Ecology

Neo, M. L., Eckman, W., Vicentuan, K., Teo, S.L.-M. & Todd, P. A. The ecological significance of giant clams in coral reef ecosystems. Biol. Conserv. 181, 111–123 (2015).Article 

Google Scholar 
Hill, R. W. et al. Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea. Biol. Lett. 14, 20180047 (2018).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Welsh, K., Elliot, M., Tudhope, A., Ayling, B. & Chappell, J. Giant bivalves (Tridacna gigas) as recorders of ENSO variability. Earth Planet. Sci. Lett. 307, 266–270 (2011).ADS 
CAS 
Article 

Google Scholar 
Elliot, M. et al. Profiles of trace elements and stable isotopes derived from giant long-lived Tridacna gigas bivalves: Potential applications in paleoclimate studies. Palaeogeogr. Palaeoclimatol. Palaeoecol. 280, 132–142 (2009).Article 

Google Scholar 
Killam, D., Thomas, R., Al-Najjar, T. & Clapham, M. Interspecific and intrashell stable isotope variation among the Red Sea giant clams. Geochem. Geophys. Geosyst. https://doi.org/10.1029/2019GC008669 (2020).Article 

Google Scholar 
Duprey, N., Galipaud, J.-C., Cabioch, G. & Lazareth, C. E. Isotopic records from archeological giant clams reveal a variable climate during the southwestern Pacific colonization ca. 3.0ka BP. Palaeogeogr. Palaeoclimatol. Palaeoecol. 404, 97–108 (2014).Article 

Google Scholar 
Batenburg, S. J. et al. Interannual climate variability in the Miocene: High resolution trace element and stable isotope ratios in giant clams. Palaeogeogr. Palaeoclimatol. Palaeoecol. 306, 75–81 (2011).Article 

Google Scholar 
Ayling, B. F., Chappell, J., Gagan, M. K. & McCulloch, M. T. ENSO variability during MIS 11 (424–374 ka) from Tridacna gigas at Huon Peninsula, Papua New Guinea. Earth Planet. Sci. Lett. 431, 236–246 (2015).ADS 
CAS 
Article 

Google Scholar 
Yan, H., Shao, D., Wang, Y. & Sun, L. Sr/Ca profile of long-lived Tridacna gigas bivalves from South China Sea: A new high-resolution SST proxy. Geochim. Cosmochim. Acta 112, 52–65 (2013).ADS 
CAS 
Article 

Google Scholar 
Warter, V. & Müller, W. Daily growth and tidal rhythms in Miocene and modern giant clams revealed via ultra-high resolution LA-ICPMS analysis—A novel methodological approach towards improved sclerochemistry. Palaeogeogr. Palaeoclimatol. Palaeoecol. 465, 362–375 (2017).Article 

Google Scholar 
Warter, V., Erez, J. & Müller, W. Environmental and physiological controls on daily trace element incorporation in Tridacna crocea from combined laboratory culturing and ultra-high resolution LA-ICP-MS analysis. Palaeogeogr. Palaeoclimatol. Palaeoecol. 496, 32–47 (2018).Article 

Google Scholar 
Wei, G., Sun, M., Li, X. & Nie, B. Mg/Ca, Sr/Ca and U/Ca ratios of a Porites coral from Sanya Bay, Hainan Island, South China Sea and their relationships to sea surface temperature. Palaeogeogr. Palaeoclimatol. Palaeoecol. 162, 59–74 (2000).Article 

Google Scholar 
Brahmi, C. et al. Effects of elevated temperature and pCO2 on the respiration, biomineralization and photophysiology of the giant clam Tridacna maxima. Conserv. Physiol. 9, 041 (2021).Article 
CAS 

Google Scholar 
Watson, S.-A. & Neo, M. L. Conserving threatened species during rapid environmental change: Using biological responses to inform management strategies of giant clams. Conserv. Physiol. 9, 082 (2021).
Google Scholar 
Armstrong, E. J., Dubousquet, V., Mills, S. C. & Stillman, J. H. Elevated temperature, but not acidification, reduces fertilization success in the small giant clam, Tridacna maxima. Mar. Biol. 167, 8 (2020).CAS 
Article 

Google Scholar 
Leggat, W., Buck, B. H., Grice, A. & Yellowlees, D. The impact of bleaching on the metabolic contribution of dinoflagellate symbionts to their giant clam host. Plant Cell Environ. 26, 1951–1961 (2003).CAS 
Article 

Google Scholar 
Zhou, Z., Liu, Z., Wang, L., Luo, J. & Li, H. Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature. Fish Shellfish Immunol. 84, 451–457 (2019).CAS 
PubMed 
Article 

Google Scholar 
Dubousquet, V. et al. Changes in fatty acid composition in the giant clam Tridacna maxima in response to thermal stress. Biol. Open 5, 1400–1407 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Blidberg, E., Elfwing, T., Plantman, P. & Tedengren, M. Water temperature influences on physiological behaviour in three species of giant clams (Tridacnidae). In Proc. 9th International Coral Reef Symposium 561–565 (2000).Junchompoo, C., Sinrapasan, N., Penpain, C. & Patsorn, P. Changing seawater temperature effects on giant clams bleaching, Mannai Island, Rayong Province, Thailand. In Proc. Design Symposium on Conservation of Ecosystem. https://doi.org/10.13140/2.1.1906.5600 (2012).Watson, S.-A., Southgate, P. C., Miller, G. M., Moorhead, J. A. & Knauer, J. Ocean acidification and warming reduce juvenile survival of the fluted giant clam, Tridacna squamosa. Molluscan Res. 32, 177–180 (2012).
Google Scholar 
Watson, S.-A. Giant clams and rising CO2: Light may ameliorate effects of ocean acidification on a solar-powered animal. PLoS ONE 10, 1–18 (2015).CAS 

Google Scholar 
Kurihara, H. & Shikota, T. Impact of increased seawater pCO2 on the host and symbiotic algae of juvenile giant clam Tridacna crocea. Galaxea J. Coral Reef Stud. 20, 19–28 (2018).Article 

Google Scholar 
Alves Monteiro, H. J. et al. Molecular mechanisms of acclimation to long-term elevated temperature exposure in marine symbioses. Glob. Change Biol. 26, 1271–1284 (2020).ADS 
Article 

Google Scholar 
Collins, M. et al. Long-term climate change: Projections, commitments and irreversibility. In Climate Change 2013—The Physical Science Basis (eds Stocker, T. F. et al.) 1029–1136 (Cambridge University Press, 2013).
Google Scholar 
Poloczanska, E. et al. Climate change and Australian marine life. Oceanogr. Mar. Biol. 45, 407 (2007).
Google Scholar 
Ganachaud, A. S. et al. Observed and expected changes to the tropical Pacific Ocean. In Vulnerability Trop. Pac. Fish. Aquac. Clim. Change Secr. Pac. Community Noumea New Caledonia 101–187 (2011).Doney, S. C., Fabry, V. J., Feely, R. A. & Kleypas, J. A. Ocean acidification: The other CO2 problem. Annu. Rev. Mar. Sci. 1, 169–192 (2009).ADS 
Article 

Google Scholar 
Meinshausen, M. et al. The RCP greenhouse gas concentrations and their extensions from 1765 to 2300. Clim. Change 109, 213–241 (2011).ADS 
CAS 
Article 

Google Scholar 
Pierrot, D., Lewis, E. & Wallace, D. MS Excel program developed for CO2 system calculations. In ORNLCDIAC-105a Carbon Dioxide Inf. Anal. Cent. Oak Ridge Natl. Lab. US Dep. Energy Oak Ridge Tenn. Vol. 10 (2006).Mehrbach, C., Culberson, C. H., Hawley, J. E. & Pytkowicx, R. M. Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol. Oceanogr. 18, 897–907 (1973).ADS 
CAS 
Article 

Google Scholar 
Dickson, A. G. & Millero, F. J. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 34, 1733–1743 (1987).ADS 
CAS 
Article 

Google Scholar 
Dickson, A. G. Standard potential of the reaction: AgCl (s) + 12H2 (g) = Ag (s) + HCl (aq), and the standard acidity constant of the ion HSO4− in synthetic sea water from 273.15 to 318.15 K. J. Chem. Thermodyn. 22, 113–127 (1990).CAS 
Article 

Google Scholar 
Wolf, R. E. & Adams, M. Multi-elemental Analysis of Aqueous Geochemical Samples by Quadrupole Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) 38. http://pubs.er.usgs.gov/publication/ofr20151010, https://doi.org/10.3133/ofr20151010 (2015).Schrag, D. P. Rapid analysis of high-precision Sr/Ca ratios in corals and other marine carbonates. Paleoceanography 14, 97–102 (1999).ADS 
Article 

Google Scholar 
Howell, D. C. Permutation Tests for Factorial ANOVA Designs (2009).Fox, J. & Weisberg, S. An R Companion to Applied Regression (Sage, 2019).
Google Scholar 
R Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, 2020).Lenth, R. emmeans: Estimated Marginal Means, aka Least-Squares Means (2020).Navarro, D. Learning Statistics with R: A Tutorial for Psychology Students and other beginners (Version 0.5) (University of Adelaide, 2015).
Google Scholar 
Zhao, L., Schöne, B. R. & Mertz-Kraus, R. Controls on strontium and barium incorporation into freshwater bivalve shells (Corbicula fluminea). Palaeogeogr. Palaeoclimatol. Palaeoecol. 465, 386–394 (2017).Article 

Google Scholar 
Bragg, W. L. The structure of some crystals as indicated by their diffraction of X-rays. Proc. R. Soc. Lond. Ser. Contain. Pap. Math. Phys. Character 89, 248–277 (1913).ADS 
CAS 

Google Scholar 
Bragg, W. L. The structure of aragonite. Proc. R. Soc. Lond. Ser. Contain. Pap. Math. Phys. Character 105, 16–39 (1924).ADS 
CAS 

Google Scholar 
Killam, D., Al-Najjar, T. & Clapham, M. Giant clam growth in the Gulf of Aqaba is accelerated compared to fossil populations. Proc. R. Soc. B Biol. Sci. 288, 20210991 (2021).CAS 
Article 

Google Scholar 
Waters, C. G. Biological Responses of Juvenile Tridacna maxima (Mollusca: Bivalvia) to Increased pCO2 and Ocean Acidification (The Evergreen State College, 2008).
Google Scholar 
Toonen, R. J., Nakayama, T., Ogawa, T., Rossiter, A. & Delbeek, J. C. Growth of cultured giant clams (Tridacna spp.) in low pH, high-nutrient seawater: Species-specific effects of substrate and supplemental feeding under acidification. J. Mar. Biol. Assoc. U. K. 92, 731–740 (2012).CAS 
Article 

Google Scholar 
Hart, A. M., Bell, J. D. & Foyle, T. P. Growth and survival of the giant clams, Tridacna derasa, T. maxima and T. crocea, at village farms in the Solomon Islands. Aquaculture 165, 203–220 (1998).Article 

Google Scholar 
Van Wynsberge, S. et al. Growth, survival and reproduction of the giant clam Tridacna maxima (Röding 1798, Bivalvia) in two contrasting lagoons in French Polynesia. PLoS ONE 12, 1–20 (2017).
Google Scholar 
Lucas, J. S., Nash, W. J., Crawford, C. M. & Braley, R. D. Environmental influences on growth and survival during the ocean-nursery rearing of giant clams, Tridacna gigas (L.). Aquaculture 80, 45–61 (1989).Article 

Google Scholar 
Schwartzmann, C. et al. In situ giant clam growth rate behavior in relation to temperature: A one-year coupled study of high-frequency noninvasive valvometry and sclerochronology. Limnol. Oceanogr. 56, 1940–1951 (2011).ADS 
Article 

Google Scholar 
Syazili, A., Syafiuddin, N. A. & Jompa, J. Effect of ocean acidification and temperature on growth, survival, and shell performance of fluted giant clams (Tridacna squamosa). IOP Conf. Ser. Earth Environ. Sci. 473, 012141 (2020).Article 

Google Scholar 
Li, J. et al. Assessment of the juvenile vulnerability of symbiont-bearing giant clams to ocean acidification. Sci. Total Environ. 812, 152265 (2022).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Li, S. et al. Cloning and expression of a pivotal calcium metabolism regulator: Calmodulin involved in shell formation from pearl oyster (Pinctada fucata). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 138, 235–243 (2004).PubMed 
Article 
CAS 

Google Scholar 
Wang, X., Li, C., Lv, Z., Zhang, Z. & Qiu, L. A calcification-related calmodulin-like protein in the oyster Crassostrea gigas mediates the enhanced calcium deposition induced by CO2 exposure. Sci. Total Environ. 833, 155114 (2022).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Rees, T., Fitt, W. & Yellowlees, D. The haemolymph and its temporal relationship with zooxanthellae metabolism in the giant clam symbiosis [Conference paper]. In ACIAR Proc.-Aust. Cent. Int. Agric. Res. Aust. (1993).Leggat, W., Rees, T. A. V. & Yellowlees, D. Meeting the photosynthetic demand for inorganic carbon in an alga-invertebrate association: Preferential use of CO2 by symbionts in the giant clam Tridacna gigas. Proc. Biol. Sci. 267, 523–529 (2000).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Ip, Y. K. et al. Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and. Gene 659, 137–148 (2018).CAS 
PubMed 
Article 

