Antioxidants, Vol. 11, Pages 2299: Reduced Fitness and Elevated Oxidative Stress in the Marine Copepod Tigriopus japonicus Exposed to the Toxic Dinoflagellate Karenia mikimotoi

4.1. SurvivalThe lethal effect of K. mikimotoi has been documented in many marine organisms, such as fish, shellfish, rotifers, and crustaceans, with copepods being one of the less sensitive lineages [19,32]. Within the group of copepods, the sensitivity to K. mikimotoi varies among species, and the benthic harpacticoid copepod T. japonicus tested in this study seems more tolerant to K. mikimoti than the pelagic calanoid copepods [19,29,31,32]. No mortality was observed in adult T. japonicus within 96 h at all tested concentrations of K. mikimotoi, ranging from 10,000 to 50,000 cells·mL−1, and they retained a high survival rate (>90%) during 14-d incubation at concentrations −1 (Figure 1a). Contrastingly, higher mortality has been reported in adult Pseudodiaptomus marinus (52–100%, 13 d) and Calanus sinicus (20%, 96 h or 22% 16 d) fed with less or comparable concentrations of K. mikimotoi [29,31,32]. It has been duly recognized that the toxicity of K. mikimotoi is target-species-specific and algal-strain-specific [4,11], which could be attributed to the physiological diversity in structure and defense mechanisms among organisms. Previous studies also postulate that benthic copepods and other benthic invertebrates have a higher degree of tolerance to toxic algae than their pelagic counterparts [27,41,42]. High survival rates are also observed in T. japonicus exposed to various environmental stressors [43,44,45]. This is probably due to their physiological acclimation and genetic adaptation to the benthic environments, where the disturbance and contaminant accumulation are high while food supply might be limited relative to pelagic environments.As expected, nauplii and copepodite T. japonicus were more vulnerable compared to adults (Figure 1), which is in concert with findings in C. sinicus fed on K. mikimotoi [31]. It has been suggested that inadequate feeding due to nonoptimal predator-to-prey size ratio may reduce the survival of nauplii T. japonicus [27]. However, the significant concentration and time-dependent decreases of survival in nauplii and copepodite T. japonicus revealed in our study indicate that the toxicity of K. mikimotoi might be the major factor influencing survival. 4.2. FeedingBoth algae were ingested by T. japonicus in the concentration-dependent pattern, but the ingestion rate on K. mikimotoi was only 5.0–14.3% of that on non-toxic control prey I. galbana. The functional response in clearance rate to both prey concentration fit Holling type III, with significantly higher clearance rates found on K. mikimotoi. Such depressed ingestion and enhanced clearance on K. mikimotoi compared to the control prey are in line with the findings in the pelagic copepod Temora longicornis [34]. Reduced grazing on K. mikimotoi has also been documented in several copepods, including Acartia omorii, Calanus helgolandicus, P. marinus, and Pseudocalanus elongatus, and in several independent studies on T. longicornis [29,30,33]. Explanations related to nutritional inadequacy, toxic, or deterrent effect on grazers have been proposed. K. mikimotoi lacks essential polyunsaturated fatty acid and amino acid for zooplankton [31], which was observed in copepod Pseudodiaptomus annandalei and other marine zooplankton [19]. Alternation of feeding behavior has been convincingly demonstrated by direct observation in several copepods fed on K. mikimotoi. Significantly reduced beating frequency of the feeding appendages have been shown for copepod C. helgolandicus and T. longicornis [30]. Copepods use appendage movements to produce feeding current and further capture food particles; therefore, reduced beating frequency mirrors the lower prey-encounter rate and consequently lower ingestion rate of prey. Moreover, deterrent effects during the process of capture, examination and rejection of viable toxic cells have been observed in copepod P. elongatus and T. longicornis, and the high rejection rate of K. mikimotoi was high compared to that of non-toxic algae [33,34]. These studies argued that the signal molecules produced by toxic algae and remote characterization of the prey by copepod collectively reduce the predation risk, which may account for the long duration of K. mikimotoi bloom, as proposed by box-model analysis [46]. 