Physiological responses (Hsps 60 and 32, caspase 3, H2O2 scavenging, and photosynthetic activity) of the coral Pocillopora damicornis under thermal and high nitrate stresses
Introduction
Global climate change has exposed corals to several threats, such as high seawater temperatures (Baker et al., 2008; Hoegh-Guldberg, 1999), intense solar radiation (Lesser and Farrell, 2004), and increased concentrations of atmospheric CO2 which cause ocean acidification (Hoegh-Guldberg et al., 2007; Kavousi et al., 2015). Furthermore, anthropogenic sources of metal pollution (Prouty et al., 2013), nutrient loads from excessive fertilizer use (Møller et al., 2015), and microplastic contamination in marine ecosystems (Moore, 2008) negatively affect the coral health. Consequently, extensive coral bleaching and diseases have caused a decline in the coral reefs worldwide (Douglas, 2003; Fitt et al., 2001; Schoepf et al., 2013). Additionally, the combination of multiple stresses which may result in synergistic effects, severely disrupt the physiology of the coral holobiont (Baker et al., 2018; Coles and Jokiel, 1978).
Optimal and sustainable concentrations of nutrients and organic matter in seawater are crucial factors to maintain the coral health (Brown, 1997). Specific concentrations of dissolved inorganic nitrogen (DIN) (the sum of nitrate, nitrite and ammonium) are utilized as a nitrogen source by the Symbiodiniaceae for primary productivity. In general, DIN concentrations in oligotrophic areas such as reef lagoons are very low, with values ranging from 0.02 to less than 1 μM (Fabricius, 2005). The question is how higher than normal DIN concentrations could affect the coral physiology. Atkinson et al. (1995) found that corals grew well at high DIN concentrations (up to 5 μM) in the Waikiki Aquarium, Honolulu, Hawaii. However, Fabricius and De'ath (2004) indicated a substantial decrease in seawater quality around the Great Barrier Reef, resulting from land-based runoff with high nitrate (NO3-) concentrations, caused macroalgal dominance and decreased the proportion of hard corals and octocorals. High nutrient concentrations generally enter the shallow lagoons through riverine inputs and spring water flashes particularly during low tide causing negative impacts on the coral ecosystem (Wittenberg and Hunte, 1992; Faxneld et al., 2010; Shilla et al., 2013). High concentrations of inorganic nutrients, such as NO3-, ammonia (NH4+), and phosphate (PO43-) are used in agricultural fertilizers. Meekaew et al. (2014) reported high NO3- concentrations (up to 11.8 μM) in Bise, Okinawa. Similarly, Shimoda et al. (1998) and Omori (2011) reported that fertilizers commonly used in sugarcane, rice, pineapple, and vegetable fields containing high NO3- concentrations, negatively affected the corals around the Okinawa Islands. It was also found that high DIN concentrations combined with elevated seawater temperature resulted in synergistic effects, which strongly affect the maximum quantum yield or the photosynthetic activity (Fv/Fm) and maximum excitation pressure (Qm) of photosystem II (PSII) in Symbiodiniaceae. This was clearly observed in the coral Pocillopora damicornis where recovery was challenging after being subjected to those combined stressors (Chumun et al., 2013). In addition, the effects of elevated seawater temperature and high light intensity in combination with high NO3- concentrations increased the damage to the symbionts and led to coral bleaching (Higuchi et al., 2015). Furthermore, other studies observed that DIN enrichment reduced thermal tolerance, increased coral sensitivity to bleaching (Wiedenmann et al., 2013; Burkepile and Burkepile, 2019), promoted cell apoptosis by activating the caspase enzyme cascade and autophagy (Dunn et al., 2007; Dunn and Weis, 2009), increased coral respiration and decreased in coral calcification (Marubini and Davies, 1996). The above studies showed how increasing nutrients inputs in reef lagoons could negatively impact coral health. Moreover, recent observations in Okinawan reefs revealed that corals are exposed to prolonged periods of thermal stress in shallow lagoons during the summer (Kayanne, 2017) most probably exacerbating the negative impacts of excess nutrient loading. Under this scenario, we wanted to explore coral physiological responses at molecular level. Our approach was to test the effect of single and combined stresses on the expression of Hsp60 and Hsp32, the regulation of antioxidant enzymes and caspase 3 of the coral Pocillopora damicornis and its symbiodiniaceae.