Google Scholar 
Armstrong, E. J., Roa, J. N., Stillman, J. H. & Tresguerres, M. Symbiont photosynthesis in giant clams is promoted by V-type H+-ATPase from host cells. J. Exp. Biol. https://doi.org/10.1242/jeb.177220 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
Sano, Y. et al. Past daily light cycle recorded in the strontium/calcium ratios of giant clam shells. Nat. Commun. 3, 761 (2012).ADS 
PubMed 
Article 
CAS 

Google Scholar 
Adams, A. L., Needham, E. W. & Knauer, J. The effect of shade on water quality parameters and survival and growth of juvenile fluted giant clams, Tridacna squamosa, cultured in a land-based growth trial. Aquac. Int. 21, 1311–1324 (2013).CAS 
Article 

Google Scholar 
Rossbach, S., Saderne, V., Anton, A. & Duarte, C. M. Light-dependent calcification in Red Sea giant clam Tridacna maxima. Biogeosciences 16, 2635–2650 (2019).ADS 
CAS 
Article 

Google Scholar 
Ip, Y. K. et al. The whitish inner mantle of the giant clam, Tridacna squamosa, expresses an apical plasma membrane Ca2+-ATPase (PMCA) which displays light-dependent gene and protein expressions. Front. Physiol. 8, 781 (2017).PubMed 
PubMed Central 
Article 

Google Scholar 
Berner, R. A. The role of magnesium in the crystal growth of calcite and aragonite from sea water. Geochim. Cosmochim. Acta 39, 489–504 (1975).ADS 
CAS 
Article 

Google Scholar 
Alibert, C. et al. Source of trace element variability in Great Barrier Reef corals affected by the Burdekin flood plumes. Geochim. Cosmochim. Acta 67, 231–246 (2003).ADS 
CAS 
Article 

Google Scholar 
McCulloch, M. et al. Coral record of increased sediment flux to the inner Great Barrier Reef since European settlement. Nature 421, 727–730 (2003).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Sinclair, D. & Mcculloch, M. Corals record low mobile barium concentrations in the Burdekin River during the 1974 flood: Evidence for limited Ba supply to rivers? Palaeogeogr. Palaeoclimatol. Palaeoecol. 214, 155–174 (2004).Article 

Google Scholar 
Fleitmann, D. et al. East African soil erosion recorded in a 300 year old coral colony from Kenya. Geophys. Res. Lett. 34, L04401 (2007).ADS 
Article 

Google Scholar 
Prouty, N. G., Field, M. E., Stock, J. D., Jupiter, S. D. & McCulloch, M. Coral Ba/Ca records of sediment input to the fringing reef of the southshore of Moloka’i, Hawai’i over the last several decades. Mar. Pollut. Bull. 60, 1822–1835 (2010).CAS 
PubMed 
Article 

Google Scholar 
Fallon, S. J., McCulloch, M. T., van Woesik, R. & Sinclair, D. J. Corals at their latitudinal limits: Laser ablation trace element systematics in Porites from Shirigai Bay, Japan. Earth Planet. Sci. Lett. 172, 221–238 (1999).ADS 
CAS 
Article 

Google Scholar 
Reuer, M. K., Boyle, E. A. & Cole, J. E. A mid-twentieth century reduction in tropical upwelling inferred from coralline trace element proxies. Earth Planet. Sci. Lett. 210, 437–452 (2003).ADS 
CAS 
Article 

Google Scholar 
Montaggioni, L. F., Le Cornec, F., Corrège, T. & Cabioch, G. Coral barium/calcium record of mid-Holocene upwelling activity in New Caledonia, South-West Pacific. Palaeogeogr. Palaeoclimatol. Palaeoecol. 237, 436–455 (2006).Article 

Google Scholar 
Ourbak, T. et al. A high-resolution investigation of temperature, salinity, and upwelling activity proxies in corals: Activity proxies in corals. Geochem. Geophys. Geosyst. 7, 1. https://doi.org/10.1029/2005GC001064 (2006).CAS 
Article 

Google Scholar 
Alibert, C. & Kinsley, L. A 170-year Sr/Ca and Ba/Ca coral record from the western Pacific warm pool: 1. What can we learn from an unusual coral record? J. Geophys. Res. 113, C04008 (2008).ADS 

Google Scholar 
Alibert, C. & Kinsley, L. A 170-year Sr/Ca and Ba/Ca coral record from the western Pacific warm pool: 2. A window into variability of the new ireland coastal undercurrent. J. Geophys. Res. 113, C06006 (2008).ADS 

Google Scholar 
Agbaje, O. B. A. et al. Architecture of crossed-lamellar bivalve shells: The southern giant clam (Tridacna derasa, Röding, 1798). R. Soc. Open Sci. 4, 170622 (2017).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Clark, M. S. et al. Deciphering mollusc shell production: The roles of genetic mechanisms through to ecology, aquaculture and biomimetics. Biol. Rev. https://doi.org/10.1111/brv.12640 (2020).Article 
PubMed 

Google Scholar 
Wilkerson, F. P. & Trench, R. K. Uptake of dissolved inorganic nitrogen by the symbiotic clam Tridacna gigas and the coral Acropora sp.. Mar. Biol. 93, 237–246 (1986).CAS 
Article 

Google Scholar 
Summons, R. E., Boag, T. S. & Osmond, C. B. The effect of ammonium on photosynthesis and the pathway of ammonium assimilation in Gymnodinium microadriaticum in vitro and in symbiosis with tridacnid clams and corals. Proc. R. Soc. Lond. B Biol. Sci. 227, 147–159 (1986).ADS 
CAS 
Article 

Google Scholar 
Onate, J. & Naguit, M. A preliminary study on the effect of increased nitrate concentration on the growth of giant clams Hippopus hippopus. In Cult. Giant Clams Bivalvia Tridacnidae Aust. Cent. Int. Agric. Res. Canberra 57–61 (1989).Hastie, L. C., Watson, T. C., Isamu, T. & Heslinga, G. A. Effect of nutrient enrichment on Tridacna derasa seed: Dissolved inorganic nitrogen increases growth rate. Aquaculture 106, 41–49 (1992).CAS 
Article 

Google Scholar 
Belda, C. A., Lucas, J. S. & Yellowlees, D. Nutrient limitation in the giant clam-zooxanthellae symbiosis: Effects of nutrient supplements on growth of the symbiotic partners. Mar. Biol. 117, 655–664 (1993).Article 

Google Scholar 
Belda-Baillie, C., Leggat, W. & Yellowlees, D. Growth and metabolic responses of the giant clam-zooxanthellae symbiosis in a reef-fertilisation experiment. Mar. Ecol. Prog. Ser. 170, 131–141 (1998).ADS 
CAS 
Article 

Google Scholar 
Calosi, P. et al. Multiple physiological responses to multiple environmental challenges: An individual approach. Integr. Comp. Biol. 53, 660–670 (2013).CAS 
PubMed 
Article 

Google Scholar 
Tanner, R. L. & Dowd, W. W. Inter-individual physiological variation in responses to environmental variation and environmental change: Integrating across traits and time. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 238, 110577 (2019).CAS 
PubMed 
Article 

Google Scholar 
Guscelli, E., Spicer, J. I. & Calosi, P. The importance of inter-individual variation in predicting species’ responses to global change drivers. Ecol. Evol. 9, 4327–4339 (2019).PubMed 
PubMed Central 
Article 

Google Scholar 
Telesca, L. et al. Biomineralization plasticity and environmental heterogeneity predict geographical resilience patterns of foundation species to future change. Glob. Change Biol. 25, 4179–4193 (2019).ADS 
Article 

Google Scholar 
Yan, H., Shao, D., Wang, Y. & Sun, L. Sr/Ca differences within and among three Tridacnidae species from the South China Sea: Implication for paleoclimate reconstruction. Chem. Geol. 390, 22–31 (2014).ADS 
CAS 
Article 

Google Scholar 
Warter, V., Mueller, W., Wesselingh, F. P., Todd, J. A. & Renema, W. Late Miocene seasonal to subdecadal climate variability in the Indo-West Pacific (East Kalimantan, Indonesia) preserved in giant clams. Palaios 30, 66–82 (2015).ADS 
Article 

Google Scholar 
Gannon, M. E., Pérez-Huerta, A., Aharon, P. & Street, S. C. A biomineralization study of the Indo-Pacific giant clam Tridacna gigas. Coral Reefs 36, 503–517 (2017).ADS 
Article 

Google Scholar 
Zhao, L. et al. A review of transgenerational effects of ocean acidification on marine bivalves and their implications for sclerochronology. Estuar. Coast. Shelf Sci. 235, 106620 (2020).CAS 
Article 

