Potential of the small cyclopoid copepod Paracyclopina nana as an invertebrate model for ecotoxicity testing

Aquatic Toxicology 180 (2016) 282–294

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Potential of the small cyclopoid copepod Paracyclopina nana as an invertebrate model for ecotoxicity testing Hans-Uwe Dahms a,b , Eun-Ji Won c , Hui-Su Kim c , Jeonghoon Han c , Heum Gi Park d , Sami Souissi e , Sheikh Raisuddin f , Jae-Seong Lee c,∗ a

Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan c Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea d Department of Marine Bioscience, College of Life Sciences, Gangneung-Wonju National University, Gangneung 25457, South Korea e Univ. Lille, CNRS, Univ. Littoral Cote d’Opale, UMR 8187, LOG, Laboratoire d’Océanologie et de Géosciences, 62930 Wimereux, France f Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard University, New Delhi 110062, India b

a r t i c l e

i n f o

Article history: Received 16 May 2016 Received in revised form 13 October 2016 Accepted 14 October 2016 Available online 14 October 2016 Keywords: Copepod Monitoring Bioassay Biomarker Aquatic pollution Gene expression Environmental OMICS approaches

a b s t r a c t Aquatic invertebrates contribute significantly to environmental impact assessment of contaminants in aquatic ecosystems. Much effort has been made to identify viable and ecologically relevant invertebrate test organisms to meet rigorous regulatory requirements. Copepods, which are ecologically important and widely distributed in aquatic organisms, offer a huge opportunity as test organisms for aquatic toxicity testing. They have a major role not only in the transfer of energy in aquatic food chains, but also as a medium of transfer of aquatic pollutants across the tropic levels. In this regard, a supratidal and benthic harpacticoid copepod Tigriopus japonicus Mori (order Harpacticoida) has shown promising characteristics as a test organism in the field of ecotoxicology. Because there is a need to standardize a battery of test organisms from species in different phylogenetic and critical ecosystem positions, it is important to identify another unrelated planktonic species for wider application and comparison. In this regard, the cyclopoid copepod Paracyclopina nana Smirnov (order Cyclopoida) has emerged as a potential test organism to meet such requirements. Like T. japonicus, it has a number of features that make it a candidate worth consideration in such efforts. Recently, the genomics of P. nana has been unraveled. Data on biochemical and molecular responses of P. nana against exposure to environmental chemicals and other stressors have been collected. Recently, sequences and expression profiles of a number of genes in P. nana encoding for heat shock proteins, xenobiotic-metabolizing enzymes, and antioxidants have been reported. These genes serve as potential biomarkers in biomonitoring of environmental pollutants. Moreover, the application of gene expression techniques and the use of its whole transcriptome have allowed evaluation of transcriptional changes in P. nana with the ultimate aim of understanding the mechanisms of action of environmental stressors. Whole-animal bioassays and gene expression studies indicate that P. nana may serve as an excellent tool to evaluate the impact of diverse disturbances in the marine environment. With a better understanding of toxicological mechanisms, ecotoxicologists will be able to understand defense mechanisms against toxicants in copepods. In this review, we illustrate the potential of P. nana as an alternative as well as a complementary invertebrate model organism for risk assessment of aquatic pollutants. © 2016 Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 1.1. Research history of P. nana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

∗ Corresponding author. E-mail address: [email protected] (J.-S. Lee). http://dx.doi.org/10.1016/j.aquatox.2016.10.013 0166-445X/© 2016 Elsevier B.V. All rights reserved.

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2.

3. 4. 5.

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1.2. RNA-seq and genome assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 1.3. Environmental issues with marine copepods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 Use of P. nana in environmental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 2.1. UV radiation responses of P. nana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 2.2. Gamma radiation responses of P. nana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 2.3. Antioxidant defense mechanisms in response to gamma radiation in P. nana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Advantages of using biomarkers from P. nana in biomonitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Research can help elucidate the role of P. nana in ecotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

1. Introduction Small invertebrates are integral to ecotoxicological risk assessment and regulatory framework (Lagadic and Caquet, 1998; Markwiese et al., 2001; Baun et al., 2008). Their commonly short life span enables study of reproductive and multi-generational effects of toxic chemicals with limited resources (Lagadic and Caquet, 1998). Therefore, invertebrates such as crustaceans and mollusks have been extensively used as sentinel species, laboratory test species, and in aquatic food chain studies. They also occupy a large biomass in their respective trophic level. A number of crustacean species have been standardized for ecotoxicity testing; Daphnia is a classic example (Papillon and Maximilian, 2007). However, some other crustaceans such as the intertidal copepod Tigriopus japonicus have shown great promise of becoming a test organism (Raisuddin et al., 2007). It is observed that having a battery of organisms with different habitats may be helpful in holistic and realistic risk assessment of aquatic pollutants. It is also desirable to study effects of toxic chemicals in trophic transfer scenarios, while considering biomagnification. Thus, exploration of new complementary models is necessary to optimize ecotoxicity testing. In terms of susceptibility in response to pollutants, the copepod T. japonicus is tolerant to certain stressors such as ultraviolet (UV) radiation, gamma radiation, and certain heavy metals (Rhee et al., 2012; Han et al., 2014). This, in other words, also suggests that more sensitive copepod species are required for a representative ecotoxicological evaluation against these stressors (Lee et al., 2012; Rhee et al., 2012; Han et al., 2014, 2015; Won et al., 2014). While comparing relative sensitivity among copepods, the cyclopoid copepod Paracyclopina nana Smirnov (order Cyclopoida) has been found to be highly susceptible to selected environmental stressors of varied classes such as copper, UV-B radiation, fluorene, phenanthrene, and gamma radiation (Fig. 1). In P. nana, LC50 and LD50 values of copper, UV-B radiation, and fluorene were about three times lower than those in T. japonicus. After gamma radiation, 96-h LD50 of P. nana was 172 Gy (Won and Lee, 2014). However, T. japonicus showed no mortality within 4 days even after exposure to 800 Gy (Han et al., 2014). Such differences in susceptibility may be helpful in complementing the data from other species from a set of organisms used in test batteries. In this regard, P. nana has recently attracted attention of ecotoxicologists for its utility as a test organism in ecotoxicological and ecophysiological investigations. In this review, we critically appraise the existing knowledge of this organism and also discuss our data with special reference to its transcriptomics and its application in aquatic biomonitoring.

