbing the integrity of the epidermis and examined for signs of erythema and oedema, at 60 min, and then at 24 h, 48 h and 72 h after EO-MRC removal. Dermal reactions are graded and recorded according to the grades in the Table 8. As per the method described by Banerjee et al.69; primary irritation index (PII) was calculated. Further, we have followed the Draize method of classification for PII scoring as non-irritant (if PII  More

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The improved performance of the mangrove LUE model considering coastal environments in this study was mainly attributed to the determination of environmental scalars. Parameters determining environmental stressors (e.g., Topt, Tmin, Tmax, VPDmin, and VPDmax) were set based on the general characteristics of mangroves worldwide. It may not be as accurate for the mangroves in our study sites, but it generally reflects the response of mangroves to environmental changes. Furthermore, as can be seen in Fig. S1, it is applicable to our study sites. Despite the specific characteristics of each mangrove ecosystem at different sites being preferred, this study first offers the possibility to estimate mangrove productivity at a larger scale to track GPP, thus emphasizing the role of mangrove ecosystems nationally or worldwide.The validation results showed that the LUE values of the mangrove model agreed well with those estimated by EC method (Fig. 3) and indicated improved performance (slope = 0.8218–1.0108, intercept = -0.0006–0.0052, R2 = 0.54–0.64, RMSE = 0.0051–0.0068, Pearson’s r = 0.73–1), compared to the MOD17 model (slope = 0.4993–0.5566, intercept = 0.0311–0.0313, R2 = 0.24–0.45, RMSE = 0.0217–0.0220, Pearson’s r = 0.45–0.49). Firstly, the RS-based LUE model for terrestrial ecosystems (MOD17) considers only the environmental stressors of Tair and VPD. The photosynthesis in mangrove forests is influenced by other unique environmental factors caused by tidal inundation. According to Fig. S3, PAR caused the most significant effect on LUE, which is consistent with previous studies14,30,32. The impact of SST has not been quantitatively assessed, however, SST is a critical control that determines the upper latitudinal range of mangrove ecosystems12,33. In our study, the effects of SST and salinity on the mangrove LUE were quantified and helped improve LUE modeling.Secondly, LUEmax was typically defined for different land covers, however, there were no specific values for mangrove forests. In this study, the LUEmax of mangroves was first determined. It is worth noting that daytime NEE responses to PAR vary depending on the Tair23,30,34 so that LUEmax was determined separately at high, optimal, and low temperatures. The results showed that LUEmax reached a maximum when Tair was within the optimal range for mangroves, which represents the high productivity of mangrove ecosystems. Furthermore, the estimated LUEmax of mangrove forests (0.057) was larger than most terrestrial forests35,36,37, which could contribute to the high production and carbon sequestration in mangrove forests.Lastly, the relatively low stomatal conductance of mangroves leads to low LSP compared with terrestrial forests, which could result in the high-irradiance stress for photosynthesis38,39. Mangrove LSP ranges from about 0.2–1.2 mmol/m2/s, depending on the species and environments40,41,42. LUE was relatively low in April and May when seasonal PAR was high, as photosynthesis is more likely to reach saturation. Therefore, we assumed the LUE of mangroves decreased with increasing PAR. In addition, we found that the downscaling effect of PAR on LUE was not constant, but varied with increasing PAR. As follows, different PAR scalars were set for mangroves according to different PAR values. This is a first attempt at refining PARscalar considering different solar radiation, which represents a significant departure from the assumption of a constant downscaling effect of PAR in RS-driven models14,43. The accuracy of the LUE model was improved by refining the PARscalar with different downscaling slopes, especially in periods of high PAR values.Compared with the results obtained from flux-tower measurements, the modeled GPP was basically within the confidence interval of the measured results. The annual averages of GPP in Zhangjiang were 1729 g C/m2/year and 1924 g C/m2/year, in 2012 and 2016, and the annual mean value of GPP in Zhanjiang was 1434 g