Google Scholar  More

Sample collection from sea turtlesIn total, 45 serum samples from 33 juvenile green turtles (C. mydas), including 6 sea turtles with tumors, 5 juvenile hawksbill turtles (Eretmochelys imbricate), and 7 olive ridley turtles (Lepidochelys olivacea) (juvenile = 5; sub-adult = 2). All turtles were sourced from: eastern Taiwan (n = 24), southern Taiwan (n = 14), central Taiwan (n = 6), and northern Taiwan (n = 1). Among the 45 sea turtle samples, 6 green turtles developed FP (n = 1 with tumor score 1; n = 1 with tumor score 2; n = 4 with tumor score 3)32, while 39 did not have FP. FP tumor tissues were collected from 6 green turtles (from shoulder/flippers/inguinal regions) with FP during surgical procedures. Regarding the collection of normal skin tissues, one normal skin tissue (from shoulder) was collected from one necropsied dead green turtles (stranding and discovered from southern Taiwan) confirmed without FP. All tissue samples were fixed in 10% neutral buffered formalin prior to further analysis. In this study, all sea turtles were discovered and rescued through the official reporting system of the Marine Animal Rescue Network (established by the Ocean Conservation Administration) and admitted to the National Museum of Marine Biology and Aquarium (NMMBA), between 2017 and 2020.Detection of ChHV5 DNA by polymerase chain reaction (PCR)Total DNA was extracted from blood of 45 sea turtles by DNeasy blood & tissue kit (Cat. No. 69504, Qiagen, Valencia, CA, USA) following manufacturer’s instructions. Subsequently, the ChHV5 infection status all 45 sea turtles was determined by PCR using primers targeting on UL18 (capsid protein gene), UL22 (glycoprotein H gene), and UL27 (glycoprotein B gene) regions4. The sequence of primer sets are: UL18F: 5′-CACCACGAGGGGGAAAATGA, UL18R:5′-TCAAATCCCCCGTTCACTCG; UL22F: 5′-ACGGCGTTGGCTAGTGAATC, UL22R: 5′-GCAGTTCGGTACACACCTCT; UL27F: 5′-TAACAAGAAAGAACCGCGCG; UL27R: 5′-ATTTTCCCGGTCAGTGCCAA. PCR amplifications were performed in a total volume of 50 μl. The reaction included 1 μl of the template DNA, 1 μl of each primer (10 μM), 22 μl of distilled water (DDW), and 25 μl of the AmpliTaq Gold® 360 Master Mix (Cat. No. 4398876, Life Technologies, Valencia, CA, USA). The thermocycle for amplification was: Initial denaturing at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, and then a final extension at 72 °C for 7 min. Results were visualized by gel electrophoresis (2% agarose) with SYBR Safe DNA Gel Stain (Cat. No. S33102, Invitrogen, Carlsbad, CA, USA).Sequence optimization of the UL27 gene for expression of the ChHV5 glycoprotein protein using E. coli
To express large quantities of ChHV5 gB, we adopted the prokaryotic Escherichia coli (E. coli) expression system. The construct (namely UL27/pUC57) containing sequences of the full length UL27 fused with FLAG tag sequence (GenBank accession no. AF035003.3) was synthesized by Allbio Science Co., Ltd, Taiwan. The sequence information of the glycoprotein (gB) datasets used and analyzed for protein expression during the current study was obtained and available from the GenBank repository [https://www.ncbi.nlm.nih.gov/nuccore/AF035003.3]. Considering the difference in tRNA-codon usages between prokaryotes and eukaryotes would possibly affect subsequent protein expression, the optimized UL27 gene sequence, without altering the translated amino acid sequences, to fit the E. coli expression system was synthesized. The codon optimized UL27 gene was further used as the template for amplification of different gene fragments by Polymerase Chain Reaction (PCR).Construction of plasmids expressing partial fragments of ChHV5 gB proteinTo determine the relative antigenicity and also to increase the expression yield, plasmids expressing various regions of gB protein were constructed. Briefly, the five regions covering different fragments of the UL27 gene were amplified from plasmid UL27/pUC57 by PCR using specific primer sets with built-in restriction enzyme sequences shown as underlined in Table 1. The thermal cycling conditions were: 98 °C (5 min) followed by 35 cycles of denaturation (98 °C, 30 s), annealing (58 °C, 1 min), and extension (72 °C, 2 min), and finished with a final extension (72 °C, 10 min). PCR amplicons with expected sizes were isolated from gel and trimmed with the restriction enzymes followed by ligation with vectors either pET24a or pET32b (Novagen, Germany) linearized with the same restriction enzymes. The resulting plasmids with expected insert sizes as confirmed by restriction enzymes were sent for automated DNA sequencing (Mission Biotech, Taipei, Taiwan).Table 1 Information on the constructs expressing the UL27 fragments. The bold characters indicate sequences recognized by restriction enzymes for the ease of further cloning procedure.Full size tableExpression of recombinant gB fragments in E. coli
In the current study, the recombinant gB protein is a key reagent that served as antigen for seroprevalence of ChHV5 as well as for the generation of ChHV5 gB antibody (conducted by Yao-Hong Biotech Inc., Taiwan). The plasmids expressing individual gB fragment were transformed into E. coli host cells, strain BL21 (DE3), Rosetta. Expression of all the recombinant gB fragments was induced by 0.8 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 28 °C for 16 h. As all the gB fragments cloned into the pET series vectors were expressed as a fusion protein with a 6-histidine tag at C-terminus end, they could be further purified by Ni–NTA column chromatography using the chelating Sepharose Fast Flow (GE Healthcare) following the method described in one previous study33. The yield and purity of recombinant gB proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, 6 M urea and 0.4 M imidazole contained in the purified protein were depleted by step-wise dialysis against 1 × PBS buffer (0.02 M phosphate, 0.15 M NaCl) with gradually decreased concentrations of urea at 4 °C. The concentration of recombinant proteins were then estimated by National Institutes of Health ImageJ software (https://imagej.nih.gov/ij/, 1997–2018.) using the standard curve established by bovine serum albumin (BSA) with known concentrations42.Western blot analysisRecombinant gB fragments were separated by 12.5% or 15% SDS-PAGE and electrotransferred to PVDF membrane by using Mini Proten III apparatus (Cat. No. 165-3301, BioRad). The filters were blocked in PBS-T buffer (0.02 M phosphate, 0.15 M NaCl, 0.05% Tween-20) containing 5% skimmed milk and reacted with mouse anti-his tag antibody (1:5,000, Cat. No. GTX40628, GeneTex) at 4 °C for overnight. After six-time wash with PBS-T buffer, the PVDF filter was then incubated with the secondary antibody, 1:5000 diluted goat anti-mouse IgG conjugated with horseradish peroxidase (HRP), or 1:500 diluted Protein A/G-HRP (Cat. No. 32400, Thermo fisher scientific™, United States) for sea turtle antibody detection, at room temperature for 1 h followed by PBS-T wash to remove the unbound antibodies. Ultimately, the signal was detected by ECL reagents (Thermo Fisher Scientific, United States) and the image was acquired by ImageQuant LAS 4000 Mini (GE Healthcare).Immunohistochemical (IHC) analysisTo establish IHC protocol, normal skin tissue from PCR-negative sea turtles served as the negative control. In total, the FP on skin tissue from six individual sea turtles that were detected positive for ChHV5 DNA (positive tissue samples), and one normal tissue detected negative (the negative tissue) were included in the IHC analysis.IHC procedure was conducted as reported in our previous study34. In brief, sections of formalin-fixed and wax-embedded skin tissues of sea turtles were made using a rotary microtome (Leica RM2245, Leica Biosystems, Germany) and were further deparaffinized and rehydrated. Antigen retrieval was carried out by heat-induced epitope retrieval method: slides immersed into boiled sodium citrate buffer (0.01 M, pH 6.0), which was preheated up to 100 °C, for 20 min and cooled at room temperature for 20 min. Subsequently, the slides were incubated with peroxidase-blocking reagent (Cat. No. S200389, Dako, Denmark) for 30 min, and then treated with or without primary antibodies (the anti-gB serum prepared from this study). In each interval of the following procedures, sections were rinsed with a mixture of TBST buffer. Tissue sections were then reacted with secondary antibody (HRP anti-rabbit/mouse, DAKO, Denmark), followed by incubation of DAB and chromogen (dilution 1 μL in 100 μL) from a commercial ChemMate EnVision detection kit (Cat. No. K5007, Dako, Denmark). Ultimately, tissue sections were counterstained with Mayer’s hematoxylin reagents (Code S3309, Dako, Denmark) for 2 min followed by wash with DDW, and reacted with 37 mM ammonia water for 5 s and rinsed with DDW.Immunofluorescent assay (IFA)Human 293 T cells were transfected with plasmids expressing full-length ChHV5 gB protein fused with FLAG tag at its C-terminus. At 24 h post transfection, 293 T cells (CRL-3216, ATCC, USA) were fixed with 2% formaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 for another 10 min. Subsequently, cells were incubated with anti-FLAG antibody (1:500) (F7425; Sigma-Aldrich), or antisera (F1, F2, F3, F2–3) at the dilution of 1:500 for 1 h at room temperature. After six times of washes with PBS containing1% bovine serum, goat anti-mouse IgG (1:2,000 fold diluted) (Cat. No. A28175, Alexa Fluor® 488, Invitrogen) was used as secondary antibody. After one-hour incubation, nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Cat. No. D9542, Sigma-Aldrich) for 10 min, followed by confocal microscopy (FV1000, Olympus, Tokyo, Japan) with Olympus FV10-ASW 1.3 viewer software.Statistical analysisTo evaluate the association between seropositivity and FP or viremia tested by PCR of UL27 gene, Fisher’s exact test was applied due to very limited number of sea turtles with FP. The statistical significance was determined by p  More

Prosopis juliflora leaves collection and processing for RibotypingProsopis juliflora species of the genus Prosopis, family of Fabaceae had its genetic variation in Doha evaluated. Seven samples of P. juliflora leaves were collected from six different locations in Doha, Qatar, during five field trips. Plant leaves were collected after proper permissions and all methods were carried out in accordance with relevant guidelines and regulations. Trees in all locations were naturally growing around urbanization areas in their normal arid habitat without artificial irrigation, samples were collected from fully mature trees. Table 1 shows the samples details. Figure 1 shows the location sites of where the samples were collected on the map of Qatar, Doha. Leaf samples were kept in sterile labeled bags until having reached the laboratory where few leaflets were washed with sterile distilled water and sterilized using 70% ethanol to be used for DNA extraction.Table 1 Location details of the collection sites of P. juliflora leaves.Full size tableFigure 1Location map of collection sites of P. juliflora leaf samples (ArcGIS software).Full size imageRibotyping analysisThe leaflets of each sample were transferred into a sterile mortar previously cooled at -20 ˚C and used for DNA extraction following the kit manufacturer instructions (DNeasy Plant Mini Kit-QIAGEN-USA).Extracted DNA of each sample were subject to PCR using ITS1 and ITS4 primers. PCR products obtained were purified using the Invitrogen Quick PCR Purification Kit (QIAGEN, Germany) as indicated by the manufacturer and sequenced using Sanger sequencer (3130/3130xl DNA Analyzers, Thermofisher Scientific, USA) as previously described22.Sanger sequencer raw data was read using BioEdit software. Basic Local Alignment Search Tool (BLAST) network services of the National Centre for Biotechnology Information (NCBI) database were used to compare the obtained sequences to the existing sequences. Sequences were submitted to NCBI for accession numbers. The various P. juliflora ribosomal sequences obtained were also uploaded on MEGA-X software and the phylogeny tree was generated using the neighbor-joining algorithm26.Minimum inhibitory concentrations of PJ-WS-LE extracts prepared using leaf samples collected from various locations against A. alternata and C. gloeosporioides
Preparation of PJ-WS-LE extractFresh, young full leaves of P. juliflora were collected from various locations as indicated in Fig. 1. Samples were washed, dried and ground into powder to be used in the preparation of PJ-WS-LE extract as previously described22. Briefly, every 20 g of the leaf powder were incubated in 200 mL of 70% ethanol for 48 h. The supernatant has its solvent evaporated, the extract was then re-dissolved in sterile distilled water. Only water-soluble phytochemicals were tested by centrifuging the final preparation tubes and excluding the pellet. Stock solution of 100 g L−1 was stored at 4 °C to be used for later experiments. PJ-WS-LE extract concentration used in treatments was 8 g L−1 which is double the highest minimum inhibitory concentration of the extract against spoiling microorganisms as previously determined22.Determination of minimum inhibitory concentrationThe MIC test was conducted in a sterile 96-well plate, with each well containing 100 μl of potato dextrose broth (PDB) (HIMEDIA-India). Every four wells made one replication, nine different concentrations of the crude extracts were tested (1:1 dilutions) ranging from 42 to 0.16 g L−1. Wells were then inoculated with one of the two tested microorganisms’ spore suspensions (A. alternata and C. gloeosporioides). The last three rows are control rows: no spores and no extract control wells, negative control with spores but no extract wells, and positive control with spores and 10 µl of the fungicidal Clatrimazole (1%) wells.Fungal spore suspensions were adjusted to the range of 104 spores L−1 using a 10 day old fungal plate and sterile distilled water, the spore concentration was calculated using a heamatocytometer.Fungal growth in each well was monitored using Resazurin (HIMEDIA-India) dye. Upon cells division, Resazurin changes its color from blue to pink and fluorescent27. Results were taken within 48 h of incubation at 25 °C. MIC was recorded as the last extract concentration that shows no change in the color of Resazurin within the incubation period.Curative and preventive effects of PJ-WS-LE extract against A. alternata and C. gloeosporioides induced infection in mangoesPathogensThe two fungal strains used C. gloeosporioides and A. alternata were obtained from our laboratory collection, Department of Biological and Environmental Sciences, Qatar University, Qatar. Both fungal isolates were previously isolated from locally collected fruit samples. Isolates were molecularly identified by sequencing the Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) that was amplified by PCR. Identified fungal isolates were given the strains code of AaltQU17 for A. alternata and CgloQU17 for C. gloeosporioides22. Preserved cultures were sub-cultured on potato dextrose agar (PDA) plates and incubated at 25 °C for 10 days. Plates were then flooded with 10 mL of sterile distilled water each, to prepare the needed spores suspension solutions. The concentrations of spores suspensions were adjusted to 106 spores L−1 using a heamatocytometer18.FruitThe mango (Mangifera indica) type known as Neelam imported from India was used in the experiments. Fruit were bought from the whole sale market upon their arrival to the country. Only undamaged mature fruit were used in the experiment. Fruits chosen were ripen but not yet soft with firmness average of 20 ± 5.1 N, weight average of 177.61 ± 0.2 g and TSS average of 70 ± 5.3%. Fruit were first washed with sink water and sterilized twice with 70% ethanol to be then washed with sterile distilled water and left to air dry.Preventive and curative effects of PJ-WS-LE extractWounded mango fruit were used during the experiment, the wounds were made through three needle pricks (2 mm deep) in three different places for each plant using a sterile syringe. A completely randomized design was used and each treatment was made of a triplicate of 10 fruit each. The experiment was repeated twice.PJ-WS-LE extract of leaves collected from Qatar university field was first tested for its efficacy in preventing fungal contamination in wounded mango fruit (preventive effect). Therefore, the wounded zone of each fruit was sprayed with 8 g L−1 PJ-WS-LE extract and then left to air-dry. Once dry the fruit were sprayed again with the extract at the same concentration and left to dry. Control fruit were only treated with sterile distilled water without the plants extract. After two hours all wounds were inoculated with 20 μL of conidia aqueous solution (106 spores mL−1) of one of the tested fungi. The extract was then tested for its ability to cure fungal contamination in wounded fruit. Therefore, wounds were inoculated first with 20 μL of conidia aqueous solution (106 spores mL−1) and left to dry. Wounds were then sprayed twice with 8 g L−1 PJ-WS-LE extract.All mangoes were stored in sterilized plastic trays inside an incubator at 25 °C and 75% humidity. Fruit were observed every 24 h for 5 days for C. gloeosporioides inoculated fruit and for 10 days for A. alternata inoculated fruit. Three parameters were recorded at the end of the experiment: disease incidence (DI), disease severity (DS), and percent plant extract efficacy (%EE). To calculate disease severity, the diameter of the infected area of each fruit was measured in two perpendicular directions and mean diameter mycelial growth was calculated28,29.$$mathrm{DI}=frac{(mathrm{Number, of, rotten, fruit})times 100}{mathrm{Total, number, of, fruit}}$$$$mathrm{DS }=frac{(mathrm{Average, lesion, diameter, of, treated, plants})times 100}{mathrm{Average, Lesion, diameter, of, control, plants})}$$$$mathrm{%EE}=frac{(mathrm{Disease, incidence, in, Control, batch}-mathrm{Disease, incidence, in, treated, batch})times 100}{mathrm{Disease, incidence, in, Control, batch}}$$End of the trial samples firmnessAt the end of the trial, remaining mango fruit were tested for their flesh quality using a penetrometer (Agriculture Solutions, USA) to test the flesh firmness. Fruit were peeled, then the stainless steel probe of the instrument was inserted in three different points towards the equator of the fruit. Firmness in Newton was recorded and compared with standard fruit firmness to judge fruit quality18.Effectiveness of PJ-WS-LE extract as long-term coating material and the preservative value of its chitosan-embedded formCoating solutions preparationChitosan solution of 1% concentration was prepared by stirring chitosan powder (CAS 9012-76-4, Himedia, India) in 1% glacial acetic acid (IsoLab, Germany) overnight. The final chitosan solution pH was adjusted to 5.6 using 0.1 M NAOH (Sigma-Aldrich, Germany). To prepare PJ-WS-LE extract chitosan-embedded coating material, filter-sterilized PJ-WS-LE extract stock solution was added to 1% chitosan to achieve a final concentration of 8 g L−130.Samples preparationEighty-four mango samples chosen as described above, were divided into four groups of 18 samples each. Samples were divided into four treatment batches and treated as following:

Batch A: non-treated fruit.