1.1. Research history of P. nana P. nana is a planktonic brackish water cyclopoid copepod species (Fig. 2). It belongs to the order Cyclopoida, which includes more than 1500 species and subspecies, and approximately half of them belong to the family Cyclopidae. Traditionally, the systematics of

cyclopoid copepods has been based on morphological characteristics (Monchenko, 1974). Cyclopoid copepods are an abundant and successful marine biota (Copepod Web Portal – http://www. luciopesce.net/copepods/index.html). They play an important role as a primary link in aquatic food chains, ranging from microscopic phytoplanktonic algae or members of the microbial loop to secondary consumers (e.g., fishes). P. nana is regarded as an important food source for many developing larvae, post larvae, juvenile fish, and other crustaceans (Sun and Fleeger, 1995; Pinto et al., 2001). It also serves as live food in aquaculture (Lee et al., 2006). P. nana has a high tolerance to wide salinity and temperature ranges (Lee et al., 2006) (Table 1). Dussart and Defaye (2006) proposed that the first taxonomic system for cyclopoid species including P. nana that could serve as a base of its systematics. A first complete mitogenome sequence discovered for P. nana provided a potential initiation point for cyclopoid molecular taxonomy (Ki et al., 2009). In the P. nana mitogenome, a highly rearranged gene order and high divergence were shown compared to other copepod mitogenomes (Jung et al., 2006; Ki et al., 2009). The P. nana mtDNA (15,981 bp in length) consists of 37 genes (13 protein-coding genes, 2 rRNAs, and 22 tRNAs), which are all typical for metazoan mitogenomes. The P. nana mitogenome has a putative long control region with a high AT content. The Cytb gene is considerably short in length compared to other crustaceans. In addition, P. nana has highly divergent mitochondrial genes, judged by their amino acid substitution (Ki et al., 2009).

1.2. RNA-seq and genome assembly Recently, analysis of a large RNA-seq database (Table 2; GCJT00000000) was published for P. nana (Lee et al., 2015). In the P. nana RNA-seq database, 21,390 National Center for Biotechnology Information (NCBI) non-redundant (NR) Basic Local Alignment Search Tool (BLAST) matches were found along with the identification of 16,159 genes from whole genome assembly (Fig. 3A), indicating that nearly all the genes are available for transcriptional analysis in response to environmental stressors. This array of information prompted use of P. nana for transcriptomicbased risk assessment of marine pollutants; the whole genome database was assembled (total length 85,806,777 bp; 275 scaffolds, N50 = 1,682,134 bp; and Core Eukaryotic Genes Mapping Approach [CEGMA], complete score 95.97% and partial score 97.58%) (Fig. 3A). After genome assembly, phylogenomic analysis of homeobox (hox) clusters was revealed (Fig. 3B), indicating that P. nana has a more compact gene structure (Kim et al., 2016) compared to Drosophila melanogaster (Kaufman et al., 1990), Tribolium castaneum (Tribolium genome sequencing consortium, 2008), Daphnia pulex (Papillon and Maximilian, 2007), Caenorhabditis elegans (C. elegans sequencing Consortium, 1998), Neanthes virens (Kulakova et al., 2008), and Strongylocentrotus purpuratus (Sea Urchin Genome Sequencing Consortium, 2006).

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Fig. 1. Dose-response of the copepods Paracyclopina nana and Tigriopus japonicus to copper, UV-B radiation, fluorene, phenanthrene, and gamma radiation.

Copepods with a small and compact genome size have merit in assessment of their promoter regions and the gene structure, as all the motifs for transcriptional regulation are closely placed to one another. To date, Tigriopus californicus and T. japonicus have a genome size of 180 Mb (PRJNA237968) and 196 Mb, respectively, whereas P. nana’s genome is 85 Mb. Thus, it would be advantageous to use P. nana for in silico analysis of promoter regions. 1.3. Environmental issues with marine copepods In recent times, development aided by emergence and application of sophisticated techniques in the field ecotoxicology has contributed to biomonitoring, ecological risk assessment, biomarkers, and toxicity testing at different levels (Snape et al., 2004; Ankley

et al., 2009; Gaytán and Vulpe, 2014). Taking into account that a single species may not be sufficient to study the complex dimensions of ecotoxicity of a chemical, a multi-species approach is widely employed in ecotoxicological investigations. Species used in ecotoxicity testing are taxonomically well defined, reared, and adapted to laboratory conditions (Mothersill and Austin, 2003; Nunes et al., 2006; Raisuddin et al., 2007). As a representative for certain habitats and environmental situations, test organisms must have defined characteristics such as ecological relevance, sensitivity to exposure to chemicals, a relatively short life cycle, cost effectiveness, and a wide geographical distribution (Nunes et al., 2006; Raisuddin et al., 2007). Different phylogenetic positions and trophic levels are also important parameters for the selection of test organisms. In this

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Fig. 2. Developmental stages of the copepod Paracyclopina nana. Adopted from Hwang et al. (2010).