C/m2/year in 2015. The previous study showed that the GPP in Zhangjiang ranged from 1763 to 1919 g C/m2/year with a mean value of 1871 g C/m2/year32,44,45, which is in good agreement with the estimated values obtained in this study. Liu and Lai46 reported that the GPP of the Mai Po mangrove reserve was 2827 g C/m2/year. Rodda, et al.20 found a GPP value of 1271 g C/m2/year for Sunderbans mangroves in India. Gnanamoorthy, et al.47 estimated a GPP of 2305 g C/m2/year for Pichavaram mangroves. Variations in these estimates across sites were possibly caused by different climate-hydrological conditions, mangrove species, and ages. Differences in the same location may be due to different time scales and different methods of data gap filling and flux partitioning.In a similar way to the GPP model for terrestrial ecosystems48, the effect of the mangrove GPP model on the accuracy of GPP estimates can vary considerably under different environmental conditions. However, in comparison with the accuracy of models built for other vegetation types, the GPP model in this study performed substantially in two sites with RMSE of 2.54–3.41 g C/m2/day. Wang et al.49 adopted different models to estimate GPP for global vegetation and validation results showed the RMSE ranged from 1.79 to 2.33 g C/m2/day. Xiao, et al.50 demonstrated that the deviation between observed and predicted GPP was about 35–282 g C/m2 in an evergreen needleleaf forest. Also, the absolute GPP errors were 7.94–20.92% and 9.97–13.70% for maize cropland and degraded grassland36. Despite the discrepancy, our results were generally consistent with previous studies and were verified by field observations near the flux towers.The comparison of MODIS GPP and EC-estimated GPP showed that the MODIS GPP had a large fluctuation and weakly reflected productivity, being overestimated in 2012 and underestimated in 2015. Different meteorological inputs, different environmental scalars and fraction of absorbed photosynthetic active radiation (fAPAR) products in MODIS GPP and our mangrove GPP model can explain their different results. However, the improvements in our GPP model may help to obtain more accurate GPP estimates. The response of mangrove productivity to Tair has not been well-calibrated in the MODIS GPP product, which may partly account for the poor correlation between the MODIS GPP and EC estimates. Besides, MODIS GPP product was developed based on the International Geosphere-Biosphere Programme (IGBP) land cover map, which doesn’t include mangroves as a specific land cover37. Therefore, LUEmax and environmental parameters were not defined for mangroves, which varied with different environments. This may lead to uncertainty in MODIS GPP product for mangrove forests14. However, the GPP model generated in our study showed similar trends to the field measurements, capturing seasonal variations. The increase in the difference between MODIS GPP and EC estimates may be due to the assumption that the increase in GPP is linear with respect to PAR. In our model, the response of GPP to PAR was suppressed, resulting in seasonal changes in GPP that better match the observations. In addition, the GPP derived from this study was in higher agreement with measured values compared with GPP estimated from the vegetation photosynthesis model (VPM), as shown in Fig. S4. The improvement of this model was more obvious in winter (December to February), which may be due to the environmental stress of SST and PAR. The VPM without considering SSTscalar and PARscalar overestimated GPP in winter. It is indicated that the performance of the mangrove GPP model in this study varied with season. It is recommended to improve the estimation of GPP in the future by considering the seasonal variation of mangrove forests when determining environmental variables.Most studies provide EC-based estimates of GPP that are measurements from a limited footprint. It is possible to extrapolate results across similar vegetation types and geographic settings, but not to areas of heterogeneous vegetation. The RS-based GPP model offers spatial-scale estimates that can be directly incorporated into ecosystem-type models. PAR, SST, and salinity are the key environmental parameters of this RS-based mangrove GPP model. SST and salinity data were derived from the satellite images, while PAR was generated from the reconstructed PAR data, since it is more accurate than the existing RS data and has