Batch B: PJ-WS-LE extract at 8 g L–1 was used to spray the fruit.

Batch C: 1% chitosan was used to spray the fruit.

Batch D: 8 g L−1 PJ-WS-LE extract embedded in 1% chitosan was used to spray the fruit.

Every experimental replicate was made up of three mango samples that were stored together in one sterile bag at 4 °C. The number of replications per treatment was six. The experiment was repeated twice31.Evaluation of sensory qualityA five-points scale was used for the evaluation of the sensory quality of the samples for overall quality, smell, and color change. The attributes were evaluated weekly using the fruit of one experimental replicate. Scores were given using the following scale: 5 points indicate “extremely liked”, 4 points indicate “liked”, 3 points indicate “acceptable” 2 points indicate “disliked” and 1 point indicates “extremely disliked”. The weekly average score per batch was also calculated32.Estimation of weight lossUpon treatment at day zero, all mango samples were weighed and their weights were recorded as initial weights. Weights of all remaining samples were measured at the end of every week. The variation between the start weight and weekly weights is calculated as weekly weight loss. The average percent of weekly weight loss of each batch was calculated32.Determination of samples firmnessThe samples of each experimental replicate evaluated on a weekly basis had their firmness measured as previously described. The weekly average samples firmness (N) of every treatment batch was also calculated33.pH measurementMango fruit of each experimental replicate were blended weekly into juice, after filtration, a digital pH meter (Jenway, UK) was used to measure pH. The weekly average fruit pH of every treatment batch was also calculated. The pH meter was calibrated using a buffer solution of pH 734.Total soluble solids (TSS) measurementTotal soluble solids of the prepared mango juice samples were measured in percent brix using a refractometer (ANTAHI, New Zealand). The weekly average fruit TSS (%) for each treatment batch was also calculated. The refractometers was calibrated using distilled water35.DPPH radical scavenging assayA 1/10 mango juice dilution was prepared using sterile distilled water. 100 μL of each dilution was mixed with 1 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (100 mg L−1) to be incubated in the dark at 37 °C for 45 min. After incubation, samples were centrifuged and the pellet was discarded. The intensity of the change in color of the supernatant was measured by spectrophotometry at 517 nm using methanol as a blank. 100 μL of methanol in 1 mL DPPH was used as the control for the experiment. Percent radical scavenging activity was calculated as per the below formula:$$ % {text{ radical scavenging activity}}, = ,left( {{text{absorbance of the control solution}} – {text{ absorbance of the juice sample}}} right)*{1}00/{text{absorbance of the control solution}}. $$The weekly average % radical scavenging activity for each treatment batch was finally calculated31.Statistical analysisThe experimental design used was Completely Randomized Design (CRD). One-way ANOVA followed by Tukey Post-Hoc test was used to evaluate the significance of the weekly percent change in weight among treatment batches at P ≤ 0.05. The significances of pH and TSS variation within different treatment batches were evaluated using One-way ANOVA test at P ≤ 0.05. Data was presented as average ± standard error of the Means (SEM). SPSS (Ver. 27, SPSS Inc. Chicago, USA) was used to perform the statistical analysis tests. More

Printzen, C. & Lumbsch, H. T. Molecular evidence for the diversification of extant lichens in the late cretaceous and tertiary. Mol. Phylogenet. Evol. 17, 379–387 (2000).CAS 
PubMed 
Article 

Google Scholar 
Kraichak, E. et al. A Tale of two Hyper-diversities: Diversification dynamics of the two largest families of lichenized fungi. Sci. Rep. https://doi.org/10.1038/srep100288 (2015).Article 
PubMed 
PubMed Central 

Google Scholar 
Leavitt, S. D., Lumbsch, H. T., Stenroos, S. & Clair, L. L. S. Pleistocene speciation in North American lichenized fungi and the the impact of alternative species circumscriptions and rates of molecular evolution on divergence estimates. PLoS ONE 8, e85240 (2013).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Gaya, E. et al. The adaptive radiation of lichen-forming Teloschistaceae is associated with sunscreening pigments and bark-to-rock substrate shift. PNAS 112, 11600–11605 (2015).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Schneider, K., Resl, P. & Spribille, T. Escape from the cryptic species trap: Lichen evolution on both sides of a cyanobacterial acquisition event. Mol. Ecol. 25, 3453–3468 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Widhelm, T. J. et al. Oligocene origin and drivers for diversification in the genus Sticta (Lobariaceae, Ascomycota). Mol. Phylogenetic Evol. 126, 58–73 (2018).Article 

Google Scholar 
Vamosi, J. C. & Vamosi, S. M. Factors influencing diversification in angiosperms: At the crossroads of intrinsic and extrinsic traits. Am. J. Bot. 98, 460–471 (2011).PubMed 
Article 

Google Scholar 
Wagner, C. E., Harmon, L. J. & Seehausen, O. Ecological opportunity and sexual selection together predict adaptive radiation. Nature 487, 366–369 (2012).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Karunarathne, P. et al. Intraspecific ecological niche divergence and reproductive shifts foster cytotype displacement and provide ecological opportunity to polyploids. Ann. Bot. 121, 1183–1196 (2018).PubMed 
PubMed Central 
Article 

Google Scholar 
Nakov, T., Beaulieu, J. & Alverson, A. Accelerated diversification is related to life history and locomotion in a hyperdiverse lineage of microbial eukaryotes (Diatoms, Bacillariophyta). New Phytol. 219, 462–473 (2018).PubMed 
PubMed Central 
Article 

Google Scholar 
Tripp, E. A. Is asexual reproduction an evolutionary dead end in lichens?. Lichenologist 48, 559–580 (2016).Article 

Google Scholar 
Tripp, E. A. & Lendemer, J. C. Twenty-seven modes of reproduction in the obligate lichen symbiosis. Brittonia 70, 1–14 (2018).Article 

Google Scholar 
Bowler, P. A. & Rundell, P. W. Reproductive strategies in lichens. Bot. J. Linn. Soc. 70, 325–340 (1975).Article 

Google Scholar 
Honegger, R. Developmental biology of lichens. New Phytol. 125, 659–677 (1993).PubMed 
Article 

Google Scholar 
Buschbom, J. & Mueller, G. M. Testing “Species Pair” hypotheses: Evolutionary processes in the lichen-forming species complex Porpidia flavocoerulescens and Porpidia melinodes. Mol. Biol. Evol. 23, 574–586. https://doi.org/10.1093/molbev/msj063 (2006).CAS 
Article 
PubMed 

Google Scholar 
Sanders, W. B. Complete life cycle of the lichen fungus Calopadia puiggarii (Pilocarpaceae, Ascomycetes) documented in situ: Propagule dispersal, establishment of symbiosis, Thallus development, and formation of sexual and asexual reproductive structures. Am. J. Bot. 101, 1836–1848 (2014).PubMed 
Article 

Google Scholar 
Poelt, J. Flechtenflora und eiszeit in Europa. Phyton (Horn) 10, 206–214 (1963).
Google Scholar 
Stofer, S. et al. Species richness of lichen functional groups in relation to land use intensity. Lichenologist 38, 331–353 (2006).Article 

Google Scholar 
Ludwig, L. R., Summerfield, T. C., Lord, J. M. & Singh, G. Characterization of the mating-type locus (MAT) reveals a heterothallic mating system in Knightiella splachnirima. Lichenologist 49, 373–385 (2017).Article 

Google Scholar 
Czarnota, P. The lichen genus Micarea (Lecanorales, Ascomycota) in Poland. Pol. Bot. Stud. 23, 1–190 (2007).
Google Scholar 
Czarnota, P. & Guzow-Krzemińska, B. A phylogenetic study of the Micarea prasina group shows that Micarea micrococca includes three distinct lineages. Lichenologist 42, 7–21 (2010).Article 

Google Scholar 
Sérusiaux, E., Brand, A. M., Motiejūnaitè, J., Orange, A. & Coppins, B. J. Lecidea doliiformis belongs to Micarea, Catillaria alba to Biatora and Biatora lignimollis occurs in Western Europe. Bryologist 113, 333–344 (2010).Article 

Google Scholar 
van den Boom, P., Brand, A., Coppins, B. & Sérusiaux, E. Two new species in the Micarea prasina group from Western Europe. Lichenologist 49, 13–25 (2017).Article 

Google Scholar 
Guzow-Krzemińska, B., Czarnota, P., Łubek, A. & Kukwa, M. Micarea soralifera sp. nov., a new sorediate species in the M. prasina group. Lichenologist 48, 161–169 (2016).Article 

Google Scholar 
Guzow-Krzemińska, B. et al. Understanding the evolution of phenotypical characters in the Micarea prasina group (Pilocarpaceae) and descriptions of six new species within the group. MycoKeys 57, 1–30 (2019).PubMed 
PubMed Central 
Article 

Google Scholar 
Kantvilas, G. & Coppins, B. J. Studies on Micarea in Australasia II. A synopsis of the genus in Tasmania, with the description of ten new species. Lichenologist 51, 431–481 (2019).Article 

Google Scholar 
Launis, A. & Myllys, L. Micarea fennica, a new lignicolous lichen species from Finland. Phytotaxa 409, 179–188 (2019).Article 

Google Scholar 
Launis, A., Pykälä, J., van den Boom, P., Sérusiaux, E. & Myllys, L. Four new epiphytic species in the Micarea prasina group from Europe. Lichenologist 51, 7–25 (2019).Article 

Google Scholar 
Launis, A. et al. Sharpening species boundaries in the Micarea prasina group, with a new circumscription of the type species M. prasina. Mycologia 111, 574–592 (2019).CAS 
PubMed 
Article 

Google Scholar 
van den Boom, P., Guzow-Krzemińska, B. & Kukwa, M. Two new Micarea species (Pilocarpaceae) from Western Europe. Plant Fungal Syst. 65, 189–199. https://doi.org/10.35535/pfsyst-2020-0014 (2020).Article 

Google Scholar 
Kantelinen, A., Hyvärinen, M., Kirika, P. & Myllys, L. Four new Micarea species from the montane cloud forests of Taita Hills, Kenya. Lichenologist 53, 81–94. https://doi.org/10.1017/S0024282920000511 (2021).Article 

Google Scholar 
Coppins, B. J. A taxonomic study of the lichen genus Micarea in Europe. Bull. Br. Mus. (Nat. Hist.) Bot. 11, 17–214 (1983).
Google Scholar 
Launis, A. & Myllys, L. Micarea byssacea new to North America and Micarea hedlundii new to Maine, Michigan and Quebec. Opusc. Philolichenum 13, 84–90 (2014).
Google Scholar 
Myllys, L. & Launis, A. Additions to the diversity of lichens and lichenicolous fungi living on decaying wood in Finland. Graphis Scr. 30, 78–87 (2018).
Google Scholar 
Yahr, R., Florence, A., Škaloud, P. & Voytsekhovich, A. Molecular and morphological diversity in photobionts associated with Micarea s. str. (Lecanorales, Ascomycota). Lichenologist 47, 403–414 (2015).Article 

Google Scholar 
Spribille, T., Thor, G., Bunnell, F. L., Goward, T. & Björk, C. R. Lichens on dead wood: Species-substrate relationships in the epiphytic lichen floras of the Pacific Northwest and Fennoscandia. Ecography 31, 741–750 (2008).Article 

Google Scholar 
Resl, P., Fernańdez-Mendoza, F., Mayrhofer, H. & Spribille, T. The evolution of fungal substrate specificity in a widespread group of crustose lichens. Proc. R. Soc. B 285, 20180640. https://doi.org/10.1098/rspb.2018.0640 (2018).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Stokland, J. N., Siitonen, J. & Jonsson, B. G. Biodiversity in Dead Wood 412 (Cambridge University Press, Cambridge, 2012).Book 