The expression of Hsps is mainly induced under several stress conditions, such as thermal stress, UV radiation, diseases, and heavy metal pollution (Tom et al., 1999; Whitley et al., 1999). This pattern has been observed in all living organisms (from bacteria to humans), and plays essential roles in protecting and repairing cell damage (recovery mechanism) (Arya et al., 2007; Lanneau et al., 2008). Hsps promote refolding of denatured proteins, protect against aggregation, and regulate stress-induced apoptosis, thus, supporting cell survivability (Kregel, 2002). Hsp60 (~60 kDa) is a chaperone that regulates protein structure under normal and stress conditions. This protein is synthesized and transported from the mitochondria and since Hsp60 promotes folding of newly synthesized proteins, it has an anti-apoptotic effect (Seveso et al., 2013, Seveso et al., 2016a). Conversely, Hsp32 (~32 kDa) is a heme oxygenase 1 (HO-1) enzyme that protects cells from oxidative stress, degrades toxic heme into free iron, and removes carbon monoxide (CO) and biliverdin from the cytoplasm (Errico et al., 2010; Seveso et al., 2016b). Coral studies on the expression of Hsps have mainly focused on isolated environmental stressors, such as increased light intensity, elevated seawater temperature, coral disease conditions, and cold seawater shock (for example Chow et al., 2009; Fitt et al., 2009; Mayfield et al., 2011; Robbart et al., 2004; Seveso et al., 2014). Most of these studies demonstrated Hsps upregulation in corals exposed to environmental stressors. However, studies on effect of combined stresses on the Hsps in corals have rarely been conducted despite constant exposure of reefs to several simultaneous stressors. Therefore, some of the effects of combined stressors that can eventually result in synergistic effects remain unknown. Apoptosis, a form of programmed cell death, occurs in different stages that are interconnected by the action of specific proteases or caspases operating in a proteolytic cascade (Dunn et al., 2007). Consequently, caspases are used as markers because they remain inactive in normal coral cells, but under specific stresses, including oxidative stress and other multiple physiological stresses, they induce cell apoptosis (Richier et al., 2006; Weis, 2008). To investigate the induced oxidative stress in coral cells, H2O2 scavenging activity was monitored since H2O2 is stable in both symbiont and host, and therefore, could easily pass through the cell membranes, especially from the symbiont to the coral cells (Palmer et al., 2009).
To better understand the cellular mechanisms underpinning coral physiological responses, particularly to high NO3- concentrations in combination with elevated seawater temperature stresses, we designed an incubation experiment with four different treatments as normal condition (control), high temperature (normal temperature plus 4 °C), high NO3- (~10 μM) and their combination along 60 h with every 12 h observations to assess the modulation of Hsps along with other biomarkers, and the variations in coral morphological parameters, such as the symbiont algal density and their pigments. Thus, we aimed to estimate 1) the photophysiological variations of Symbiodiniaceae associated with P. damicornis regarding changes in the maximum quantum yield (Fv/Fm), photosynthetic pigment (Chl a) concentration, and cell density; 2) differential expression of Hsp60 and Hsp32 under experimental stresses; 3) regulation of caspase 3 and its relationship with oxidative stress and apoptosis; and 4) morphological variations in P. damicornis during the 60 h experiment involving different responses of the coral to the stress conditions.
Section snippets
Sampling site and coral collection
Pocillopora damicornis was sampled at Sesoko reef, Sesoko Island, Okinawa, Japan (26°38′N 127°51′E), at low tide (depth 0.5–1 m) on September 9 and 10 of 2018. Seawater temperature was examined at the low ebb of a spring tide from 12:00 to 16:00 in the sampling site and it ranged between 26.2 °C and 31.4 °C (DEFI2-T, JFE Advantech Co. Ltd., Hyogo, Japan) during the sampling period (Table S1). The maximum monthly mean (MMM) of the seawater surface temperature (SST) during September 2018 was
Coral morpho-physiological conditions
Coral fragments in the control (28T + AN) and high NO3- stress (28T + HN) treatments did not show considerable morphological changes during the 60 h of incubation. In contrast, the fragments were discolored after 48 h of exposure and partially bleached after 60 h of incubation under high-temperature stress (32T + AN) (Fig. 1). However, the coral fragments were severely discolored in the form of partial bleaching under the combined stresses (32T + HN) at 36 h, and the tissues were detached at
Discussion and conclusions
As benthic sessile organisms, corals are incapable of migrating to healthy environments to evade the multiple environmental stresses that they are exposed to. However corals can tackle these stresses by their adaptive physiological responses, which represent the first line of defense against unfavorable conditions (Rosic et al., 2014; Seveso et al., 2016a). Modulation of molecular chaperones such as Hsps, apoptotic proteases like caspase 3, and scavenging enzymes for reactive oxygen species
Ethical statement
Permission to collect P. damicornis samples was granted by Moritake Tomika, Governor of Okinawa Prefecture (permit no. 30–55) in July 2018. The permit period was extended from July 20, 2018 to July 19, 2019. The study was reviewed by the Okinawa Prefectural Government following established protocols.
Funding
This research was supported by the Environmental Leaders Program of Shizuoka University (ELSU) through a Research Promotion Grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the 50th Anniversary Grant of the Mitsubishi Corporation on Global Coral Reef Conservation Project (GCRCP).
Coral sampling in the field and preparation of the incubation system was supported by the volunteer program of the Mitsubishi Corporation.
CRediT authorship contribution statement
Montaphat Thummasan: conceptualization, methodology, formal analysis, investigation, writing–original draft, writing–review & editing, visualization. Beatriz E. Casareto: Conceptualization, methodology, investigation, resource, validation, writing–original draft, writing–review & editing, supervisor. Chitra Ramphul: Validation, data curation, writing–original draft, writing–review & editing. Toshiyuki Suzuki: Investigation, visualization, formal analysis, data curation Keita Toyoda:
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors are grateful to the Tropical Biosphere Research Center staff, University of the Ryukyus, Japan, for providing the necessary incubation facilities. We would like to thank K S. Uehara for his support during the field survey and coral collection. The authors are also grateful to all members of the Marine Biogeochemistry Laboratory, Shizuoka University. Montaphat Thummasan is grateful to the ENKEI Foundation scholarship awarded in the fiscal years 2018 and 2019. We thank Editage
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