historical year data. However, PAR products from Hamawari-8, MERIS, and SeaWiFS are available now, which provide an opportunity to obtain large-scale PAR data using RS in the future. In addition to this, GPP of two mangrove forests was assessed and validated with three-year measurements. Validation at different sites and years showed similar results, which indicated the model has similar performance across mangrove forests. Nonetheless, these estimates need to be corroborated with EC databases, which are relatively accurate and provide many additional variables that are currently beyond the scope of higher spatial-resolution RS estimates. The proposed GPP model considering coastal environments was well suited to extend the study area by incorporating RS information and meteorological data. Currently, there are still few mangrove carbon flux towers worldwide. The LUE and GPP models proposed in this study are difficult to validate with measurements from flux towers in other countries. However, local measurements are available in many countries with large mangrove forests, such as Thailand, Vietnam, India, and Bangladesh. Therefore, it is expected that comparisons with measurements from previous studies can be conducted to show the consistency and applicability.The LUE model considering the effects of SST, salinity, and PAR performed well, however, the GPP estimated from the LUE, fAPAR, and PAR showed discrepancies and were generally lower than the measured values. Although the results are better than MODIS GPP products, limitations exist still.Firstly, the effects of salinity and SST on mangrove productivity were directly related to tidal activities. The soil pore water and surface water salinity could affect the osmotic pressure of mangroves especially for the submerged parts which would control the stomatal conductance. In the same way, SST could influence the temperature of mangrove root systems and soil sediments which has impacts on mangrove roots’ respiration and transpiration. Although, theoretically, salinity and SST should be considered as environmental variables affecting mangrove LUE, our results (Fig. S3) indicated that salinity and SST have little influence on mangrove productivity51. To date, the quantitative impact of SST has not been comprehensively unfolded, but it is a global control that determines the upper limit of the latitudinal range of mangroves12,33. The weak relationships between salinity, SST, and mangrove GPP could be due to the uncertainty caused by tidal inundation. Tide duration, tide height, and tide cycle would determine the effect of salinity and SST on the mangrove LUE and GPP. However, quantifying the influence from the tidal cycle remains a challenging task, which could result in the relatively poor performance of Salinityscalar and SSTscalar as shown in Fig. S3. Quantifying the soil temperature and surface water salinity considering the tidal cycle will contribute to model the LUE and GPP of mangrove forests.Secondly, mangroves of different species and ages exhibit diverse structural and physical conditions, resulting in different LUEmax, and optimal growing conditions such as Topt and VPDmin. The environmental settings would also vary from region to region. Liu and Lai46 found that LUE increased slightly with the increasing salinity below 15 ppt (R2 = 0.16). However, it was noted that photosynthetic activity of mangroves would be inhibited when the surface water salinity was high30,51,52,53. Probably, the mutual relationship between LUE and salinity depends on the salinity level and mangrove species. However, we have not specified the variables for different mangrove species, ages and locations which could be improved in the future. Besides, there are multicollinearities between different environmental variables. For example, Tair may have effects on SST and VPD, but as shown in Fig. S5, they are all important for mangrove photosynthesis. However, the correlations between them are not clear and need to be quantified in the future.Thirdly, the relatively low spatial and temporal resolution of the environmental data from RS would influence the accuracy of the model. The datasets have a relatively coarse resolution (usually 500 m–1 km and daily) and are thereby less suitable for smaller nature reserves, especially in the narrow patches of mangrove areas that are rapidly being exploited in coastal China. Moreover, the variability in LUE decreases with