Temperature response
In the present study, the isolate of D. acuminata from the Yellow Sea was characterized by relatively low growth rates when prey was not limited, ranging from 0.09 to 0.29 d-1 during the exponential phase. Other cultured D. acuminata grew either at a similar level, i.e. the D. acuminata isolate from Inokushi Bay, Japan, with growth rates of 0.14 d-1 at 10°C and 0.28 d-1 at 22°C under a 12-h light : 12-h dark cycle (Kamiyama et al. 2010), or higher, i.e. D. acuminata isolates from North America, the USA, with growth rates of 0.23 d-1 at 10°C (Tong et al. 2010), 0.37 d-1 at 15°C under a 14-h light:10-h dark cycle (Tong et al. 2015a) and from Masan Bay, Korea, with growth rates of 0.91 d-1 at 20°C under continuous light (Park et al. 2006). Interestingly, a significantly low growth (0.09 d-1) was observed at 10°C in the present study, when there was still sufficient prey in the mixed culture, indicating that this geographical isolate of D. acuminata was able to survive, but not feed and/or divide actively at low temperature. The insufficient growth and predation of Dinophysis at 10°C might be a survival strategy of cells exposed to lower temperature, which may be the same response of Dinophysis cells to other environmental pressures in natural seawater. As a cosmopolitan species, different strains of D. acuminata were revealed to adapt to a wide range of conditions and were capable of growing under temperatures as low as 6°C in North America (Tong et al. 2010) or 8°C in the northeast of Japan (Maestrini 1998). A strain of D. acuminata from Inokushi Bay, Japan (32.80°N, 131.90°E) was reported to grow exponentially at a wide range of temperatures (from 10 to 22°C), even though cultures were originally maintained at 18°C (Kamiyama et al. 2010).
Temperature of all the environmental factors may not be identified as the “weighted” factor for certain physiological and toxigenic characteristics of Dinophysis (Alves-de-Souza et al. 2013). However, field studies revealed the succession of Dinophysis on a seasonal basis (Alves-de-Souza et al. 2013; Jiang et al. 2014; Fabro et al. 2016). Meanwhile, the positive effect of temperature on the phytoplankton growth has been confirmed by numerous studies. A warmer habitat leads to many ecological variations in an organism, such as frequency of division, active motility and metabolic activities (Wotton 1995). Therefore, it is a rational assumption that the higher temperature in the present experiment (20°C) stimulated the growth of D. acuminata by enhancing the encounter rates of the predator and prey, which in turn, activated the phagotrophy of Dinophysis.
Prey quantity or biovolume
The type of prey had a significant effect on the population growth and cellular biomass of D. acuminata. Both the cell size and biovolume of the two ciliate strains were different. Strains from Japan (JAMR) were three times larger than AND-A0711. Although AND-A0711 was used as prey in three times larger amounts than JAMR, the growth rate and biomass of D. acuminata were still significantly higher when fed on JAMR through the entire growth period. It is possible that factors other than biomass, such as pigment or the type of chloroplast, may affect the growth. Park et al. (2010) investigated the fate of “kleptoplasts” in one isolate of D. caudata and found that CR-MAL01-type plastids stayed longer than CR-MAL11-type plastids in D.caudata cells with the increased starvation time. This indicated that Dinophysis treats plastids taken up from different cryptophytes via its ciliate prey M. rubrum in different ways. Unfortunately, we could not investigate the effect of the plastid type on the growth of Dinophysis, because the plastids of the T. amphioxeia strain JATA and the strain AND-A0710 were identical according to their 16S rDNA and psbA sequencing. Furthermore, genetic differences in D. acuminata may have led to differentiating physiological characteristics within this species. D. acuminata from North America (DAMV01) had a growth rate of 0.37 d-1 at 15°C when fed on the same prey (Tong et al. 2015b). Dinophysis (D. caudata, D. acuta and D. tripos) from Northwest Spain had growth rates of 0.27-0.40 d-1 under high light illumination (Rial et al. 2013).
Toxin profile of Dinophysis
Environmental factors, such as temperature, light intensity, dissolved inorganic nitrate and phosphate,
do not directly affect the toxin profile and the content of Dinophysis in batch cultures (Kamiyama & Suzuki 2009; Tong et al. 2011; Nielsen et al. 2013; Hattenrath & Gobler. 2015; Hattenrath et al. 2015; Tong et al. 2015b). Tong et al. (2011) investigated the toxin production of D. acuminata under two temperatures (4 and 6°C) and three light intensities (65, 145 and 284 µmol photons m-2 s-1), and showed that the toxin content of Dinophysis was not significantly altered by changes in these environmental conditions. Kamiyama et al. (2010) found that the cellular PTX2 content was greater at lower temperatures, but no clear differences in OA and DTX1 were observed in relation to temperature. Although relatively higher amounts of OA per ml of culture were observed at higher temperature in both feeding regimes in the present study, the differences were not statistically analyzed. The cellular toxin content was unaffected by irradiance (Tong et al. 2011; Nielsen et al. 2012; Nielsen et al. 2013), but light was required for the growth and toxin production of D. acuminata (Tong et al. 2011). Dissolved nitrate and phosphate do not have a direct effect on the toxin production of D. acuminata, but these nutrient pools may contribute to prey growth and biomass, thereby indirectly promoting overall toxin concentrations in the D. acuminata culture system (Hattenrath & Gobler 2015; Hattenrath et al. 2015; Tong et al. 2015b). Additionally, the cellular toxin content of Dinophysis isolates varies greatly due to the variability associated with different growth stages (Tong et al. 2011; Tong et al. 2015a).
