Creseis acicula belongs to the Pteropoda order, Cavoliniidae family, and Creseis genus. This pteropod is the largest in the Creseis genus. C. acicula lives in the upper portion of the water column (less than 500 m), and is abundant in the Atlantic, Indian, and Pacific Oceans (Albergoni, 1975). The temporal dynamics and spatial distribution patterns of pteropods are well studied. C. acicula outbreaks have been reported in the coastal waters of India (Sakthivel and Haridas, 1974; Peter and Paulinose, 1978; Pillai and Rodrigo, 1984; Naomi, 1988), Japan (Nishimura, 1965; Morioka, 1980), Gulf of Mexico (Hutton, 1960), and in the Mediterranean Sea (Burgi and Devos, 1962; Albergoni, 1975; Kokelj et al., 1994) between the 1960s and the 1990s, with the highest abundance recorded at about 500 inds m^–3. In Daya Bay, South China Sea, C. acicula generally appear from March to November each year, with average abundances ranging from 0.04 to 1.9 inds m^–3 (Xu, 1989). Since the 1990s, there was no report of C. acicula bloom in the world. However, a massive aggregation occurred from June to July 2020 near the thermal discharge area of Daya Bay Nuclear Power Plant (DNPP), with the highest abundance reaching 4525–5595 inds m^–3 (Dai et al., 2020; Zhong et al., 2021). Large amount of C. acicula gathered around the thermal discharge area of DNPP and seriously affected the normal operation of the plant.

The outbreak mechanism of C. acicula is very complex, like any other species (Wishner et al., 1995; Baliarsingh et al., 2020; Maas et al., 2020). The distribution of C. acicula is restricted by various physical and chemical environmental parameters, such as temperature, salinity, food, oxygen, and water depth (Herman, 1998; Dai et al., 2020; Zeng et al., 2021). The potential mechanisms for the bloom of C. acicula include optimum temperature and salinity (or the temperature and salinity are not at the most optimal levels for C. acicula, but they might be worse for the competitors or predators of C. acicula) and an adequate food supply for C. acicula. As Dai et al. (2020) suggested that the initiation of C. acicula bloom in Daya Bay well-matched a sharp increase of temperature and chlorophyll a, as well as an abrupt decrease of salinity attributed to a heavy rainfall happened before the bloom which lasted for more than 20 days. However, till now, there is very little information on the causes of C. acicula blooms from a physiological point of view. A further understanding of the physiological mechanisms regulating the blooms will contribute to our knowledge of the survival tolerance of this species.

The objective of the present study was to investigate the effects of temperature and salinity on the physiological responses of C. acicula, and to provide the useful information on the outbreak reasons of C. acicula around the thermal discharge area of DNPP.

At the end of the incubation, the survival rates of C. acicula were > 90% for the 17, 20, 23, 26, 29, and 32°C treatments, and the 28, 33, and 38 ppt treatments. The survival rate of C. acicula was ∼50% for the 23 ppt treatment. Unfortunately, no C. acicula survived in the 35°C and 18 ppt treatments. The survival results suggest that C. acicula can tolerate a temperature range between 17 and 32°C and a salinity range between 23 and 38 ppt.

The results of the present study suggest that C. acicula has the ability to regulate its metabolisms, e.g., respiration, ammonia excretion, and calcification rates, within a relatively broad range of water temperature and salinity. This could be an advantage for its worldwide distribution. The results showed that the OCR of C. acicula increased by 145%, as the experimental temperature increased from 17 to 32°C, and its value decreased at 35°C. The respiration rates of pteropod species increased with increasing temperature (Seibel et al., 2007; Comeau et al., 2010; Maas et al., 2012; Bednaršek et al., 2016). However, the correlation between respiration and temperature was not linear in C. acicula. This is consistent with previous findings reported for copepod species, usually rising more steeply at the upper end and falling when the lethal temperature is approached (Krause et al., 2003). This indicated that C. acicula has a metabolic regulation ability. The decrease in OCR here reported suggests that when the water temperature reached 35°C, C. acicula entered a state of physiological stress and paid relatively higher energy cost. On the other hand, AER in C. acicula increased with increasing temperature, with the highest value observed at 35°C. It is well-known that ammonia is the major form of nitrogen waste–derived from protein metabolism–excreted by marine zooplankton (Butler et al., 1970; Corner and Davies, 1971). Therefore, the high ammonia production at 35°C indicates that C. acicula uses more protein as its energy source in higher water temperature than in lower water temperature conditions.