Google Scholar 
Russell, M. B., Woodall, C. W., Fraver, S. & D’Amato, A. W. Estimates of downed woody debris decay class transitions for forests across the eastern United States. Ecol. Model. 251, 22–31 (2013).Article 

Google Scholar 
Russell, M. B. et al. Residence times and decay rates of downed woody debris biomass/carbon in eastern US Forests. Ecosystems 17, 765–777 (2014).CAS 
Article 

Google Scholar 
Zoller, S., Lutzoni, F. & Scheidegger, C. Genetic variation within and among populations of the threatened lichen Lobaria pulmonaria in Switzerland and implications for its conservation. Mol. Ecol. 8, 2049–2059 (1999).CAS 
PubMed 
Article 

Google Scholar 
Honegger, R., Zippler, U., Gansner, H. & Scherrer, S. Mating systems in the genus Xanthoria (lichen forming Ascomycetes). Mycol. Res. 108, 480–488 (2004).CAS 
PubMed 
Article 

Google Scholar 
Honegger, R. & Zippler, U. Mating systems in representatives of the Parmeliaceae, Ramalinaceae and Physciaceae (Lecanoromycetes, lichen-forming ascomycetes). Mycol. Res. 11, 424–432 (2007).Article 
CAS 

Google Scholar 
Ament-Velásquez, S. L. et al. The plot thickens: Haploid and triploid-like thalli, hybridization, and biased mating Type Ratios in Letharia. Front. Fungal Biol. 2, 656386. https://doi.org/10.3389/ffunb.2021.656386 (2021).Article 

Google Scholar 
van den Boom, P. & Coppins, B. J. Micarea viridileprosa sp. nov., an overlooked lichen species from Western Europe. Lichenologist 33, 87–91 (2001).Article 

Google Scholar 
Simon, J.-C., Rispe, C. & Sunnucks, P. Ecology and evolution of sex in aphids. Trends Ecol. Evol. 17, 34–39 (2002).Article 

Google Scholar 
Silvertown, J. The evolutionary maintenance of sexual reproduction: Evidence from the ecological distribution of asexual reproduction in clonal plants. Int. J. Plant Sci. 169, 157–168 (2008).Article 

Google Scholar 
Gomez-Mestre, I., Pyron, R. A. & Wiens, J. J. Phylogenetic analyses reveal unexpected patterns in the evolution of reproductive modes in frogs. Evolution 66, 3687–3700. https://doi.org/10.1111/J.1558-5646.2012.01715.X (2012).Article 
PubMed 

Google Scholar 
Dańko, A., Schaible, R. & Dańko, M. J. Salinity effects on survival and reproduction of hydrozoan Eleutheria dichotoma. Estuaries Coasts 43, 360–374. https://doi.org/10.1007/s12237-019-00675-2 (2020).CAS 
Article 

Google Scholar 
Coppins, B. J. & Tønsberg, T. A new xanthone-containing Micarea from Northwest Europe and the Pacific Northwest of North America. Lichenologist 33, 93–96 (2001).Article 

Google Scholar 
Konoreva, L., Chesnokov, S., Kuznetsova, E. & Stepanchikova, I. Remarkable records of Micarea from the Russian Far East and significant extension of Micarea laeta and M. microareolata range. Botanica 25, 186–201 (2019).Article 

Google Scholar 
Weber, L., Printzen, C., Bässler, C. & Kantelinen, A. Seven Micarea (Pilocarpaceae) species new to Germany and notes on deficiently known species in the Bavarian forest. Herzogia 34, 5–17 (2021).Article 

Google Scholar 
van den Boom, P. Some interesting records of lichens and lichenicolous fungi from The Netherlands VI. Osten. Z. Pilzk. 12 (2003).Orange, A., James, P. W. & White, F. J. Microchemical Methods for the Identification of Lichens 101 (British Lichen Society, London, 2010).
Google Scholar 
Meyer, B. & Printzen, C. Proposal for a standardized nomenclature and characterization of insoluble lichen pigments. Lichenologist 32, 571–583 (2000).Article 

Google Scholar 
Culberson, C. F. & Kristinsson, H. D. A standardized method for the identification of lichen products. J. Chromatocraphy A 46, 85–93 (1970).CAS 
Article 

Google Scholar 
Myllys, L. et al. Phylogeny of the genus Bryoria. Lichenologist 43, 617–638 (2011).Article 

Google Scholar 
Myllys, L., Lohtander, K., Källersjö, M. & Tehler, A. Sequence insertion and ITS data provide congruent information in Roccella canariensis and R. tuberculata (Arthoniales, Euascomycetes) phylogeny. Mol. Phylogenetics Evol. 12, 295–309 (1999).CAS 
Article 

Google Scholar 
White, T. J., Bruns, T., Lee, S. & Taylor, J. W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to the Methods and Applications (eds Innis, M. A. et al.) 315–322 (Academic Press, Cambridge, 1990).
Google Scholar 
Zoller, S., Scheidegger, C. & Sperisen, C. PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31, 511–516 (1999).Article 

Google Scholar 
Leavitt, S. D., Johnson, L. A., Goward, T. & Clair, L. L. S. Species delimitation in taxonomically difficult lichen-forming fungi: an example from morphologically and chemically diverse Xanthoparmelia (Parmeliaceae) in North America. Mol. Phylogenetics Evol. 60(3), 317–332 (2011).Article 

Google Scholar 
Schmitt, I. et al. New primers for promising single-copy genes in fungal phylogenetics and systematics. Persoonia 23, 35–40 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Edgar, R. C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. https://doi.org/10.1093/nar/gkh340 (2004).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Kauff, F. & Lutzoni, F. Phylogeny of the Gyalectales and Ostropales (Ascomycota, Fungi): Among and within order relationships based on nuclear ribosomal RNA small and large subunits. Mol. Phylogenet. Evol. 25, 138–156 (2002).CAS 
PubMed 
Article 

Google Scholar 
Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003).CAS 
PubMed 
Article 

Google Scholar 
Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Huelsenbeck, J. P., Larget, B. & Alfaro, M. E. Bayesian phylogenetic model selection using reversible jump markov chain monte carlo. Mol. Biol. Evol. 21, 1123–1133. https://doi.org/10.1093/molbev/msh123 (2004).CAS 
Article 
PubMed 

Google Scholar 
Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior Summarization in bayesian phylogenetics using tracer 1.7. Syst. Biol. 67, 901–904 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Maddison, D. R. & Maddison, W. P. Mesquite: a modular system for evolutionary analysis. Version 3.40 http://mesquiteproject.org (2018).Pagel, M. Detecting correlated evolution on phylogenies: A general method for the comparative analysis of discrete characters. Proc. R. Soc. B. 255, 37–45 (1994).ADS 
Article 

Google Scholar  More

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.This is a summary of: Van der Stocken, T. et al. Mangrove dispersal disrupted by projected changes in global seawater density. Nat. Clim. Change https://doi.org/10.1038/s41558-022-01391-9 (2022). More

Gao, Y., Schofield, O. M. & Leustek, T. Characterization of sulfate assimilation in marine algae focusing on the enzyme 5′-adenylylsulfate reductase. Plant Physiol. 123, 1087–1096 (2000).CAS 
PubMed 
PubMed Central 

Google Scholar 
Huang, C.-W., Walker, M. E., Fedrizzi, B., Gardner, R. C. & Jiranek, V. Hydrogen sulfide and its roles in Saccharomyces cerevisiae in a winemaking context. FEMS Yeast Res. 17, 058 (2017).
Google Scholar 
Kopriva, S., Calderwood, A., Weckopp, S. C. & Koprivova, A. Plant sulfur and big data. Plant Sci. 241, 1–10 (2015).CAS 
PubMed 

Google Scholar 
Shibagaki, N. & Grossman, A. The state of sulfur metabolism in algae: From ecology to genomics. In Sulfur Metabolism in Phototrophic Organisms (eds Hell, C. D. R. et al.) 231–267 (Springer, 2008).
Google Scholar 
Fakhraee, M. & Katsev, S. Organic sulfur was integral to the Archean sulfur cycle. Nat. Commun. 10, 1–8 (2019).CAS 

Google Scholar 
Ho, T. Y. et al. The elemental composition of some marine phytoplankton 1. J. Phycol. 39, 1145–1159 (2003).CAS 

Google Scholar 
Jørgensen, B. B. Unravelling the sulphur cycle of marine sediments. Environ. Microbiol. 21, 3533–3538 (2019).PubMed 

Google Scholar 
El Mahrad, B. et al. Social-environmental analysis for the management of coastal lagoons in North Africa. Front. Environ. Sci. 8, 37 (2020).
Google Scholar 
Srarfi, F. Etude géochimique et état de pollution de la lagune de Bizerte. These de doctorat, Univ. Tunis el Manar 122 (2007).FAO. La Situation Mondiale Des Pêches et de L’aquaculture 2020 (Food & Agriculture Organisation, 2020).
Google Scholar 
Soto, D. & Wurmann, C. The Future of Ocean Governance and Capacity Development 379–384 (Brill Nijhoff, 2019).
Google Scholar 
Ran, W. et al. Storage of starch and lipids in microalgae: Biosynthesis and manipulation by nutrients. Bioresour. Technol. 291, 121894 (2019).CAS 
PubMed 

Google Scholar 
Aikawa, S. et al. Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering. Biotechnol. J. 10, 886–898 (2015).CAS 
PubMed 

Google Scholar 
Klok, A., Lamers, P., Martens, D., Draaisma, R. & Wijffels, R. Edible oils from microalgae: Insights in TAG accumulation. Trends Biotechnol. 32, 521–528 (2014).CAS 
PubMed 

Google Scholar 
Yuan, Y. et al. Enhancing carbohydrate productivity of Chlorella sp. AE10 in semi-continuous cultivation and unraveling the mechanism by flow cytometry. Appl. Biochem. 185, 419–433 (2018).CAS 

Google Scholar 
Rodríguez, M. C., Matulewicz, M. C., Noseda, M., Ducatti, D. & Leonardi, P. I. Agar from Gracilaria gracilis (Gracilariales, Rhodophyta) of the Patagonic coast of Argentina-Content, structure and physical properties. Biores. Technol. 100, 1435–1441 (2009).
Google Scholar 
Lee, W.-K. et al. Factors affecting yield and gelling properties of agar. J. Appl. Phycol. 29, 1527–1540 (2017).
Google Scholar 
Fethi, M. & Ghedifa, A. B. Optimum ranges of combined abiotic factor for Gracilaria gracilis aquaculture. J. Appl. Phycol. 31, 3025–3040 (2019).
Google Scholar 
Friedlander, M. Inorganic nutrition in pond cultivated Gracilaria conferta (Rhodophyta): Nitrogen, phosphate and sulfate. J. Appl. Phycol. 13, 279–286 (2001).CAS 

Google Scholar 
Lee, W.-K., Namasivayam, P. & Ho, C.-L. Effects of sulfate starvation on agar polysaccharides of Gracilaria species (Gracilariaceae, Rhodophyta) from Morib, Malaysia. J. Appl. Phycol. 26, 1791–1799 (2014).CAS 

Google Scholar 
Carfagna, S. et al. Impact of sulfur starvation in autotrophic and heterotrophic cultures of the extremophilic microalga Galdieria phlegrea (Cyanidiophyceae). Plant Cell Physiol. 57, 1890–1898 (2016).CAS 
PubMed 

Google Scholar 
Collén, P. N., Camitz, A., Hancock, R. D., Viola, R. & Pedersén, M. Effect of nutrient deprivation and resupply on metabolites and enzymes related to carbon allocation in gracilaria tenuistipitata (rhodophyta) 1. J. Phycol. 40, 305–314 (2004).
Google Scholar 
Collier, J. L. & Grossman, A. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria. EMBO J. 13, 1039–1047 (1994).CAS 
PubMed 
PubMed Central 

Google Scholar 
Richaud, C., Zabulon, G., Joder, A. & Thomas, J.-C. Nitrogen or sulfur starvation differentially affects phycobilisome degradation and expression of the nblA gene in Synechocystis strain PCC 6803. J. Bacteriol. 183, 2989–2994 (2001).CAS 
PubMed 
PubMed Central 

Google Scholar 
Lloyd, A. G., Dodgson, K. S. & Rose, F. A. Infrared studies on sulphate esters I. Polysaccharide sulphates. Biochim. Biophys. Acta 46, 108–115 (1961).CAS 
PubMed 

Google Scholar 
Kawachi, M. & Noël, M.-H. Sterilization and sterile technique. In Algal Culturing Techniques (ed. Anderson, R. A.) 65–81 (Academic Press, 2005).
Google Scholar 
Harrison, P. J. & Berges, J. A. Marine culture media. In Algal Culturing Techniques (ed. Anderson, R. A.) 21–34 (Academic Press, 2005).
Google Scholar 
Guiry, M. & Cunningham, E. Photoperiodic and temperature responses in the reproduction of north-eastern Atlantic Gigartina acicularis (Rhodophyta: Gigartinales). Phycologia 23, 357–367 (1984).
Google Scholar 
Kolmert, Å., Wikström, P. & Hallberg, K. B. A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures. J. Microbiol. Methods 41, 179–184 (2000).CAS 
PubMed 