Prey availability affected the total amounts of OA, DTX1 and PTX2. In the presence of prey, the number of D. acuminata cells increased, resulting in elevated total toxin concentrations (Kamiyama et al. 2010; Nagai et al. 2011; Tong et al. 2011; Tong et al. 2015a). Active toxin production by Dinophysis required the presence of ciliate prey (Tong et al. 2015b). In the absence of prey, there were no changes in cellular, dissolved or total OA, DTX1 or PTX2 over one month of incubation (Smith et al. 2012; Tong et al. 2015b).
Prey isolates or prey nutritional quality might be a driver of toxin content in Dinophysis. Two nutritionally different ciliates were supplied as prey for D. acuminata DAYS01. Although more Spanish ciliates were added to maintain an equivalent biomass/ biovolume, the toxin content, especially PTX2, in the two feeding chain systems varied greatly, suggesting that inherent differences in the prey influenced the PTX2 toxin quota of Dinophysis. No obvious differences in PTX2 content were observed at 10°C, possibly due to the non-growth of Dinophysis at such a low temperature. Therefore, Dinophysis cultures at 10°C may have maintained their initial toxin concentration. However, toxin analysis was not performed for the Dinophysis inoculum, which prevented us from testing this assumption.
Intrinsic differences in Dinophysis strains may lead to variations in the toxin profile and production. In a batch culture study, OA, DTX1 and PTXs toxin content of northwestern Atlantic D. acuminata isolates (DAEP01, DAMV01 and DABOF02) from the northwestern Atlantic was typically 0.01−1.80 pg cell-1 of OA or DTX1 in batch culture (Tong et al. 2015a), a value at the lower end of the D. acuminata isolates from Japan (0.2−12.2 pg cell-1, Kamiyama & Suzuki 2009; Kamiyama et al. 2010; Nagai et al. 2011) and Brazil (3.2−18.0 pg cell-1, Mafra et al. 2014). Compared to those studies, the extracted toxins were not from isolated cells, but from the culture (including cell and medium) in the present study. Therefore, the OA or DTX1 content of our Chinese D. acuminata, nd-0.54 pg cell-1, were overestimated. However, the content of OA or DTX1 was still low. The overestimated PTX2 content of the isolates (9.63-18.49 pg cell-1) in the present study was within the range of quotas reported for many other regions (Nielsen et al. 2012). The relatively low OA and DTX1 toxin content of the Yellow Sea isolates is consistent with scarce harvesting closures in that region due to OA-group toxins, but the potential risk of moderate or high PTX toxin exposure cannot be ignored.
Individual Dinophysis strains appear to be able to produce only one type of toxin profile (Reguera et al. 2012). Dinophysis cf acuminata cells from northern and southern Chile (Blanco et al. 2007; Fux et al. 2011) produced only PTX2; D. cf ovum cells from Texas contained only OA (Deeds et al. 2010; Fux et al. 2011; Tong et al. 2015a). The toxin PTX2 was the dominant toxic component in D. acuminata, D. norvegica and D. infundibulus from Hokkaido, Japan (Suzuki et al. 2009), and D. fortii was reported to produce DTX1 and PTX2 (Kamiyama & Suzuki 2009; Suzuki et al. 2009). The toxin profiles and content of D. caudata varied seasonally and geographically. The cellular toxin content was extracted from the isolated cell, and low concentrations of OA were detected in field populations of D. caudata from Singapore (0.07-0.14 pg cell-1) (Holmes et al. 1999; Holmes & Teo 2002). Moderate to high values of OA (7.9-56.5 pg cell-1) and DTX1 (7.2-53.9 pg cell-1) were reported in the Philippines, where D. caudata populations were seasonally dominant in phytoplankton communities at intervals higher than 1000 cell l-1 (Marasigan et al. 2001). Dinophysis caudata isolates from Northwest Spain were reported to contain PTX2, ranging from 5 to 130 pg cell-1 (Fernandez et al. 2006; Pizarro et al. 2013), and trace amounts of OA and/or DTX2 (Pizarro et al. 2013). A field investigation of D. caudata in China (Gouqi Island, the East China Sea) found low PTX2 (0.58 pg cell-1) and trace amounts of OA and DTX1 using LC-MS/MS (Li et al. 2015), suggesting an urgent need to investigate the toxin-producing capacity of local species and to assess the potential risk to local aquaculture industries.