Table 1 Ecotoxicological studies with Paracyclopina nana. Species

Stressors tested

Endpoint measured

Type of test

Reference

P. nana

UV

life table

Won et al. (2014)

P. nana

Food (5 microalgae)

diet

Lee et al. (2006)

P. nana

mRNA expression

Jeong et al. (2015)

P. nana P. nana

Temperature, salinity, density, LPS Gamma radioisotope Heavy metals, EDCs

Mortality, reproductive parameters Fecundity, mortality, growth Gene expression

radiation EDCs, HM exposure

Won and Lee (2014) Hwang et al. (2010)

P. nana

UV

UV exposition

Won et al. (2015a,b)

P. nana

Light intensity

light exposure

Lee et al. (2011)

P. nana

Density, antioxidants

culture density

Lee et al. (2012)

Growth, fecundity Molecular, Vg expression Clutch number, growth pattern, newly hatched nauplii, ingestion rate, assimilation of diet, DNA repair, heat shock protein Survival, growth, productivity Naupliar production, gene expression

Table 2 Assembly and annotation statistics of the Paracyclopina nana RNA-seq (modified from Lee et al., 2015). Assembly and annotation statistics Assembled bases (bp)

Number of contigs

Mean contig length (bp)

Median contig length (bp)

N50(bp)

GC content

44,635,065

32,165

1387

999

1869

48.08

Annotation statistics NCBI NR Blast

SignalP

InterProScan

GO (annotated)

21,390

4197

23,188

16,861

review, we examined the suitability of P. nana as a test organism based on these criteria. One of the most challenging issues in ecotoxicological studies is to find an appropriate model for acute and chronic toxicity assessment of pollutants in different species, genders, and development stages. Such a test organism should also facilitate understanding of

the mechanism of toxic action at various levels of biological organization (i.e., the molecular, cellular, whole organism, and population level) (Won et al., 2015a,b). In a way, a test organism could be used for a number of purposes such as the generation of standardized and comparative ecotoxicity tests to elucidate how organisms cope with toxicants and to understand the linkage between sub-

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Fig. 3. (A) Genome browse of the copepod Paracyclopina nana, and (B) Phylogenomic comparison of P. nana Hox clusters with other species. Adopted from Kim et al. (2016).

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Fig. 4. An animal model Paracyclopina nana allows several ecotoxicological measurements.

organismal responses to toxicants at individual, population, and community levels (Fig. 4). As for the marine environment, a suitable model organism might provide insights into physicochemical regulations and adaptations to challenging circumstances (Cassidy et al., 2007). A number of copepods that meet regulatory requirements have been used for bioassays for regulatory toxicology (EDMAR, 2002). A review by the Organization for Economic Cooperation and Development (OECD, 2011) on crustacean species in toxicity testing indicated that copepods are suitable candidates for bioassays. Although T. japonicus emerged as a strong candidate fulfilling these criteria (2007), copepod species such as Tisbe battagliai, Amphiascus tenuiremis, and Nitocra spinipes have also shown promise (see Table 3). Also, geographical distribution became important criterion for ecotoxicity risk assessment. For example, T. japonicus is a representative for the Northwest Pacific region (Raisuddin et al., 2007). Because the habitat of P. nana is vulnerable to anthropogenic pollution and a substantial ecotoxicological database is emanating from laboratory and field investigations, it could be a promising candidate for screening and testing of chemical pollutants and other emerging environmental stressors (Hwang et al., 2010; Won et al., 2014; Han et al., 2015; Won and Lee, 2014). It could also be employed for testing of specific stressors of the water column of brackish waters such as UV-B radiation, gamma radiation, heavy oil spillage, heavy metals, etc. However, concerted inter-laboratory efforts are needed to test its efficacy in different geographical locations.

2. Use of P. nana in environmental studies Recently, P. nana has been used in toxicity assessment of diverse environmental chemicals. In each of these studies it demonstrated appreciable consistency in response to exposure (Ki et al., 2009; Hwang et al., 2010; Won et al., 2014; Han et al., 2015; Won and Lee, 2015). Such as heavily researched T. japonicus, P. nana has several suitable characteristics, such as a small size (∼600 ␮m), a short generation cycle (∼2 weeks), and ease of rearing and handling. The recent addition of several molecular endpoints and omics data (such as genomics, transcriptomics, and proteomics) support its