increasing temporal scale54. In our study, we determined the PARscalar based on the response of LUE to hourly-scale PAR and found the different down-regulation effects with increasing PAR. However, this phenomenon is not obvious in previous studies. Most RS-based LUE models were developed at a daily or 8-day temporal scale6,50,55,56,57. In terrestrial forests, the light saturated effect caused by increasing PAR was neglectable with coarse temporal scale because the average PAR was usually lower than the LSP. However, as the time scale increases, the effect of light saturation on LUE becomes more pronounced32,58,59. More importantly, this effect is more obvious in mangroves due to their lower LSP18,38, which makes it important in mangrove LUE modeling. The results in Fig. 3 show similar performances of LUE model on hourly and daily scale. Thus, we suggested that our model can be adopted in hourly and daily temporal resolution. However, the PARscalar developed in this study was based on the mangrove forests in one study site which may be influenced by the mangrove species with different LSP and light conditions. What’s more, VPD was on a monthly scale, which cannot reflect environmental dynamics. However, the hourly and daily VPD data are currently not available for coastal areas in China. Therefore, we used monthly averages to represent daily VPD, which may lead to uncertainty in the derived GPP estimates (Figs. 6 and 7). Besides, porewater salinity is controlled by sea surface salinity, precipitation, and river discharge. However, currently, pore water salinity was expressed in terms of sea surface salinity, which may lead to an underestimation of Salinityscalar. More systematic study is necessary to make it more applicable and accurate on a large scale, of which modeling the LUE for different mangrove species and locations is inevitable. However, serving as a fundamental and preliminary step, our study aims to provide a framework for RS-based mangrove GPP modeling. Recently, with the advancement of satellite imagery, hourly-scale RS data for PAR, temperature and SST are available. It can be expected that our current work could be further improved by investigating the light saturation effects in different mangrove forests and adopt higher temporal resolution RS products such as Himawari-8 and GCOM-C in the future.Lastly, the overall underestimation of GPP was mainly caused by the underestimation of fAPAR. Even though the fAPAR computed from Sentinel-2 had higher resolution and accuracy than MODIS fAPAR products, future improvements are still needed. Sentinel-2 fAPAR products (fAPAR-S2) was calculated as the instantaneous fAPAR obtained at 10:00 local solar time which only roughly represented the daily average but was not accurate. Besides, RS-derived fAPAR only considers the absorptions by living green vegetation elements, whereas the ground measured fAPAR refers to the contributions from all absorbing components60. The lower fAPAR-S2 values in mangrove forests may be due to the exposed-to-air root systems which absorb the radiation. Moreover, the spatial distribution of PAR was determined by Co-Kriging interpolation. The elevation was taken as the covariate to estimate spatial PAR. There are many other variables affecting the incoming PAR (e.g., slope and clearness)61. A more comprehensive set of variables needs to be included in the Co-kriging interpolation to improve the PAR estimation.The spatial and seasonal variations of the mangrove GPP were related to environmental changes along the shoreline. The low summer GPP was explained by the lower fAPAR in summer compared with other seasons, which was principally due to the underestimation of fAPAR in summer. Furthermore, PARscalar took a mean value of LSP as 1 mmol/m2/s, however, LSP varied with different species and environmental conditions. In summer, mangroves are more likely to obtain light saturation, and thus PARscalar may lead to an underestimation of LUE and thus GPP. On the contrary, PAR values in winter were relatively low but increased slightly with decreasing latitude. Thus, the inhibitory effect of PAR on LUE was not significant, and GPP increased with decreasing latitude. Salinity and VPD were more stable across years and locations and had no noticeable effect on the mangrove LUE and GPP. The seasonal latitudinal patterns and effects on mangrove productivity were similar for Tair and SST. Tair and SST were lower in