4.3. Oxidative StressOxidative stress is one of the toxicological consequences of exposure to harmful algae in a variety of aquatic organisms [11,47,48], which refers to the imbalance between pro-oxidant and antioxidant homeostatic cellular conditions or a disruption of redox signaling and control [49]. Such oxidative stress and subsequent damage could be caused by algal-borne ROS, which is also speculated as one of the substances involved in K. mikimotoi-induced toxicity [12,13,28], besides hemolytic toxins [7,8,50] and cytotoxins [9,10]. The ROS production of K. mikimotoi varies among strains, and some strains such as NGU04 could generate ROS at a level nearly equal to that of raphidophyte Chattonella marina, which produces the highest level of superoxide per cell among microalgae [12,51]. However, several studies have suggested that ROS might not play a major role in the toxic effect of K. mikimotoi, as shown in rotifers [12,32,52,53].In spite of this, K. mikimotoi could induce significant modulation on ROS production and the antioxidant defense system in aquatic organisms, as many other toxic algae do [47,48,54]. In our study, K. mikimotoi stimulated the ROS production in adult T. japonicus with a concentration-dependent pattern (Figure 3). ROS production is considered as a metabolic response to toxic algal exposure, which could be induced in minutes to hours [55,56,57], and it even exhibited a high level after a 14-day exposure in our case. Although the mechanism of algae-induced ROS generation remains elusive, recent evidence showed that brevitoxin produced by K. brevis inhibits mammalian thioredoxin reductase, a component of the thioredoxin system [58,59,60]. The thioredoxin system is a major cellular antioxidant system that is responsible for maintaining redox homeostasis and is present in all living organisms [61]. Given that cytotoxic polyethers, gymnocin produced by K. mikimoti, are structurally analogous to brevetoxins [9,10,62,63], K. mikimotoi could potentially disturb the cellular oxidative status in copepod by inhibiting thioredoxin reductase.Along with the rise in ROS level, K. mikimotoi also activated the antioxidant defense system in T. japonicus, expressed as enhanced T-AOC, and the activities of SOD, CAT, and GPx (Figure 3), which indicates active ROS scavenging [36]. Oxidative stress indicated by the alteration of antioxidant enzymes is commonly found in T. japonicus and other aquatic organisms under various chemical and physical stresses, such as heavy metals, ocean acidification, climate change, and toxic algae [32,64,65]. The effects of K. mikimotoi on the antioxidant enzymes have been reported in diatom Thalassiosira pseudonana [18], abalone Haliotis discus hannai [14,17], zebrafish [15], and medaka Oryzias melastigma [16]. Such effects include inhibition or activation followed by inhibition, and the pattern with respect to algal concentration and exposure time varies among enzymes. Although no direct evidence is available, we cannot rule out the possibility that nutritional inadequacy of K. mikimotoi may be also a stressor to activate antioxidants in copepods. Here, we used T-AOC to describe the cumulative action of all the antioxidants present in T. japonicus, which was significantly induced by K. mikimotoi with least variation among concentrations, whereas the activities of SOD, CAT, and GPx fluctuated. In comparison with the control, no inhibition was found in antioxidant enzyme activity, indicating that the oxidative stress imposed by K. mikimotoi was still within the antioxidative capacity of T. japonicus. However, with the increase of algal concentrations, the elevated enzyme activity of SOD and GPx was followed by a reduction, which points to the possibility that a minimum threshold concentration may be required to depress the enzyme activity. A similar bell-shape pattern has been detected in the SOD activity of abalone exposed to K. mikimotoi [14] and several antioxidant biomarkers of zebrafish exposed to microcystins [66]. It has been suggested that activation of antioxidant is energy- and nutrient-demanding [36]. Therefore, exceeding the threshold of concentration or exposure time would exhaust antioxidative capacity and lead to decreased antioxidant levels [67]. Moreover, a positive correlation among antioxidant biomarkers (Table 5) highlighted the cooperation in the antioxidant defense system of T. japonicus to counteract K. mikimotoi toxicity, in concert with the findings in T. japonicus exposed to nickel [38].Interestingly, a strongly positive correlation was also found between AChE and all oxidative-status-related variables (ROS level and antioxidant activity). We found that with the increase of K. mikimotoi concentrations, AChE activity significantly increased, followed by a decrease (Figure 3). Activated AChE activities have been also found in copepods fed on toxic cyanobacteria and several other microcrustaceans under stress [37,67]. AChE has been identified as a biomarker of neurotoxic contaminants in benthic copepods [68]. In concert with previous findings on zebrafish larvae and rotifers [69,70], we observed tetany and hypoactivity