Google Scholar 
Destombe, C., Godin, J., Nocher, M., Richerd, S. & Valero, M. In Fourteenth International Seaweed Symposium (eds Brown, M. T. & Lahaye, M.) 131–137 (Springer, 1993).
Google Scholar 
Rueness, J. & Tananger, T. In Eleventh International Seaweed Symposium (eds Bird, C. J. & Ragan, M. A.) 303–307 (Springer, 1984).
Google Scholar 
Shea, R. & Chopin, T. Effects of germanium dioxide, an inhibitor of diatom growth, on the microscopic laboratory cultivation stage of the kelp, Laminaria saccharina. J. Appl. Phycol. 19, 27–32 (2007).CAS 

Google Scholar 
Dawes, C., Orduna-Rojas, J. & Robledo, D. Response of the tropical red seaweed Gracilaria cornea to temperature, salinity and irradiance. J. Appl. Phycol. 10, 419–425 (1998).
Google Scholar 
Yaphe, W. & Arsenault, G. Improved resorcinol reagent for the determination of fructose, and of 3, 6-anhydrogalactose in polysaccharides. Anal. Biochem. 13, 143–148 (1965).CAS 

Google Scholar 
Mensi, F., Ksouri, J., Seale, E., Romdhane, M. S. & Fleurence, J. A statistical approach for optimization of R-phycoerythrin extraction from the red algae Gracilaria verrucosa by enzymatic hydrolysis using central composite design and desirability function. J. Appl. Phycol. 24, 915–926 (2012).CAS 

Google Scholar 
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T. & Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356 (1956).CAS 

Google Scholar 
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).CAS 
PubMed 

Google Scholar 
Sörbo, B. Sulfate: Turbidimetric and nephelometric methods. Methods Enzymol. 143, 3–6 (1987).PubMed 

Google Scholar 
Redmond, S., Green, L., Yarish, C., Kim, J. & Neefus, C. New England Seaweed Culture Handbook (University of Connecticut Sea Garent, 2014).
Google Scholar 
Kakita, H. & Kamishima, H. Effects of environmental factors and metal ions on growth of the red alga Gracilaria chorda Holmes (Gracilariales, Rhodophyta). J. Appl. Phycol. 18, 469–474 (2006).CAS 

Google Scholar 
Berges, J. A., Franklin, D. J. & Harrison, P. J. Evolution of an artificial seawater medium: Improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37, 1138–1145 (2001).
Google Scholar 
Shpigun, L. K., Kolotyrkina, I. Y. & Zolotov, Y. A. Experience with flow-injection analysis in marine chemical research. Anal. Chim. Acta 261, 307–314 (1992).CAS 

Google Scholar 
Cosano, J., de Castro, M. & Valcarcel, M. Flow injection analysis of water. Part 1: Automatic preconcentration determination of sulphate, ammonia and iron (II)/iron (III). J. Autom. Chem. 15, 141–146 (1993).CAS 

Google Scholar 
Van Staden, J. & Taljaard, R. Determination of sulphate in natural waters and industrial effluents by sequential injection analysis. Anal. Chim. Acta 331, 271–280 (1996).
Google Scholar 
Petersen, S. P. & Ahring, B. K. Analysis of sulfate in sewage sludge using ion chromatographic techniques. J. Microbiol. Methods 12, 225–230 (1990).CAS 

Google Scholar 
Rand, M., Greenberg, A., Taras, K. & Franson, M. Standard Methods for the Examination of Water and Waste Water (American Public Health Association, 1975).
Google Scholar 
Wanner, G., Henkelmann, G., Schmidt, A. & Köst, H.-P. Nitrogen and sulfur starvation of the cyanobacterium Synechococcus 6301 an ultrastructural, morphometrical, and biochemical comparison. Zeitschrift Naturforschung C 41, 741–750 (1986).CAS 

Google Scholar 
Molloy, F. & Bolton, J. The effect of season and depth on the growth of Gracilaria gracilis at Lüderitz, Namibia. Bot. Mar. 39, 407–414 (1996).
Google Scholar 
Mensi, F., Nasraoui, S., Bouguerra, S., Ben Ghedifa, A. & Chalghaf, M. Effect of lagoon and sea water depth on Gracilaria gracilis growth and biochemical composition in the northeast of Tunisia. Sci. Rep. 10, 1–12 (2020).
Google Scholar 
Mensi, F., Ksouri, J., Hammami, W. & Romdhane, M. État des connaissances et perspectives de recherches sur la culture de Gracilariales (Gracilaria et Gracilariopsis): Application a la lagune de Bizerte. Bull. Inst. Natn. Scien. Tech. Mer Salammbô 41, 101–119 (2014).
Google Scholar 
Sugimoto, K., Sato, N. & Tsuzuki, M. Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii. FEBS Lett. 581, 4519–4522 (2007).CAS 
PubMed 

Google Scholar 
Cakmak, T. et al. Nitrogen and sulfur deprivation differentiate lipid accumulation targets of Chlamydomonas reinhardtii. Bioengineered 3, 343–346 (2012).PubMed 
PubMed Central 

Google Scholar 
Ostaszewska-Bugajska, M., Rychter, A. M. & Juszczuk, I. M. Antioxidative and proteolytic systems protect mitochondria from oxidative damage in S-deficient Arabidopsis thaliana. J. Plant Physiol. 186, 25–38 (2015).PubMed 

Google Scholar 
Zhang, L. et al. Sulfur deficiency-induced glucosinolate catabolism attributed to two β-glucosidases, BGLU28 and BGLU30, is required for plant growth maintenance under sulfur deficiency. Plant Cell Physiol. 61, 803–813 (2020).CAS 
PubMed 

Google Scholar 
Takahashi, H., Kopriva, S., Giordano, M., Saito, K. & Hell, R. Sulfur assimilation in photosynthetic organisms: Molecular functions and regulations of transporters and assimilatory enzymes. Annu. Rev. Plant biol. 62, 157–184 (2011).CAS 
PubMed 

Google Scholar 
Butterfield, N. J. Bangiomorpha pubescens n. gen., n. sp.: Implications for the evolution of sex, multicellularity, and the Mesoproterozoic/Neoproterozoic radiation of eukaryotes. Paleobiology 26, 386–404 (2000).
Google Scholar 
Collier, J. L. & Grossman, A. R. Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: Not all bleaching is the same. J. Bacteriol. 174, 4718–4726. https://doi.org/10.1128/jb.174.14.4718-4726.1992 (1992).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Kaur, H. et al. Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition. Biochimie 93, 175–186. https://doi.org/10.1016/j.biochi.2010.09.008 (2011).CAS 
Article 
PubMed 

Google Scholar 
Said, R. B. et al. Effects of depth and initial fragment weights of Gracilaria gracilis on the growth, agar yield, quality, and biochemical composition. J. Appl. Phycol. 30, 2499–2512 (2018).
Google Scholar 
Bird, K. T. Agar production and quality from Gracilaria sp. strain G—16: Effects of environmental factors. Bot. Mar. 31, 33–38 (1988).
Google Scholar 
Cote, G. & Hanisak, M. Production and properties of native agars from Gracilaria tikvahiae and other red algae. Bot. Mar. 29, 359–366 (1986).CAS 

Google Scholar 
Lahaye, M. & Yaphe, W. Effects of seasons on the chemical structure and gel strength of Gracilaria pseudoverrucosa agar (Gracilariaceae, Rhodophyta). Carbohydr. Polym. 8, 285–301 (1988).CAS 

Google Scholar 
Yaphe, W. Eleventh International Seaweed Symposium 171–174 (Springer, 1984).
Google Scholar 
Duckworth, M., Hong, K. & Yaphe, W. The agar polysaccharides of Gracilaria species. Carbohydr. Res. 18, 1–9 (1971).CAS 

Google Scholar 
Rotem, A., Roth-Bejerano, N. & Arad, S. Effect of controlled environmental conditions on starch and agar contents of Gracilaria sp. (Rhodophyceae) 1. J. Phycol. 22, 117–121 (1986).CAS 

Google Scholar 
Arad, S. M., Lerental, Y. B. & Dubinsky, O. Effect of nitrate and sulfate starvation on polysaccharide formation in Rhodella reticulata. Bioresour. Technol. 42, 141–148 (1992).CAS 

Google Scholar  More

Carpenter, K. E. et al. One-third of reef-building corals face elevated extinction risk from climate change and local impacts. Science 321, 560–563 (2008).CAS 
PubMed 
Article 

Google Scholar 
Pollock, F. J. et al. Coral-associated bacteria demonstrate phylosymbiosis and cophylogeny. Nat. Commun. 9, 4921–4932 (2018).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Vega Thurber, R. et al. Metagenomic analysis of stressed coral holobionts. Environ. Microbiol. 11, 2148–2163 (2009).PubMed 
Article 
CAS 

Google Scholar 
Rosenberg, E. & Zilber-Rosenberg, I. Microbes drive evolution of animals and plants: the hologenome concept. mBio 7, e01395 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
van Oppen, M. J. H. & Blackall, L. L. Coral microbiome dynamics, functions and design in a changing world. Nat. Rev. Microbiol. 17, 557–567 (2019).PubMed 
Article 
CAS 

Google Scholar 
Reshef, L., Koren, O., Loya, Y., Zilber-Rosenberg, I. & Rosenberg, E. The coral probiotic hypothesis. Environ. Microbiol. 8, 2068–2073 (2006).CAS 
PubMed 
Article 

Google Scholar 
Ainsworth, T. D., Thurber, R. V. & Gates, R. D. The future of coral reefs: a microbial perspective. Trends Ecol. Evol. 25, 233–240 (2010).PubMed 
Article 

Google Scholar 
Lee, S. M. et al. Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501, 426–429 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Freter, R. The fatal enteric cholera infection in the guinea pig, achieved by inhibition of normal enteric flora. J. Infect. Dis. 97, 57–65 (1955).CAS 
PubMed 
Article 

Google Scholar 
Corr, S. C. et al. Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118. Proc. Natl. Acad. Sci. USA 104, 7617–7621 (2007).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Kamada, N. et al. Regulated virulence controls the ability of a pathogen to compete with the gut microbiota. Science 336, 1325–1329 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Khosravi, A. et al. Gut microbiota promote hematopoiesis to control bacterial infection. Cell Host Microbe 15, 374–381 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Li, J., Kuang, W. Q., Long, L. J. & Zhang, S. Production of quorum-sensing signals by bacteria in the coral mucus layer. Coral Reefs 36, 1235–1241 (2017).Article 

Google Scholar 
Alagely, A., Krediet, C. J., Ritchie, K. B. & Teplitski, M. Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens. ISME J. 5, 1609–1620 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Krediet, C. J., Ritchie, K. B., Alagely, A. & Teplitski, M. Members of native coral microbiota inhibit glycosidases and thwart colonization of coral mucus by an opportunistic pathogen. ISME J. 7, 980–990 (2013).CAS 
PubMed 
Article 

Google Scholar 
Thompson, F. L., Hoste, B., Thompson, C. C., Huys, G. & Swings, G. The coral bleaching Vibrio shiloi Kushmaro et al. 2001 is a later synonym of Vibrio mediterranei Pujalte and Garay 1986. Syst. Appl. Microbiol. 24, 516–519 (2001).CAS 
PubMed 
Article 

Google Scholar 
Santoro, E. P. et al. Coral microbiome manipulation elicits metabolic and genetic restructuring to mitigate heat stress and evade mortality. Sci. Adv. 7, eabg3088 (2021).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Tang, K. H. et al. Antagonism between coral pathogen Vibrio coralliilyticus and other bacteria in the gastric cavity of scleractinian coral Galaxea fascicularis. Sci. China-Earth Sci. 63, 157–166 (2020).CAS 
Article 

Google Scholar 
Zhou, Y. Q. et al. Identification of bacteria-derived urease in the coral gastric cavity. Sci. China-Earth Sci. 63, 1553–1563 (2020).CAS 
Article 

Google Scholar 
Chen, B. et al. Microbiome community and complexity indicate environmental gradient acclimatisation and potential microbial interaction of endemic coral holobionts in the South China Sea. Sci. Total Environ. 765, 142690 (2021).CAS 
PubMed 
Article 

Google Scholar 
Tout, J. et al. Increased seawater temperature increases the abundance and alters the structure of natural Vibrio populations associated with the coral Pocillopora damicornis. Front. Microbiol. 6, 432 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
Savary, R. et al. Fast and pervasive transcriptomic resilience and acclimation of extremely heat-tolerant coral holobionts from the northern Red Sea. Proc. Natl. Acad. Sci. USA 118, e2023298118 (2021).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Vezzulli, L. et al. Vibrio infections triggering mass mortality events in a warming Mediterranean Sea. Environ. Microbiol. 12, 2007–2019 (2010).CAS 
PubMed 
Article 