winter, especially at high latitudes where mangroves were more sensitive to cold weather. Therefore, the GPP of mangroves at high latitudes in winter was the lowest throughout the year. However, hot weather in summer also limited the photosynthesis in mangroves, especially at low latitudes, where Tair and SST were higher. Nevertheless, there were some correlations among these environmental constants. For example, the Tair affects the vapor pressure and SST. There was a positive correlation between PAR and Tair. The multicollinearity among these variables and the various conditions of mangroves may affect the performance of the model and show variations along the coastline, which would be improved in future studies.Additionally, the GPP of mangroves increased from 2007 to 2018, which was mainly due to the expansion of mangrove forests in the coastal areas. As mangroves grow, canopy size and tree density increase, which may lead to higher LUE and less underestimation of fAPAR, thus contributing to high productivity. However, Zhejiang province (27° 02′ N–31° 11′ N) experienced extremely cold weather in January 2016 caused by the East Asia cold wave62,63, and large areas of mangrove forests died or became sick, leading to a decline in the mangrove GPP at high latitudes in 2018. More

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Evidence for biological and chemical isoprene consumption in coastal seawaterThe time course of isoprene concentration in coastal seawater samples incubated in closed glass bottles at the in situ temperature and in the dark demonstrated sustained loss for at least 45 h (Fig. 1a). Enclosure without headspace prevented isoprene loss by ventilation, and darkness was assumed to arrest all or most of the biological production25 and any photochemical production15 or degradation. Thus, the measured loss was considered the result of microbial degradation and chemical oxidation. In most cases an exponential function fitted better the decay than a linear function (Supplementary Table 1), indicating first-order (concentration-dependent) kinetics for isoprene loss.Fig. 1: Isoprene loss in dark incubations of coastal seawater.a Time course of isoprene concentration in 2 L dark incubations of non-filtered seawater samples from the back-reef lagoon of Mo’orea in April (blue) and the coastal Mediterranean in March (red) and May (green). Filled and open symbols correspond to duplicate incubations. Exponential fits to the data are shown by lines. See Supplementary Table 1 for fit equations and metrics, water temperatures and chlorophyll a concentrations. b Time course of isoprene concentration in series of 30 mL dark incubations of coastal Mediterranean seawater. Dark blue: non-filtered; red: filtered through 0.2 µm; green: filtered + 10 µmol L−1 H2O2; purple: filtered + 0.0025 units mL−1 bromoperoxidase (BrPO); light blue: filtered + H2O2 + BrPO. Exponential fit results in Supplementary Table 2.Full size imageIncubation of microorganism-devoid (filtered through 0.2 µm) coastal seawater sampled next to seaweeds showed an isoprene loss (0.12 d−1) that was half the loss in non-filtered water (0.20 d−1; Fig. 1b and Supplementary Table 2), implying that chemical oxidation accounted for half the total loss. Oxidation by OH·, the fastest amongst isoprene reactions with oxidative transients for which reaction rate data exist19, could account for the observed chemical loss. However, the possibility of oxidation by hitherto overlooked, pervasive oxidants like H2O2 deserved consideration. The addition of unrealistically high concentrations of either H2O2 or the enzyme bromoperoxidase (BrPO), substantially speeded up the chemical loss (0.91 d−1 with 10 µmol H2O2 L−1, 0.31 d−1 with 0.0025 units BrPO mL−1; Fig. 1b and Supplementary Table 2). Isoprene could have reacted with H2O2 in seawater as it does in acidic aerosols26. Besides, should dissolved27 BrPOs from seaweeds or outer-membrane-bound28 BrPOs from phytoplankton occur, they would have reacted with added H2O2 to produce hypobromous acid (HOBr), a strong oxidant29 that would further remove isoprene. Indeed, the addition of BrPO consumed isoprene because it produced HOBr by reaction with the naturally occurring H2O2. Confirming this interpretation, large HOBr production by simultaneous addition of BrPO and H2O2 caused complete isoprene removal in less than 4 h (Fig. 1b). Therefore, the results shown in Fig. 1b indicate that isoprene