inclusion in the battery of invertebrate test organisms for marine environmental risk assessment. Compared to other zooplankton species, P. nana has shown a higher mortality rate in response to heavy metals (e.g., Cd and Cu), UV-B radiation, and gamma radiation (Lee et al., 2007; Hwang et al., 2010; Rhee et al., 2012; Won and Lee, 2014; Han et al., 2014; Won et al., 2015a,b) (Tables 1 and 4). The changes at sub-individual levels show that P. nana is relatively more useful in ecotoxicological studies in response to wide ranges of environmental stressors and exposure conditions. Some of these aspects are discussed in the following sections. 2.1. UV radiation responses of P. nana The effects of UV radiation on aquatic organisms have been a great concern in recent years, as it negatively influences the relationship of primary producers and consumers in the food web resulting in subsequent effects on ecosystem functioning and biogeochemical cycling (Dahms and Lee, 2010; McKenzie et al., 2011). UV radiation can directly cause alterations in protein biosynthesis and DNA through absorption of high-energy photons and can indirectly generate reactive oxygen species (ROS). ROS, which are chemically reactive chemical species containing oxygen, may cause wide ranging damage to proteins, nucleic acids, and lipids (Rhee et al., 2012). To evaluate the effects of UV radiation on the reproductive physiology and macromolecules in marine zooplankton, P. nana was exposed to a range of doses of UV radiation to analyze life cycle parameters such as mortality and reproductive parameters. Additionally, biomarkers of oxidative stress such as ROS level, antioxidant enzyme activities (e.g., glutathione S-transferase [GST] and superoxide dismutase [SOD]), and lipid peroxidation were also studied (Won et al., 2014). The survival rate of P. nana was significantly reduced after UV radiation. Egg sac damage and a reduction in the hatching rate of offspring were observed in UV-irradiated ovigerous females. Activities of GST and SOD increased in response to UV radiation. The increase in lipid peroxidation (LPO) was accompanied by a reduction in the composition of essential fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Won et al., 2014). These findings indicate that UV radiation has a cascading effect on reproductive physiology, which may affect the fecundity of P. nana at the population level (Won et al., 2015a,b). In addition, UV-B radiation induced a significant reduction of the

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Table 3 Ecotoxicological studies with aquatic copepods. Species

Chemical(s) tested

Endpoint measured

Type of test

Reference

Acartia tonsa

Diflubenzuron

Life-cycle (part.)

Tester and Costlow (1981)

Acartia tonsa

TBT

Morphological abnormalities, development, reproductive rate Mortality, development, acute toxicity

Life-cycle (part.)

Ole Kusk and Petersen (1997)

Acartia tonsa Acartia tonsa Acartia tonsa

Methyltestosterone, fenarimol Nickel Zinc Synthetic musks

Acartia tonsa

Amphiascus tenuiremis

Polybrominated diphenyl ether, 2,4,6-tribromophenol, 20-hydroxyecdysone, tetrabromobisphenol A Atrazine

Amphiascus tenuiremis

Fipronil

Amphiascus tenuiremis Amphiascus tenuiremis Amphiascus tenuiremis

Fipronil Fipronil, vitellin (VTN) Single-walled carbon nanotubes Cadmium, copper TBT PAHs, PCBs and OCPs PAHs, PCBs Microcystin (MC-LR)

Eurytemora affinis Eurytemora affinis Eurytemora affinis Eurytemora affinis Eurytemora affinis Eurytemora affinis Eurytemora affinis

Eurytemora affinis

PAHs and PCB 17b-estradiol (E2), benzo(a)pyrene (BaP), 4-nonylphenol (NP), di(ethyl-hexyl)phthalate (DEHP), and atrazine (A) (PCBs, PAHs, APs

Nitocra spinipes Nitocra spinipes

Copper 17-Estradiol, 17-ethinylestradiol, diethylstilbestrol

Nitocra spinipes

Synthetic musks

Nitocra spinipes

Polybrominated diphenyl ether Weathered polyvinyl chloride Brominated flame–retardants (BFRs) 20-Hydroxyecdysone, diethylstilbestrol

Nitocra spinipes Nitocra spinipes

Tisbe battagliai

Tisbe battagliai

Estrone, 17-estradiol, 17-ethinylestradiol

Tisbe battagliai Tisbe battagliai T. brevicornis T. brevicornis

Cadmium, copper Nonylphenol Arsenic, cadmium Cadmium, copper, atrazine, carbofuran, dichlorvos, malathion Cadmium, copper, mercury, nickel, silver, zinc Sediment leachate

T. brevicornis

T. brevicornis

Exposition Mortality Mortality Mortality, development, acute toxicity Mortality, development, acute toxicity

Acute Acute Partial life-cycle (larval development) Partial life-cycle (larval development)

Taylor (1981a) Taylor (1981b) Wollenberger et al. (2003)

Reproduction, malformation, population growth rate multi-generation Mortality, reproduction

Acute

Bejarano and Chandler (2003)

Population growth rate, acute Full life-cycle Full life-cycle Full life-cycle

Chandler et al. (2004)

Acute Acute Exposure Exposure Exposure

Sullivan et al. (1983) Bushong et al. (1988) Lesueur et al. (2015) Cailleaud et al. (2009) Ger et al. (2009)

Exposure Exposure

Michalec et al. (2016) Forget-Leray et al. (2005)

Exposure

Lesueur et al. (2013)

Acute Full life-cycle

Bengtsson (1978) Breitholtz and Bengtsson (2001)

Full life-cycle

Breitholtz et al. (2003)

Full life-cycle

Breitholtz and Wollenberger (2003)

Acute

Bejgarn et al. (2015)

Mortality, development

Exposure

Breitholtz et al. (2008)

Mortality, development, reproduction rate Mortality, development, reproduction rate Mortality Population growth rate Mortality Mortality, acetylcholinesterase (AChE) activity Mortality, metallothionein induction Mortality

Full life-cycle

Hutchinson et al. (1999a)

Full life-cycle

Hutchinson et al. (1999b)

Acute Full life-cycle Acute Acute

Hutchinson et al. (1994) Bechmann (1999) Forget et al. (1998) Forget et al. (1999)

Acute

Barka et al. (2001)

Acute

Geffard et al. (2005)

Reproductive rate

Mortality Mortality Mortality Gene expression Toxicity, post-exposure effects Behavior Survival, development, reproduction