Q₁₀ has been recognized as a common parameter that reflects the adjustments related to the enzymatic and physiological requirements for energy when temperature increases within natural range (Kita et al., 1996; Manush et al., 2004; Kim et al., 2005). As for most animals, the normal Q₁₀ value for pteropods is approximately 2–3 (Fry, 1971). In the present study, the Q₁₀ for excretion did not clearly change with the increase in temperature. Whereas, the increase in oxygen consumption with rising temperature is fully accounted for by the basal metabolism (Hirche, 1987). Thus, in C. acicula the Q₁₀ for respiration ranged from 2.23 to 1.61 at the 17–32°C range, which indicated that this pteropod is well adapted to these temperatures. However, when the temperature increased to 32–35°C, the Q₁₀ for respiration was lowered to 0.79, indicating that C. acicula is no longer sensitive to temperature variation when temperature exceeded the optimum value.

The principle of osmotic adjustment holds that when osmotic pressure is at a minimum, the metabolic level is the lowest; when salinity is higher or lower than the isotonic point, more energy is consumed to maintain homeostasis, and the metabolic level increases (Cao and Wang, 2015). In this study, the OCR of C. acicula increased as the salinity increased from 18 to 28 ppt. A turning point was observed at 28 ppt, beyond which the OCR decreased as salinity increased from 28 to 38 ppt. Although AER in C. acicula did not significantly vary with salinity, it firstly decreased to the lowest level at 33 ppt, and then increased at 38 ppt, showing an opposite pattern to that observed for OCR. This supports the theory that the energetic cost associated with ionic and osmotic regulation is minimal within the normal salinity range tolerated by a species at species life stages (Morgan and Iwama, 1991). And at the same time, neither OCR nor AER at 28 ppt significantly differed from the corresponding values both at 23 and 33 ppt, indicating that C. acicula could well adapt to salinity range from 23 to 33 ppt. The decrease in OCR at salinities of 18 and 38 ppt is also reflected in the observed retraction of the pteropod body inside its shell–which is similar to valve closure in bivalves–that occurs when salinity either drops or increases excessively (Berger and Kharazova, 1997).

The O:N ratio can be considered as representative of the overall metabolic balance of an organism. It is usually used as an index of lipid versus protein catabolism that reflects the effects of environmental stressors on zooplankton energy reserves (Mayzaud and Conover, 1988). Typically, an O:N ratio lower than 7 indicates a protein-only catabolism; values between 7 and 17, indicating a protein-oriented catabolism; and values >17, indicating a lipid/carbohydrate catabolism (Mayzaud and Conover, 1988; Ikeda et al., 2000). In the present study, the O:N ratio values calculated for C. acicula fall into a narrow range (from 3.24 to 5.13). Previous studies also reported a low O:N ratio for some adult pteropod species, such as Clio pyramidata (4.4) and Limacina helicina antarctica (2–9) (Thibodeau et al., 2020). Thus, pteropod species, including C. acicula in this study, use protein as the major metabolism substrate.