Google Scholar 
Rosenberg, E. & Falkovitz, L. The Vibrio shiloi/Oculina patagonica model system of coral bleaching. Annu. Rev. Microbiol. 58, 143–159 (2004).CAS 
PubMed 
Article 

Google Scholar 
Gibbin, E. et al. Vibrio coralliilyticus infection triggers a behavioural response and perturbs nutritional exchange and tissue integrity in a symbiotic coral. ISME J. 13, 989–1003 (2019).CAS 
PubMed 
Article 

Google Scholar 
Kimes, N. E. et al. Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus. ISME J. 6, 835–846 (2012).CAS 
PubMed 
Article 

Google Scholar 
Banin, E., Vassilakos, D., Orr, E., Martinez, R. J. & Rosenberg, E. Superoxide dismutase is a virulence factor produced by the coral bleaching pathogen Vibrio shiloi. Curr. Microbiol. 46, 418–422 (2003).CAS 
PubMed 
Article 

Google Scholar 
Meron, D. et al. Role of flagella in virulence of the coral pathogen Vibrio coralliilyticus. Appl. Environ. Microbiol. 75, 5704–5707 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Rubio-Portillo, E. et al. Virulence as a side effect of interspecies interaction in Vibrio coral pathogens. mBio 11, e00201-20 (2020).Rubio-Portillo, E., Yarza, P., Penalver, C., Ramos-Espla, A. A. & Anton, J. New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities. ISME J. 8, 1794–1807 (2014).PubMed 
PubMed Central 
Article 

Google Scholar 
Bourne, D. G. et al. Microbial disease and the coral holobiont. Trends Microbiol. 17, 554–562 (2009).CAS 
PubMed 
Article 

Google Scholar 
Ben-Haim, Y., Zicherman-Keren, M. & Rosenberg, E. Temperature-regulated bleaching and lysis of the coral Pocillopora damicornis by the novel pathogen Vibrio coralliilyticus. Appl. Environ. Microbiol. 69, 4236–4242 (2003).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Gavish, A. R., Shapiro, O. H., Kramarsky-Winter, E. & Vardi, A. Microscale tracking of coral–vibrio interactions. ISME Commun. 1, 18 (2021).Shapiro, O. H. et al. Vortical ciliary flows actively enhance mass transport in reef corals. Proc. Natl. Acad. Sci. USA 111, 13391–13396 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Shapiro, O. H., Kramarsky-Winter, E., Gavish, A. R., Stocker, R. & Vardi, A. A coral-on-a-chip microfluidic platform enabling live-imaging microscopy of reef-building corals. Nat. Commun. 7, 10860 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Chen, D. D. et al. Identification and characterization of microsatellite markers for scleractinian coral Galaxea fascicularis and its symbiotic zooxanthellae. Conservation. Genet. Resour. 5, 741–743 (2013).Article 

Google Scholar 
Parks, D. H. et al. A complete domain-to-species taxonomy for bacteria and archaea. Nat. Biotechnol. 38, 1079–1086 (2020).CAS 
PubMed 
Article 

Google Scholar 
Liu, X. et al. Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens. Environ. Microbiol. 21, 4212–4232 (2019).CAS 
PubMed 
Article 

Google Scholar 
Wang, P. et al. Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system. J. Antimicrob. Chemother. 74, 2559–2565 (2019).CAS 
PubMed 
Article 

Google Scholar 
Zeng, Z. et al. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis. ISME J. 10, 2787–2800 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
Wang, X. et al. Cryptic prophages help bacteria cope with adverse environments. Nat. Commun. 1, 147 (2010).Bardwell, J. C., McGovern, K. & Beckwith, J. Identification of a protein required for disulfide bond formation in vivo. Cell 67, 581–589 (1991).CAS 
PubMed 
Article 

Google Scholar 
Wang, X., Kim, Y. & Wood, T. K. Control and benefits of CP4-57 prophage excision in Escherichia coli biofilms. ISME J. 3, 1164–1179 (2009).CAS 
PubMed 
Article 

Google Scholar 
Wood, T. K., Gonzalez Barrios, A. F., Herzberg, M. & Lee, J. Motility influences biofilm architecture in Escherichia coli. Appl. Microbiol. Biotechnol. 72, 361–367 (2006).CAS 
PubMed 
Article 

Google Scholar 
Song, S., Guo, Y., Kim, J. S., Wang, X. & Wood, T. K. Phages mediate bacterial self-recognition. Cell Rep. 27, 737–749 (2019).CAS 
PubMed 
Article 

Google Scholar 
Krediet, C. J., Carpinone, E. M., Ritchie, K. B. & Teplitski, M. Characterization of the gacA-dependent surface and coral mucus colonization by an opportunistic coral pathogen Serratia marcescens PDL100. FEMS Microbiol. Ecol. 84, 290–301 (2013).CAS 
PubMed 
Article 

Google Scholar 
Guo, Y., Lin, J. & Wang, X. Rapid detection of temperate bacteriophage using a simple motility assay. Environ. Microbiol. Rep. 13, 728–734 (2021).CAS 
PubMed 
Article 

Google Scholar 
Tang, K. et al. Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment. Nucleic Acids Res. 49, e128 (2021).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Ding, J. Y., Shiu, J. H., Chen, W. M., Chiang, Y. R. & Tang, S. L. Genomic insight into the host–endosymbiont relationship of Endozoicomonas montiporae CL-33(T) with its coral host. Front. Microbiol. 7, 251 (2016).PubMed 
PubMed Central 

Google Scholar 
Yang, C. S. et al. Endozoicomonas montiporae sp. nov., isolated from the encrusting pore coral Montipora aequituberculata. Int. J. Syst. Evol. Microbiol. 60, 1158–1162 (2010).CAS 
PubMed 
Article 

Google Scholar 
Schreiber, L., Kjeldsen, K. U., Obst, M., Funch, P. & Schramm, A. Description of Endozoicomonas ascidiicola sp nov., isolated from Scandinavian ascidians. Syst. Appl. Microbiol. 39, 313–318 (2016).CAS 
PubMed 
Article 

Google Scholar 
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Lu, S. N. et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 48, D265–D268 (2020).CAS 
PubMed 
Article 

Google Scholar 
Mai-Prochnow, A. et al. Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several Gram-negative bacteria. J. Bacteriol. 190, 5493–5501 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Campillo-Brocal, J. C., Lucas-Elio, P. & Sanchez-Amat, A. Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity. MicrobiologyOpen 2, 684–694 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Chacon-Verdu, M. D., Gomez, D., Solano, F., Lucas-Elio, P. & Sanchez-Amat, A. LodB is required for the recombinant synthesis of the quinoprotein l-lysine-epsilon-oxidase from Marinomonas mediterranea. Appl. Microbiol. Biotechnol. 98, 2981–2989 (2014).CAS 
PubMed 
Article 

Google Scholar 
Gomez, D., Lucas-Elio, P., Solano, F. & Sanchez-Amat, A. Both genes in the Marinomonas mediterranea lodAB operon are required for the expression of the antimicrobial protein lysine oxidase. Mol. Microbiol. 75, 462–473 (2010).CAS 
PubMed 
Article 

Google Scholar 
Piewngam, P. et al. Pathogen elimination by probiotic Bacillus via signalling interference. Nature 562, 532–537 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Zipperer, A. et al. Human commensals producing a novel antibiotic impair pathogen colonization. Nature 535, 511–516 (2016).CAS 
PubMed 
Article 

Google Scholar 
Selva, L. et al. Killing niche competitors by remote-control bacteriophage induction. Proc. Natl. Acad. Sci. USA 106, 1234–1238 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Regev-Yochay, G., Trzcinski, K., Thompson, C. M., Malley, R. & Lipsitch, M. Interference between Streptococcus pneumoniae and Staphylococcus aureus: in vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae. J. Bacteriol. 188, 4996–5001 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Paul, J. H. Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas? ISME J. 2, 579–589 (2008).CAS 
PubMed 
Article 

Google Scholar 
Frazao, N., Sousa, A., Lassig, M. & Gordo, I. Horizontal gene transfer overrides mutation in Escherichia coli colonizing the mammalian gut. Proc. Natl. Acad. Sci. USA 116, 17906–17915 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Yu, M. et al. Purification and characterization of antibacterial compounds of Pseudoalteromonas flavipulchra JG1. Microbiology-SGM 158, 835–842 (2012).CAS 
Article 

Google Scholar 
James, S. G., Holmstrom, C. & Kjelleberg, S. Purification and characterization of a novel antibacterial protein from the marine bacterium D2. Appl. Environ. Microbiol. 62, 2783–2788 (1996).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Lucas-Elio, P., Gomez, D., Solano, F. & Sanchez-Amat, A. The antimicrobial activity of marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188, 2493–2501 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Imlay, J. A. & Linn, S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J. Bacteriol. 169, 2967–2976 (1987).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Los, J. M., Los, M., Wegrzyn, G. & Wegrzyn, A. Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents. Microb. Pathog. 47, 289–298 (2009).CAS 
PubMed 
Article 

Google Scholar 
Knowles, B. et al. Lytic to temperate switching of viral communities. Nature 531, 466–470 (2016).CAS 
PubMed 
Article 

Google Scholar 
Howard-Varona, C., Hargreaves, K. R., Abedon, S. T. & Sullivan, M. B. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. ISME J. 11, 1511–1520 (2017).PubMed 
PubMed Central 
Article 

Google Scholar 
Arkin, A. P. et al. KBase: The United States Department of Energy Systems Biology Knowledgebase. Nat. Biotechnol. 36, 566–569 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Luo, P., He, X. Y., Liu, Q. T. & Hu, C. Q. Developing universal genetic tools for rapid and efficient deletion mutation in Vibrio species based on suicide T-vectors carrying a novel counterselectable marker, vmi480. PLoS ONE 10, e0144465 (2015).Wang, P. et al. Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas. Microb. Cell Fact. 14, 11 (2015).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Bertani, L. E. & Bertani, G. Preparation and characterization of temperate, non-inducible bacteriophage P2 (host: Escherichia coli). J. Gen. Virol. 6, 201–212 (1970).CAS 
PubMed 
Article 

Google Scholar 
Garneau, J. R., Depardieu, F., Fortier, L. C., Bikard, D. & Monot, M. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci Rep. 7, 8292 (2017).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Pratt, L. A. & Kolter, R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol. Microbiol. 30, 285–293 (1998).CAS 
PubMed 
Article 

Google Scholar 
Page, A. J. et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31, 3691–3693 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Brynildsrud, O., Bohlin, J., Scheffer, L. & Eldholm, V. Rapid scoring of genes in microbial pan-genome-wide association studies with Scoary. Genome Biol. 17, 238 (2016).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Callahan, B. J. et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Yilmaz, P. et al. The SILVA and ‘All-species Living Tree Project (LTP)’ taxonomic frameworks. Nucleic Acids Res. 42, D643–D648 (2014).CAS 
PubMed 
Article 

Google Scholar 
Chong, J., Liu, P., Zhou, G. & Xia, J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data. Nat. Protoc. 15, 799–821 (2020).CAS 
PubMed 
Article 

Google Scholar 
Nagpal, S., Singh, R., Yadav, D. & Mande, S. S. MetagenoNets: comprehensive inference and meta-insights for microbial correlation networks. Nucleic Acids Res. 48, W572–W579 (2020).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar  More