is reactive to pervasive H2O2 either directly or through the formation of enzymatically derived HOBr. All in all, first-order total isoprene loss (Fig. 1a) is expected to depend on photochemically-produced oxidants30 like H2O2, OH· and 1O2 as well as on microbiota through (a) microbial uptake and catabolism11 and (b) reaction with biologically produced oxidants26,31,32 like HOBr, H2O2 or superoxide.Variability of isoprene loss rate constants in the open oceanTen of the eleven offshore experimental sites were located in the open ocean, and one was located on the Southwestern Atlantic Shelf. Altogether they covered wide ranges of latitude (40°N–61°S), sea surface temperature (−0.8–28.6 °C), daily-averaged wind speed (3–12 m s−1), fluorometric chlorophyll-a (chla) concentration (0.1–5.8 mg m−3), and isoprene concentration (4–104 nmol m−3) (Fig. 2, Table 1 and Supplementary Table 3). Unfiltered seawater samples from the surface ocean were incubated in glass bottles for 24 h, at the in situ temperature and in the dark, and first-order loss rate constants were determined from initial and final isoprene concentrations (see Methods). Note that loss was determined under the assumption that isoprene production was arrested in the dark25. There is published evidence that residual isoprene production may occur in the dark33, but in our incubations, it was insufficient to counteract loss. Thus, isoprene losses caused by processes other than ventilation may have been underestimated.Fig. 2: Geographical distribution of the offshore experiments.Location of the sampling and incubation sites are shown by circles, coloured for isoprene concentration.Full size imageTable 1 Measured biological variables and isoprene process rate constants.Full size tableLoss rate constants (kloss = kbio + kchem) varied over an order of magnitude, ranging 0.03–0.64 d−1 with a median of 0.08 d−1 (Table 1). They did not show any significant relationship to sea surface temperature (SST) (Supplementary Fig. 1) but showed proportionality to the chla concentration (Fig. 3a) that was best described by the following linear regression equation:$${k}_{{{{{{rm{loss}}}}}}}=0.10; (pm 0.01),{{{{{rm{x}}}}}}, [{{{{{rm{chl}}}}}}a]+0.05; (pm 0.01)$$
(1)
Fig. 3: Isoprene processes and their main drivers.a Rate constant of isoprene loss in dark incubations (kloss, considered to be microbial and chemical consumption) vs. chlorophyll-a concentration. The linear regression equation is kloss = 0.10 × [chla] + 0.05 (R2 = 0.96, p = 10−7, n = 11). The standard error of the slope is 0.01 L mg−1 d−1, and the standard error of the intercept is 0.01 d−1. Error bars represent the experimentally determined standard error of kloss. The colour scale of the circles indicates bacterial abundances. b Specific (chla-normalised) rate of isoprene production vs seawater temperature (SST) across the sample series. The dashed line is the general smoothed trend. The blue line is the exponential adjustment at SST , 1000)$$
(2)
Substitution in Eq. (1) results in:$${k}_{{{{{{rm{loss}}}}}}}=0.14,{{{{{rm{x}}}}}}, {[{{{{{rm{chl}}}}}}{a}_{{{{{{rm{sat}}}}}}}]}^{1.28}+0.05$$
(3)
which is our recommended equation for kloss prediction from satellite chla. Note that only the variable term (kbio) changes from Eq. (1), while the intercept (kchem) is maintained at 0.05 d−1.Comparison of isoprene sinks and total turnover timeThe change of isoprene concentration ([iso]) in the surface mixed layer over time can be described as the budget of sources and sinks:$$varDelta [{{{{{rm{iso}}}}}}]/varDelta {{{{{rm{t}}}}}}=[{{{{{rm{iso}}}}}}]cdot ({k}_{{{{{{rm{prod}}}}}}} – {k}_{{{{{{rm{loss}}}}}}} – {k}_{{{{{{rm{vent}}}}}}} – {k}_{{{{{{rm{mix}}}}}}})$$
(4)
where kprod, kvent and kmix are the rate constants of isoprene production, ventilation to the atmosphere and vertical downward mixing by turbulent diffusion, respectively.We calculated kvent from our sampling sites over a period of 24 h (Table 1). Ventilation has been considered the main isoprene sink from the upper mixed layer of the ocean18. In our sampling sites, kloss was 0.4 to 10 times the kvent (median factor: 1.2). That is, loss through microbial + chemical consumption was of the same order as ventilation, sometimes considerably faster. Vertical mixing, kmix, was estimated to be one order of magnitude lower than the other process rates (Table 1), and in all cases but one it was calculated or assumed not to be a loss term but an