CaCO₃ occurs in several solid forms, including aragonite and calcite, and between these two, the former is less stable than the latter (Fransson et al., 2016). C. acicula is sensitive to chemical changes in seawater due to its highly soluble aragonite shells (Tunçer et al., 2021). The CaCO₃ saturation (Ω) is used as a chemical indicator for the dissolution potential of this compound (Fransson et al., 2016). Increasing temperature leads to increased Ω (Chierici et al., 2011), while freshwater supply causes a decrease in this parameter (Sejr et al., 2011; Fransson et al., 2015). Severe shell dissolution of aragonite-forming organisms takes place when Ωa < 1.4 (Bednaršek and Ohman, 2015). In this study, the CR of C. acicula firstly increased to the highest level at 26°C and 33 ppt, but then decreased at 29–35°C and 38 ppt; CR values were negative at high temperature (35°C) and at low salinities (18–23 ppt). The Ωa increased from 17 to 35°C and from 18 to 38 ppt, with all Ωa values being > 1.4, except at 26 ppt after incubation (Table 2). Thus, it is easy to understand that the CR was negative at low salinities (18 and 23 ppt) and the salinity had a stronger effect on the CR than temperature. While, the negative CR values observed at 35°C may have been caused by the presence of physiological stress due to higher temperature–which increases the risk of dissolution and thinning of the aragonitic shells in C. acicula (Fransson et al., 2016)–rather than by the variation of Ωa. For example, higher energetic costs may derive from necessary repair calcification processes in response to shell dissolution (Lischka and Riebesell, 2012; Blachowiak-Samołyk et al., 2015; Vader et al., 2015). In this study, for the negative CR, C. acicula could not meet the needs for increased energetic costs, and responded with metabolic depression to save energy and increase its chance of survival. This is consistent with the findings of previous studies (Bibby et al., 2007; Rosa and Seibel, 2008; Navarro et al., 2013).

Studies of various marine species suggest that a number of large swarms or aggregations are due to biological forces (i.e., development time, bloom timing, species interactions) (Wishner et al., 1995; Ji et al., 2009; Baliarsingh et al., 2020). In this study, the experimental C. acicula were at mature stage with ovulating. After this study, we also observed the reproductive development of C. acicula. But the metamorphosis to juveniles with shells was absent after 20 days of veliger stage. Most studies confirm that the early stages of pteropod development are the most vulnerable (Kurihara, 2008; Comeau et al., 2010; Lischka et al., 2011), as the organisms do not have fully formed shells (Kobayashi, 1974; Fabry et al., 2008; Howes et al., 2014; Thabet et al., 2015). Meanwhile this study showed that the protein was the major catabolism substrate of C. acicula. Due to a strong depletion of lipids suffered during the spawning phase in the summer, active lipid storage in eggs may occur in pteropods (Gannefors et al., 2005). And female pteropods generally die shortly after spawning (Gannefors et al., 2005), which may explain the extinction of C. acicula after 1 month of the bloom. Thus, this massive aggregation of C. acicula may explain to be a reproductive bloom.

Our results indicate that seawater temperature has a stronger influence on OCR and AER of C. acicula, compared to salinity. On the other hand, CR was more affected by salinity than by temperature. The preferable temperature for C. acicula ranged from 29 to 32°C and the preferable salinity ranged from 28 to 33 ppt. The warmer water (30.92 ± 0.86°C) around the thermal discharge area of DNPP may favor the physiological performances for reproduction. In addition, it is well-known that water temperature shows a regular seasonal cycle in Daya Bay (Wu et al., 2016). The C. acicula bloom occurred only in 2020. This impenetrable phenomenon may be mainly related to the tidal current which drives the horizontal migration of C. acicula from the inside to the outside of the bay during the ebb tide period (Zeng et al., 2021). Thus, it is contingency for this bloom (Zhong et al., 2021). However, it must be pointed out that the bloom formatting mechanism of C. acicula is very complex and almost be combination of many factors. Further studies are in need for a fully understanding on the bloom forming mechanism of C. acicula.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

TH and HH conceived the study. TH and ZQ designed the experiments, analyzed the data, and wrote the manuscript. RS and QL conducted the research and collected the data. MD and HH provided materials and interpreted the data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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