Mortality, growth in nauplii Mortality Mortality, development, reproductive success, acute toxicity Mortality, development, population growth rate, acute toxicity Development, population growth rate Mortality

Wollenberger et al. (2005)

Cary et al. (2004) Volz and Chandler (2004) Templeton et al. (2006)

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Table 4 List of selected genes studied in Paracyclopina nana. Species

Genes

References

P. nana P. nana P. nana

Dorsal, Dorsal-like Vitellogenin (Vg) Hsp10, Hsp40, Hsp60, Hsp70, Polymerase ␤, Uracil glycosylase, UIT-mismatch DNA glycosylase, RAD23 UV excision repair, UV resistance GR, CAT, MnSOD, CuZnSOD, PHGPx, SeGPx, Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, GST-mu, GST-kappa, GST-omega, GST-zeta, Vg1, Vg2 CuZn-SOD, Mn-SOD, Catalase, GR, GST-Omega, GST-mu, GST-zeta, Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, se-GPx, p53, DNA-PK, Ku70, Ku80

Jeong et al. (2015) Hwang et al. (2010) Won et al. (2015a,b)

P. nana P. nana

re-brooding rate of ovigerous P. nana (Won et al., 2015a,b). UVinduced egg sac damage was closely correlated with a reduction in the naupliar hatching rate of UV-irradiated ovigerous females. Using chlorophyll ␣ and stable carbon isotope incubation experiments, a dose-dependent decrease (P < 0.05) in food ingestion and the rate of nutrient assimilation was found in response to UV radiation. This observation implied that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation (Won et al., 2015a,b). Expression of base excision repair (BER) and hsp chaperoning genes were significantly increased in response to UV radiation at the 1 kJ m−2 exposure level in P. nana, implying that even a low level of exposure was detrimental to survival or adaptation under stressed conditions. The 24-h LD50 dose of UV radiation of the copepod P. nana was 6.9 kJ m−2 (Won et al., 2014), while the 48-h LD50 dose of the harpacticoid copepod Tigriopus japonicus was 26.4 kJ m−2 (Rhee et al., 2012) (Fig. 1). A dose-dependent increase of egg sac injury was found in response to UV radiation in P. nana (Won et al., 2015a,b), indicating that UV radiation can induce a striking effect on the reproduction of copepods. Reproductive impairment and reduced fecundity in ovigerous P. nana females were often observed after UV radiation, suggesting that UV radiation interferes with the copepod hatching mechanisms, likely due to increased ROS levels (Rhee et al., 2012). In P. nana, the enhanced formation of lipid peroxides and a subsequent reduction of EPA and DHA were demonstrated by oxidative stress after UV radiation (Won et al., 2014). However, in UV irradiated P. nana, reduction in EPA and DHA was not accompanied by change in the fatty acid composition (Won et al., 2015a,b). This may be explained by the generation of LPO byproducts. Finally, we conclude that in vivo endpoints (the life cycle, including mortality and reproductive parameters) can be linked to biomarker responses in P. nana after UV radiation. Furthermore, changes in energy metabolism or nutrient may adversely affect the food web. A species that is critical in the food web such as P. nana offers valuable insight into the dynamics of aquatic pollutants. Overall, the endpoints including fecundity and various macromolecules were found to be good indicators of the effects of UV radiation on reproductive physiology and community structure in the copepod P. nana. 2.2. Gamma radiation responses of P. nana Accidental nuclear radioisotope release from nuclear power plants into the ocean is of concern due to associated ecological and health risks. An understanding of the effects of gamma radiation on aquatic invertebrates is helpful in determining its effects on aquatic ecosystems and also human health (Kryshev and Sazykina, 1995). The adverse outcomes of radioisotopes on marine organisms were examined by assessing the effects of gamma radiation on growth and fecundity in P. nana (Won and Lee, 2014). Upon exposure to gamma radiation, mortality (LD50–96 h = 172 Gy) was significantly increased in a dose-dependent manner in ovigerous P. nana females. In the over 30 Gy-irradiated group, growth retardation was found. Thus, offspring did not grow to adults (Won and

Lee et al. (2012) Won et al. (2014)