Study siteThe study was conducted at the Center for Applied Research on the Environment and Sustainability (CARES) at The American University in Cairo, New Cairo, Egypt (30°01′11.7″N 31°29′59.8″E) from 12/Nov/2019 until 31st/March/2020. The experiment was carried out in a greenhouse-controlled environment with temperatures ranging from 18 to 23 °C and relative humidity between 60 and 70% during the growing period.Experimental designThe proposed design starts by treating brackish water using RO membrane separation technology, powered by an on-grid 10 kW photovoltaic solar panel as shown in Fig. 1. The permeate (freshwater) from the RO facility is directed to the aquaculture units of capacity of 1 m3, where the fish effluents are used as irrigation water and as the sole source of fertilizers for the crops.Figure 1Schematic Integrated model design. T1 Deep water culture system without sand, T2 Sandponics system with sand from October, T3 Sandponics system with sand from Beni suef, T4 Sandponics system with sand from Fayoum.Full size imageThe study followed a completely randomized design with four variants, i.e., an aquaponic deep-water culture system (T1) and three sandponics systems (T2–T4). The three sandponics systems were established with different sand collected from different sand locations in Egypt during the period between September and October 2019.Initially, an exploratory field trip was set to six different locations in Egypt to collect sand samples for lab analysis aimed at sourcing the most suitable sand for the system under study with regards to both the physical and chemical parameters. These areas include Ismailia Governorate; 30°34′55.2″N 31°50′08.1″E, 6th October governorate; 29°54′49.8″N 31°05′51.5″E, Benu Suef governorate; 28°53′18.4″N 30°45′12.9″E, Al-Minya governorate; 28.725799, 30.630305, and two sites from Fayoum governorate; 29°05′07.4″N 30°49′39.9″E.From the six locations in Egypt, preliminary sand analysis was carried out, and sand samples were also collected for both physical and chemical lab analysis at the Soil and Water Lab at the Agricultural Research Center in Dokki, Egypt. Following a thorough technical, field, mechanical, and lab chemical evaluation of the six sand samples from six locations, three sand locations/types were selected for experimentation that seemed fit and suitable for the current study. The criteria parameters for the shortlisting of sand included water retention potential of the sand by the percolation process, testing the carbonates level in the soil, the turbidity of the sand, porosity percentage and drainage potential of the sand. The three locations included 6th October (T2), Benu Suef (T3), and Fayoum site 2 (T4). In the second week of November 2019, ten cubic meter tracks of sand from the three above locations were set to collect sand from these areas to the research facility at CARES where the experiment was carried out.The study was carried out with two systems/setups, i.e., an aquaponic Deep Water Culture (DWC) and SP systems. The DWC model comprises a 1 m3 fish tank, a settlement tank, a mechanical filter, a biological filter, three grow beds, and a drainage tank. This system being the most practiced aquaponics technique was considered as the control. Fish effluent water flowed from the fish tank to the settlement tank to filter big solid wastes through the mechanical filter to remove the smaller solid wastes and the biological filter for the nitrification process. Then filtered water continues to the grow beds, where overflow drains into the drainage tank and back to the fish tank in a closed system.On the other hand, the variable in the three IAVS systems is the sand source. This system comprises three independent set-ups: a 1 m3 fish tank, three grow beds, and a drainage tank. Fish effluents flowed from the fish tank directly to the sand grow beds where water was supplied through irrigation drip lines using diaghram emitters connected with valves to ensure uniformity of water application to each grow bed.All the fish tanks were installed with the same fish stock size of 30 Nile tilapia (Oreochromis niloticus) from an existing fish stock at the research center with an average initial weight of 244 g and the same amount of water, initially 850L per tank. The fish was sourced from an already existing aquaponics system at the research center to avoid any transportation stress effects and related shocks on the small fish, leading to a lot of mortality cases. The fish were fed 3–4 times daily with commercial pellets containing 30% proteins, 5% crude lipid, 6% crude fiber, 13% Ash, and 9% moisture content supplied by Skretting Egypt. The feeding pattern and frequency were according to the fish body biomass percentage of 2–3% depending on the growth stage and upon reaching satiation.DesalinationThe experiment was entirely run with desalinated water produced from a desalination facility at the center. The desalination technology used was Reverse Osmosis (RO); in batch mode; using a Sea Water Pump with Energy Recovery Unit (model Danfoss-APP1.0/APM1.2). The RO membrane used is Hydraunatic SWC5-4040, from Lenntech company with an average salt rejection of 99.7%. Three modules were connected in a series arrangement (3 Pressure Vessels each equipped with a single module). Synthesized brackish water was prepared by dissolving industrial grade sodium chloride (sea salt) from El-Arish Governorate, Egypt. The salt chemical properties are presented in Table 1. Feedwater salinity was 10 mg/L, with an equivalent osmotic pressure equal to 8.61 bars. The osmotic pressure was calculated using Van’t Hoff relation. Permeate Total Dissolved Solids (TDS) was 192 mg/L, and brine TDS was 13.1 g/L as shown in Table 2.Table 1 Chemical properties of the used salt.Full size tableTable 2 Chemical properties of water samples used.Full size tableThe average pure water flux is 9.5 LMH and was calculated by dividing the permeate volume by the product of membrane surface area and time. Each batch run produced around 4 m3 of permeate, which was enough to irrigate the designated plant beds. The estimated average permeate recovery for the RO process is 22% and salt rejection exceeded 98.7%. The differential pressure between membrane inlet and outlet was equal to 1 bar, where membrane inlet pressure was 16 bars, and the outlet was 15 bars. The RO process operated at an average transmembrane pressure equal to 16 bars and an average permeate and brine flow rates equivalent to 3.49 and 12.41 Lpm, respectively. All experiment runs were performed at 25 °C.Plant materials and cultivation practiceSwiss chard bright lights (Beta vulgaris subsp. cicia) seeds were imported from Seed kingdom seed company in the USA. Seeds were sown in ¼ inch holes in a seed starting mix containing perlite and vermiculite and irrigated with a hand mist sprayer daily to keep the growing media always moist. Sowing was done on the 12th of November 2019, and seedlings were transplanted when they were 40 days old. Seedlings were transplanted into raised grow beds made of fiberglass material measuring 1.8 × 1.2 × 0.6 m for each of the four systems. The beds were raised off the ground by 0.5 m to allow drainage water from the bed to be collected and circulated back to the fish tank. Each bed was constructed with a drainage pipe at the bottom covered with a mesh net to prevent water blockage by the sand. Also, a 5 cm layer of small gravel was uniformly laid at the bottom of the beds to facilitate drainage, followed by sand with a height of 50 cm.In the IAVS systems, plants were irrigated using manually punched diaphragm emitters, and the irrigation flow rate was controlled using small plastic valves at the start of every irrigation tube. Emitters were installed in drip tubing at a 30 cm distance as well the tubing lines were also placed 30 cm between each other. Seedlings were transplanted 5 cm away from the emitters at 30 cm between rows and 30 cm within the row. Since the water was pumped with submersible pumps to the grow beds, regulatory pressure valves were installed in between the pump and the main irrigation line, and then water flows through the emitters into the row furrows. Water would then saturate in the sand and eventually drain at the bottom into drainage tanks and pumped back to the fish tanks.To maintain the water quality, two full cycles of water recirculation were run every day. Every irrigation cycle recirculated 25% of the fish tank, and complete drainage was allowed for a maximum of two hours. Plants were harvested upon reaching maturity for three cuts, except with the T1, which could not grow back after the second cut. Plants took 52 days from transplanting to reach the first cut, 20 days from cut 1 to cut 2, and as well 23 days from cut 2 to reach cut 3. Measurable crop parameters included plant height at harvesting/cutting, leaf area, number of leaves per plant, chlorophyll content, fresh weight per plant, and nutrient composition. Since the focus of SP is on the crops, fish were only measured to monitor their relative growth in terms of weight gained at harvesting/cutting time.Measurement of crop parametersPlants were cut 5 cm above the soil surface, and agronomical trait measurements from a representative sample of 12 plants per replicate were taken as follows.Plant heights were taken using a foot ruler and averages determined. Leaf number was obtained as the number of leaves counted per plant and averages determined. Leaf area was calculated according to the equation reported by Yeshitila and Taye16.$${text{Leaf}} , {text{ Area }}left( {{text{cm}}^{{2}} } right) = , – {422}.{973} + { 22}.{752}0{text{L }}left( {{text{cm}}} right) , + { 8}.{text{31W }}left( {{text{cm}}} right)$$where L and W represent the leaf length and Leaf width respectively, − 422.973 is a constant relating to the shape of the leaf of Swiss chard developed by the author under citation.Chlorophyll content was measured using MC-100 chlorophyll meter from Apogee Instruments, Inc, and data was expressed as SPAD averages. Fresh weight was measured using a digital weighing balance and data expressed as g/plant.Sand testSand samples were obtained and sent for analysis at the Soils, Water and Environment Research Institute, Agricultural Research Center, Giza, Egypt. The Electrical conductivity (EC) values were measured from the sand paste extract; pH values were taken from sand suspensions at ratio of 1:2.5 as described by Estefan17. The available nitrogen in the sand sample was extracted using potassium chloride (KCl) as an extractable solution with the ratio of (5gm sand to 50 ml KCl) and determined using the micro- kjeldahl method. Available potassium was determined using a flame photometer, and the other elements in the sand sample were determined by using inductively coupled plasma (ICP) Spectrometry (model Ultima 2 JY Plasma)18,19. The physical and chemical characteristics of the used sand are presented in Table 3.Table 3 (a): Chemical analysis of field sand samples, (b): Available macro, micronutrients, and heavy metals content of the sand samples.Full size tableWater analysisEvery 15 days, a measured amount of desalinated water was added to a standard mark of 850L in the fish tanks to compensate for the consumed amount of water in the system. Fish water quality parameters such as water temperature, pH, and dissolved oxygen (DO) was closely monitored using automated digital Nilebot technologies by Conative labs to fit the ideal required levels as reported by Somerville et al.20. In contrast, ammonia, nitrite, and nitrate were adjusted using an API test kit every week. These parameters’ recorded values were as follows: water temperature ranged between 25 and 28 °C, DO range between 6–7 mg/L, and pH between 6.5 and 7.0. Ammonia levels were kept below 1 mg/L. Elements in water samples were determined according to EPA methods18 using inductively coupled plasma (ICP) Spectrometry (model Ultima 2 JY Plasma) as presented in Table 4.Table 4 Water sample analysis for the different systems’ fish tanks and sump tanks.Full size tableNutritive composition analysisAccording to Official methods of analysis from the association of official analytical chemists (A.O.A.C) (1990), moisture content and Vitamin C were determined. Vitamin A was determined according to the procedures described by Aremu and Nweze21. Briefly, 100 g of the sample were homogenized, from which 1 g was obtained and soaked in 5 mL methanol for two hours at room temperature in the dark for complete extraction of a pro-vitamin A carotenoid, β-carotene. Separation of the β-carotene layer was achieved through the addition of hexane to the sample, and moisture was removed using sodium sulphonate. The absorbance of the layer was measured at 436 nm using hexane as a blank. β-carotene was calculated using the formula:$$beta {text{-carotene }}left( {{mu g}/{1}00{text{ g}}} right) , = {text{ Absorbance }}left( {text{436 nm}} right) , times {text{ V }} times {text{ D }} times { 1}00 , times { 1}00/{text{W }} times {text{ Y}}$$where: V = total volume of the extract; D = Dilution factor; W = Sample weight; Y = Percentage dry matter content of the sample.Vitamin A was then determined according to the concept of Retinol Equivalent (RE) of the β-carotene content of the vegetables using the standard conversion formula. Total hydrolyzable carbohydrates were determined as glucose using phenol–sulfuric acid reagent as described by Michel22.Vitamin C content was determined using dichlorophenol indophenol reagent. As such, 10 g of fresh leaf tissues, were crushed using a motor and pestle in the presence of 10 ml metaphosphoric acid 6% (Merck). This was followed by centrifugation at 4000×g for 5 min at 4 °C. Five mL of the supernatant were transferred into an Erlenmeyer flask, and 20 mL of 3% metaphosphoric acid were added. The extract was titrated by dichlorophenol indophenol (Sigma-Aldrich) until a rose color was observed. Vitamin C (mg/100 g FW) was then calculated and based on the standard curve of l-Ascorbic acid (Merck) concentrations.For the determination of protein and mineral content, 0.5 g of dried samples were digested using sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) as described by Cottenie23. From the extracted sample, the following minerals were determined:Nitrogen was determined according to the procedures described by Plummer24. Briefly, 5 mL of the digestive solution was distilled with 10 mL of sodium hydroxide (NaOH) for 10 min to obtain ammonia. Back titration was then used to determine the amount of nitrogen present in ammonia. Protein content was calculated by multiplying total nitrogen by 6.25 according to methods of AOAC25.Phosphorus content was determined calorimetrically (660 nm) according to the procedures described by Jackson26. Potassium, Calcium, and Sodium were determined against a standard using a flame-photometer (JEN way flame photometer) as described by Piper27. Magnesium (Mg), Copper (Cu), Manganese (Mn), Zinc (Zn), and Iron (Fe) content were determined using Atomic Absorption Spectrophotometer, Pyeunican SP1900, according to methods described by Liu28.The moisture percentage of leaf samples was determined by weighing the fresh weight for each sample (Fw), then dried for 72 h at 80 °C. The dry matter weight was record as Dw. The leaf water content was then calculated as the following:$${text{Moisture}};{text{ content }}left( % right) , = , left( {{text{Fw}} – {text{Dw}}} right) , /{text{ Fw}} * {1}00$$Statistical analysisStatistical comparisons among means of more than two groups were performed with analysis of variance (ANOVA) using SPSS V22, and the difference in means was analyzed by Tukey’s test at α = 0.05. Statistical differences were considered significant at P ≤ 0.05 in triplicates and data expressed as mean ± S.D.Plant materialAll plant materials and related procedures in this study were done in accordance with the guidelines of the Institutional Review Board of the American University in Cairo and the Ministry of Agriculture and Land Reclamation in Egypt.Ethics approvalThis study followed the guidelines and approval of Committee of Animal Welfare and Research Ethics, Faculty of Agriculture, Kafrelsheikh University, Egypt. More