import term into the mixed layer, because vertical profiles generally show maximum isoprene concentrations below the mixed layer and turbulent diffusion causes upward transport14,17. Altogether, the microbial, chemical, ventilation, and, where relevant, mixing losses resulted in total turnover times (1/(kloss + kvent + kmix)) of isoprene between 1.4 and 16 days, median 5 days (Table 1).Isoprene productionAssuming steady-state for isoprene concentrations over 24 h (Supplementary Fig. 2), i.e. Δ[iso]/Δt = 0 in Eq. (4), the sum of the daily rate constants of all sinks (kloss + kvent) equals the rate constant of isoprene production (kprod), with kmix adding to either side depending on whether it is an import to or an export from the mixed layer (Table 1). Note that kprod was the highest coinciding with higher [chla]. This is consistent with a recent study44 where measurement of the net biological isoprene production (i.e. production — consumption rates) across seasons in the open ocean was attempted; net production rates increased in May, coinciding with a large increase in [chla] and phytoplankton cell abundance.The product of kprod by the isoprene concentration gives the daily isoprene production rate, which can be normalised by dividing it by the chla concentration. In our study, this specific isoprene production rate varied between 1 and 38 nmol (mg chla)−1 d−1 (Table 1), median 8 nmol (mg chla)−1 d−1. These values are within the broad range reported across phytoplankton taxa from laboratory studies with monocultures41,45 (0.3–32, median 3 nmol (mg chla)−1 d−1, n = 124). Five of the eleven sites gave values >13 nmol (mg chla)−1 d−1, i.e. in the higher end of the laboratory data range. This is not unexpected, since measurements in monoculture experiments are typically conducted before reaching nutrient limitation, below light saturation and in the absence of UV radiation, to mention three stressors commonly occurring in the surface open ocean. If isoprene biosynthesis and release is enhanced by any of these stressors, as is the case in vascular plants7,10, then monoculture-derived results will easily render underestimates of isoprene production in the open ocean. Production by heterotrophic bacteria46 could have also contributed to increase apparent specific isoprene production rates, but the occurrence and importance of this process in the marine environment is unknown.When plotted against the SST, which was also the temperature of the incubations, specific isoprene production rates increased exponentially between −0.8 and 23 °C and dropped drastically at higher SST (Fig. 3b). Several studies with phytoplankton monocultures have reported positive dependence of specific isoprene production rates on temperature45,47,48,49,50. One of these studies45 described that the increase with temperature reaches an optimum for production that varies among phytoplankton strains and with light intensity, but falls around 23–26 °C. The most detailed study47 was conducted with a Prochlorococcus strain; remarkably, the shape of the specific production rate vs. temperature curve for this cyanobacterium strain was almost identical to that of Fig. 3b, with an exponential increase until 23 °C and a drop thereafter. This is the canonical curve type of enzymatic activities, but the thermal behaviour of the enzymes for isoprene synthesis in marine unicellular algae has not yet been characterised12.Revising the magnitude and players of the marine isoprene cycleOur results allow redrawing the isoprene cycle in the surface mixed layer of the ocean. Figure 4 sketches the magnitude of the rate constants for production and sinks presented in Table 1, averaged according to a chla concentration threshold: the blue and green arrows correspond to the experiments in waters with [chla] lower and higher than 0.4 mg m−3, respectively. Isoprene production in productive (chla-richer) waters is faster than in oligotrophic (chla-poorer) waters. Vertical mixing is assumed to majorly constitute an input into the mixed layer, yet very small. Photochemical production and emission from surfactants15 in the surface microlayer of productive waters is depicted as uncertain. Among sinks, the microbiota-dependent consumption is much faster in productive waters; actually, the statistical uncertainty of Eq. (1) and the uneven distribution of incubation results along the [chla] axis hamper resolving kbio in phytoplankton-poor waters ( More