Lee, 2014). Particularly, ovigerous P. nana females showed abnormal bilateral egg sacs, and their offspring did not develop normally to adulthood at more than 50 Gy. Also, a dose-dependent increase in oxidative levels with elevated antioxidant enzymatic activities and DNA repair activities were found at levels higher than 50 Gy. These findings indicate that gamma radiation can induce oxidative stress and DNA damage in P. nana, resulting in growth retardation and impaired reproduction. These findings, again, signify the role of P. nana in risk assessment of radiation in marine biota. P. nana is more susceptible to gamma radiation than other copepods (e.g., T. japonicus, see Han et al., 2014) (Won and Lee, 2014). However, it was found to be more resistant than vertebrates, indicating that P. nana is suitable for monitoring the effects of gamma radiation in marine environments, particularly in coastal regions with high levels of radionuclide leaks. Different susceptibilities between organisms are likely due to various DNA content, size, repair processes, and cell cycle kinetics (Huang et al., 2005; Cassidy et al., 2007), resulting in different sensitivities in response to gamma radiation. After gamma radiation, reproductive success of P. nana declined significantly as indicated by reduced egg sac production (Won and Lee, 2014). In the eggs, the damageinducing dose (EC50-48 h, 110 Gy) was lower than that in adult P. nana (LD50-96 h, 172 Gy). Regarding reproductive success, P. nana required more than four days to obtain new egg sacs after spawning, but failed to hold normal bilateral egg sacs in response to 50 and 100 Gy gamma radiation. Untreated P. nana females generated bilateral egg sacs again within 24 h after spawning of the previous egg sac. This indicates that gamma radiation negatively affected the developmental process from the egg to the adult stage in ovigerous P. nana females. Adult P. nana females become infertile in response to 150 Gy gamma radiation, suggesting that this dose induced severe damage to the reproductive system. Previous studies suggest that the sperm population, stored in the urosome of the female, is affected by gamma radiation (Thorp and Covich, 2010), leading to reduced fecundity. These findings highlight the significance of gamma radiation to P. nana at various life stages. In the naupliar phase of P. nana, perturbation of development by gamma radiation was observed (Won and Lee, 2014). Generally, the growth rate of organisms has been considered a good parameter to show the underlying physiological conditions in a long-term exposure test (Dahlhoff, 2004). In copepods, the molting system is highly associated with growth, as distinctive developmental stages of copepods occur during molting (Lee et al., 2012; Mugnier and Soyez, 2005). Along with the retarded growth rate in P. nana, Won and Lee (2014) observed impaired reproduction (e.g., a low rate of egg production) in the gamma-irradiated group. Copepod reproduction is one of the main factors of population recruitment (Camus et al., 2009), as it is closely associated with egg hatching rate, naupliar and copepodid survival, and development rate. Growth damage and impaired reproduction in P. nana can be of great concern, as reproductive failure is an important factor for both aquaculture production and ecosystem restoration. The findings on UV- and gamma-radiation reveal that P. nana is a suitable test organism for assessment of radiation effects. Its use as a sen-

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tinel bioindicator species for adverse effects of radiation in critical biota may be considered. 2.3. Antioxidant defense mechanisms in response to gamma radiation in P. nana Study of gene transcription levels in aquatic invertebrates provides reliable early signals for detecting physiological changes under environmental stress (Morgan et al., 2009). In gammairradiated P. nana, increased ROS and GSH levels were accompanied by elevated activities of SOD, CAT, GR, and GST to cope with the oxidative stress (Won and Lee, 2014). Similarly, the cyclopoid copepod Mesocyclops hyalinus showed enhanced activities of SOD and CAT under gamma radiation exposure with fewer changes in morphology and survival than the calanoid copepod Allodiaptomus satanus, which showed decreased activities of SOD and CAT (Mukherjee et al., 2010). This indicates that antioxidant enzymatic activities play an important role in protecting organisms against gamma radiation-induced cellular oxidative stress. GSH is associated with other antioxidative enzymes and acts as a substrate or co-factor for antioxidant enzymes (Freedman et al., 1989). Taken together, GSH and the antioxidant enzymes (SOD, CAT, GR, GST) protect P. nana from the destructive effects of ROS and their measurement may provide critical insight into survival and adaptation traits of P. nana and other copepods. Oxidative stress has been reported to be associated with reduced survival rates including other sublethal effects (Ryter et al., 2007). When D. magna was exposed to alpha radiation, there was an increased metabolic cost of protecting the maintenance mechanisms from radiological stress (Alonzo et al., 2006). In P. nana, growth rate and reproduction were negatively affected by gamma radiation (Won and Lee, 2014), and oxidative stress has been implicated in this effect. In P. nana, GR, SOD, CAT, GST, Hsp, and GPx genes together with antioxidant enzymatic activities were all up-regulated in response to gamma radiation (Won and Lee, 2014). Because gene transcripts and their expression levels may provide early signals for physiological changes under environmental stress, the modulated patterns of specific genes could provide a better understanding of the underlying molecular mechanisms of the detoxification upon exposure to gamma radiation. As shown by Won and Lee (2014), antioxidants play a central role in resisting ROS-induced cytotoxicity in response to gamma radiation in P. nana. Additionally, P. nana Hsp genes were expressed at high levels upon gamma radiation (Won and Lee, 2014), as they are responsible for protecting key cellular proteins through chaperoning of proteins denatured by environmental stressor-induced oxidative damages. Thus, gamma radiation-induced Hsp gene expression in P. nana provides valuable clues as to how P. nana responds to cellular damages in response to gamma radiation. Upon exposure to gamma radiation, dose-dependent effects were observed on mRNA expression of the P. nana p53 gene (Won and Lee, 2014). In human lymphoblastoid cell lines, p53 regulates the transcription of DNA repair genes in diverse signal transduction pathways (Jen and Cheung, 2005). In gamma-irradiated P. nana, dose-dependent increases of DNA-PK, Ku70, and Ku80 genes were shown (Won and Lee, 2014). In human cells, DNA-PK, Ku70, and Ku80 genes are key components in recognizing double strand breaks (DSBs), repaired by ligating the broken ends without homologous templates (Mahaney et al., 2009). Thus, elevated expression of P. nana DNA-PK, Ku70, and Ku80 genes implies that those genes are closely related to the enhanced DNA repair processes to recover oxidative stress-induced cellular damage in gamma-irradiated P. nana (Won and Lee, 2014). The synthesis of GSH and DNA repair systems were induced by gamma radiation to mitigate free radicals and to repair DSBs (Won and Lee, 2014). Additionally, the growth retardation and reproductive impairment of P. nana in response to

gamma radiation provides a suitable life-cycle indicator for a better understanding of gamma radiation-induced impairment. However, there are still no data on the effects of gamma radiation at the level of population and community dynamics in aquatic ecosystems. The research on P. nana may provide impetus to better understanding of these effects.