in T. japonicus during exposure to K. mikimotoi (data not shown). Although a neurotoxin has not been identified in K. mikimotoi, they produced brevetoxin-like polyethers [9,10,62,63], and genes related to polyketide synthase and saxitoxin synthesis were identified in its transcriptomes [71]. Niu et al. [15] investigated potential K. mikimotoi neurotoxicity in zebrafish larvae and found an association between AChE, SOD, and CAT activity and the differential expression of neurodevelopment genes. Whether K. mikimotoi exerts neurotoxicity in copepods needs further experimental validation. However, given the remarkable diversity of AChE functions [72], the alternation of AChE activity could be the response to various external stimuli other than neurotransmission, which might be associated with physiological stress, such as oxidative stress [67]. 4.4. Development and ReproductionIt has been commonly found that toxic algae and their toxins exert adverse effects on the development and reproduction of copepods, but multigeneration toxicity of K. mikimotoi in copepods has not been covered in previous studies [29,30,31]. We found that in F1, although K. mikimotoi at concentrations −1 could support both nauplii and copepodites of T. japonicus to complete their development with high survival, the development time of both stages were significantly prolonged under all tested concentrations (Figure 4). In agreement with a multigeneration study on T. japonicus exposed to mercury, the response of F1 at the highest concentration predicted that of future generations [23]. Survival rates of two stages in F1 were significantly reduced at the highest concentration (4000 cells·mL−1), and the inhibitory impact of K. mikimotoi on development increased in F2, indicating the potential accumulation effect with generations. Remarkably, 70% of F2 nauplii exposed to 4000 cells·mL−1K. mikimotoi successfully developed to copepodite, none of which could survive to adult stage. This suggests that copepodite could be more vulnerable under continuous exposure, in accordance with the results of chronic test in T. japonicus exposed to the biocide triphenyltin [24]. Contrastingly, our acute test results support the commonly accepted idea that early life stage is more sensitive to environmental stresses, with 96 h-LC50 for nauplii being about half of that for copepodite. Such inconsistency may be due to the cumulative damage from repeated or long-term continual exposure, which could ultimately produce more severe effects in the late juvenile stage.We further found that the exposure to K. mikimotoi significantly reduced the egg production and number of clutches in T. japonicus, which is in concert with previous reports in the calanoid copepods A. omorii, C. helgolandicus, C. sinicus, P. marinus, and T. longicornis [29,30,31]. Although in some cases the hatching success of copepods may not be affected by K. mikimotoi [23], and due to the hormesis effect [73], low concentrations of some toxic algae such as cyanobacteria Nodularia spumigena and Colichlodinium polykrikoides may be beneficial to copepod reproduction and development [74,75], our findings confirmed that K. mikimotoi suppressed the reproduction of T. japonicus, even at the lowest tested concentration (500 cells·mL−1). Copepods exposed to toxic algae for a long period may lack enough energy and nutrients to complete their development and reproduction, due to the high maintenance cost for detoxification [60,76]. The situation may get worse when the algae is nutritionally inadequate, as with K. mikimotoi [19,31], and ATP synthesis is inhibited by some environmental stress [77]. Although Wang et al. [27] argued that the egg sac of T. japonicus can protect eggs from external environmental disturbances, the detrimental effect of K. mikimotoi on the reproduction of benthic copepods is comparable to that on pelagic copepods. Moreover, the number of successfully developed adult females in F2 was too low to conduct subsequent evaluation on egg production, which indicates that K. mikimotoi may also affect the sex determination of copepods. Sex determination in copepods is under strong environmental control [78], such as temperature [79], food quality and quantity [80], and toxic algae [81]. In our study, the chronic exposure to K. mikimotoi in both parents and offspring may have disturbed the sex determination in F2, and the decreased number of adult females was also observed in F2 T. japonicus exposed to biocide [24]. The prolonged development time, the decrease of development success rate, egg production, number of clutches, and female adults in T. japonicus collectively demonstrate that population recruitment of copepods could be adversely affected by K. mikimotoi, particularly in long-lasting blooms.

留言 (0)

沒有登入
gif