3. Advantages of using biomarkers from P. nana in biomonitoring Several molecular biomarker genes have been used in ecophysiological and ecotoxicological investigations in P. nana (Table 4). These genes are of interest for the early warning of pollution. For example, induction of the vitelline exposed to a “no observed effect concentration” (NOEC) of trace metals in P. nana demonstrated that vitelline may be a sensitive early warning indicator for metal exposure (Hwang et al., 2010). Hwang et al. (2010) provided the first report on the characterization of vitelline genes from P. nana and showed its expression to be developmental stage- and sex-specific. The upregulation of the vitelline gene in response to exposure to Cd, Cu, and As suggests that P. nana vitelline may be a promising biomarker for assessing and monitoring the toxic effect of heavy metals in brackish waters. In P. nana, downregulation of vitelline genes after UV-B radiation was also correlated with a reduced reproduction rate (Won et al., 2015a,b). Reproduction is generally inhibited by environmental stressors, as environmental stressors lead to the reallocation of cellular energy from reproduction to cellular defenses (Michalek-Wagner and Willis, 2001; Gomes et al., 2015; Won et al., 2015a,b). However, more detailed molecular studies are necessary to elucidate the role of each vitelline isoform and the modulatory mechanisms of heavy metals and UV-B radiation on vitelline expression. Vitellogenesis is an essential process in reproduction (Tsutsui et al., 2005). In crustaceans, vitelline mRNA can be used as an index of vitellogenesis in the kuruma prawn Marsupenaeus japonicus (Tsutsui et al., 2005) and vitelline mRNA of Penaeus merguiensis showed upregulation during the vitellogenic stage (Phiriyangkul et al., 2007). In addition, abiotic stressors such as heavy metals (e.g., Cu, As, and Cd) are known to induce vitelline mRNA expression in copepods. For instance, cadmium caused vitelline gene induction in T. japonicus (Lee et al., 2008), and two isoforms, vitelline 1 and vitelline 2 genes, in P. nana were induced by copper, arsenite, and cadmium (Hwang et al., 2010). Vitellogenin (Vg) is a precursor protein of vitellins in the egg yolk and provides free amino acids, lipids, carbohydrates, and ecdysteroids for normal development before hatching (Wahli et al., 1981; Arukwe and Goksøyr, 2003). Although there was no distinct density-dependent increase, mRNA expression of the vitelline gene was affected by higher population density compared to the lower density. This reduction of vitelline mRNA expression at higher density conditions could lead to a decrease in the naupliar production of copepods (Hwang et al., 2010). GST genes are promising biomarkers that show physiological alterations under oxidative stress conditions induced by xenobiotics and other environmental stressors (UV-B radiation, salinity, density, temperature, etc.) in aquatic organisms (Cailleaud et al., 2007; Lee et al., 2012; Won et al., 2014). Also, the water accommodated fraction (WAF) of crude oil-exposed P. nana and T. japonicus show a significant expression of GST genes following a dose-dependent increase in expression of CYP450 genes. This indicates that the components of WAF (mainly low molecular weight polycyclic aromatic hydrocarbons [PAHs] and alkylated PAHs) can be transformed by phase I and phase II reaction, of xenobiotics (Han et al., 2014, 2015). In P. nana, Hsp genes have also been considered suitable early warning biomarkers in response to environmental stress. In case

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Fig. 5. Genome wide identification provides mechanistic insight into this species and the environment: Wide range of endpoints for ecotoxicological studies.

of UV-B exposure, expression of four isoforms of Hsp genes (i.e., Hsp10, Hsp40, Hsp60, and Hsp70) were significantly increased (Won et al., 2015a,b). Similarly, P. nana Hsp genes in response to gamma radiation were highly expressed in a dose-dependent manner with subcellular alterations including ROS generation and several antioxidant enzyme activities (Won and Lee, 2014). Hsps play an important role in the chaperoning and degradation of denatured and damaged proteins (Parsell and Lindquist, 1993; Nollen and Morimoto, 2002). Thus, subsequent results of gamma radiationinduced damage by ROS generation are closely related to the increased expression of Hsp in recovering growth and reproduction in P. nana. 4. Research can help elucidate the role of P. nana in ecotoxicology Recent findings and future research directions on P. nana are summarized in Fig. 5. Briefly, sensitive in vivo endpoints of the whole life cycle parameters including molting, reproduction, short life cycle, and high fecundity are useful in assessing the ecotoxicological effects in copepods. Particularly, the biological indices covering molecules to the population level in response to environmental stressors and/or pollutants are highly valuable in the case of P. nana (Fig. 5). Rarely, such a huge data set is available for a copepod species. Ecotoxicological testing involving ethical concerns, the maintenance cost, and relative inefficiency of animal research has caused the development of alternative methods for ecotoxico-

logical testing. Alternatives to in vitro studies (cell cultures, other in vitro studies) can provide biomarkers that can also be used in the development of biosensors and offer an advantage that preserves the physiology of the living cell; these approaches ultimately do not require the sacrifice of an animal for observational or mechanistic studies. Recent advances in molecular approaches (omics studies) can identify stressor-associated genes and their metabolic products in situ. Several animal models serving as test subjects in ecotoxicological research, however, may be selectively tolerant to various stressors. This would lead an investigator to directly observe the key outcomes of interest. Furthermore, the complex nature of ecotoxicological interactions makes it difficult to translate these observations into meaningful interpretations and regulatory recommendations (Depledge, 1998). This may confound the use of such animals in ecotoxicological testing. According to several publications of the National Institutes of Health (NIH) in the United States, ecotoxicological protocols using animal models should be based on the principles of Replacement, Reduction, and Refinement (the 3 Rs). “Replacement” refers to efforts to engage alternatives to animal use. This includes the use of computer models, non-living tissues and cells, and replacement of “higher-order” animals such as mammals and primates with “lower” order animals (e.g., cold-blooded vertebrates, invertebrates, bacteria) where this is possible. “Reduction” refers to efforts to minimize the number of animals used during the course of an experiment, as well as prevention of unnecessary replication

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of previous experiments. “Refinement” refers to efforts to make experimental design as efficient as possible in order to minimize suffering. Using invertebrate models such as crustaceans (e.g., P. nana) is advantageous because they replace the need for vertebrate models. There are several areas in need of more research that would allow P. nana to become a more recognized model invertebrate for aquatic ecotoxicology. Firstly, there is a need for more ecotoxicity, gene profiling, and environmental genomic studies using P. nana. In addition, more studies are necessary at the proteomic and metabolomic levels to understand the mechanisms of stressor impacts on this organism. There is also a need to harmonize and standardize testing methodologies using P. nana and characterize sources of the test animals in updated barcoding technologies that are not fraught with taxonomic problems. This implies that more studies have to be undertaken to look into the interspecific and intraspecific differences (i.e., between genetic populations and geographic populations) with subsequent population differences in ecotoxicological sensitivities of P. nana. There is a need to identify standardized strains of P. nana that could be used in inter-laboratory harmonization exercises. Variability should not be ignored as the magnitude of variation is comparable to other sources of variability in laboratory tests. Ultimately, the aim is to establish a standardized strain of a P. nana species for laboratory cultures (OECD, 2011). P. nana is ecologically relevant and its position in aquatic food webs is central. As mentioned earlier, tests based on copepods like P. nana are cost-effective. This holds particularly true for the small body size that, among other characteristics, allows for whole animal testing. Their short life cycle allows multi-generation tests with an intrinsic population growth rate and biological fitness as endpoints. P. nana is easy to obtain from the field and simple to culture and maintain in the laboratory. Because several stressors are reported to co-occur with P. nana in the field (e.g., oil, heavy metals, EDCs, radiation), ecotoxicological reactions at all integration levels can be tested under field conditions. These also include omics approaches with sublethal endpoints (gene expression including the whole transcriptome, biochemical, and metabolic endpoints) (Fig. 5). Toxicity studies conducted in different laboratories have shown reproducible data and high sensitivity of P. nana to a wide range of chemicals (Tables 1 and 4). Thus, P. nana meets these animal model criteria and could be used as a model in risk assessment with intensive standardization and regulatory validation.

5. Summary and conclusion A comparison among copepods revealed that P. nana has a large database on its genomics and biological response profile. Ecotoxicological tests with P. nana were performed using standardized methodology. P. nana shows favorable biological attributes (small size, high fecundity, short life-cycle, sexual reproduction, distinctive life stages, ease of culture) that would usher its use as a marine model organism in ecophysiological and ecotoxicological investigations. Its environmental representation, including a wide range of distribution, high abundance, and the available of a broad knowledge base regarding its cultivation, facilitates is use in laboratories. Available knowledge on its sensitivities towards common stressors from the electromagnetic spectrum as well as xenobiotics and base-line knowledge on its genome further strengthens its use as a sentinel organism. A number of important genes have been studied for their sequence and expression matrix in P. nana. It is noteworthy that accurate interpretation of gene expression may only be possible when experiments are conducted as part of an integrated approach to understand observed responses at molecular and physiological levels. An integrated approach involving classical as well

as omics approaches may offer more relevant information about the overall environmental effects than specialized studies. Because of a short life-cycle and relatively simple culture under laboratory conditions, P. nana offers useful traits for evaluating the effect of toxic chemicals on biological fitness. Multi-generation tests may provide valuable insight into how stressors may affect the biological fitness of a species and how a species may adapt to toxic stress. Multi-generation tests are not common in ecotoxicology because of logistic issues regarding animal husbandry and culture. P. nana shows great promise in this respect. Chronic studies in P. nana, evaluating its life-cycle and intrinsic population growth rate are feasible. These studies highlight that P. nana with its favorable biological attributes, its wide geographic and environmental representation, large conventional and omics database present itself to the scientific community as a small invertebrate model in aquatic toxicology. This holds particularly true for risk assessment studies involving polluted marine/estuarine habitats and aquaculture settings. In contrast to other copepod species that are already suggested as model species, e.g., T. japonicus, P. nana belongs to a different phylogenetic group (i.e., Cyclopoida) and is of particular ecological relevance being a suspension feeder on primary producers and a central prey in pelagic food webs (Nunes et al., 2006; Raisuddin et al., 2007).

Acknowledgements This work was supported by a grant from the Development of Techniques for Assessment and Management of Hazardous Chemicals in the Marine Environment of the Ministry of Oceans and Fisheries, Korea funded to Jae-Seong Lee. H.G. Park acknowledges the support of National Research Foundation (2013R1A1A2013070). H.-U. Dahms acknowledges the support of a grant from the Research Center for Environmental Medicine, Kaohsiung Medical University (KMU), the Asia-Pacific Ocean Research Center of the Department of Oceanography (No. 76211194) in the frame of the KMU/NSYSU cooperation, and MOST104-2621-M-037001 to T.H. SHIH. Mr. Guenole Alizard is acknowledged for helpful literature support.

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