Plankton Benthos Res 12(1): 25–33, 2017 Plankton & Benthos Research © The Plankton Society of Japan Nucleic acid ratio as a proxy for starvation of coastal and pelagic copepods in the North Pacific Ocean 1, 2 3 4 5 TORU KOBARI *, SACHI MIYAKE , WILLIAM T. PETERSON , JAY PETERSON & TRACY SHAW 1 Aquatic Sciences, Faculty of Fisheries, Kagoshima University, 4–50–20 Shimoarata, Kagoshima 890–0056, Japan 2 Aquatic Sciences, Graduate School of Fisheries, Kagoshima University, 4–50–20 Shimoarata, Kagoshima 890–0056, Japan 3 Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration, 2030 SE Marine Science Drive, New- port, OR 97365, USA 4 Office of Science and Technology, National Oceanic and Atmospheric Administration, 1315 East-West Hwy., Silver Spring, MD 20910, USA 5 University of South Florida, College of Marine Science, 140 7th Avenue S., St. Petersburg, FL 33701, USA Received 1 August 2016; Accepted 1 November 2016 Responsible Editor: Koichi Ara Abstract: We investigated the nucleic acid ratio (RNA/DNA) as a proxy for starvation in copepods collected from coastal and pelagic sites across the North Pacific Ocean. While RNA/DNA ratios were variable among copepod species and their life stages, lower ratios were found for the copepods collected from the Kuroshio region compared to ones from the western subarctic site of the North Pacific Ocean. Log-transformed RNA/DNA ratios showed a negative cor- relation to log-transformed mean temperature in the sampling layer and a positive correlation to log-transformed chlo- rophyll a concentrations. Copepod incubation experiments demonstrated that RNA/DNA ratios significantly declined after exposure to starvation and the decline was greater for the copepods collected from the subtropical sites compared with those from the subarctic. However, no significant decline was found for some species and stages accumulating lip- ids. These findings suggest that copepod RNA/DNA ratios are associated with food availability but are not sensitive to starvation for some copepods accumulating lipids. Key words: copepods, DNA, RNA, starvation, lipids mined by their growth and mortality. For the last half cen- Introduction tury, a great deal of effort has been dedicated to increasing Pelagic calanoid copepods are crustacean zooplankton knowledge on zooplankton growth, estimated by tech- showing a high species diversity, with more than 2000 spe- niques such as frequent field sampling (e.g., Heinle 1966, cies world-wide (Razouls et al. 2005–2013). They are a Liang et al. 1996, Kobari et al. 2010) and ship-board or significant component of the zooplankton community in laboratory incubations (e.g., Burkill & Kendall 1982, Kim- terms of both abundance (Longhurst 1985) and biomass in merer & McKinnon 1987, Berggreen et al. 1988). On the the world’s oceans (Verity & Smetacek 1996). The material other hand, zooplankton mortality is difficult to estimate and energy flows within aquatic food webs are influenced due to the population variables being strongly influenced by their feeding on not only phytoplankton, but also pro- by the diffusion and advection of water masses (field sam- tozoans and sinking particles (e.g., Dagg 1993, Kobari et pling) and difficulty excluding predators in the incubation al. 2003a). The pelagic calanoid copepods are also major techniques (see Hirst & Kiørboe 2002). While cohort anal- food resources for fishes, mammals and sea birds (e.g., ysis with frequent sampling provides information on mor- Hunt et al. 1998, Moku et al. 2000, Yamamura et al. 2002). tality (Ohman & Hirche 2001), the practical application to Thereby, their population dynamics are important for un- pelagic zooplankton is logically much more difficult. derstanding trophodynamics in marine ecosystems. Biochemical approaches have been used for under- Population dynamics of copepods are largely deter- standing physiological processes of zooplankton for the last two decades (e.g., Oosterhuis et al. 2000, Sastri & * Corresponding author: Toru Kobari; E-mail, kobari@fish.kagoshima-u. Roff 2000, Wagner et al. 2001, Yebra & Hernández-Léon ac.jp 2004). Among the biochemical approaches, nucleic acid- 26 T. KOBARI et al. based indices have been most commonly applied in vari- Materials and Methods ous metazoan species as proxies of individual physiologi- cal condition, reproductive activity and somatic growth Oceanographic observations and sample collections (see Yebra et al. in press). The nucleic acid ratio (RNA/ DNA) is suggested as a proxy of feeding history, includ- Oceanographic observations and sample collections ing starvation, because nucleic acids are functioning in were conducted during cruises on the R/V Elakha from the first steps of protein synthesis and cellular contents May to August 2012 off the Oregon coast in the eastern of nucleic acids vary with protein synthesis activity (e.g., North Pacific (NH5, HN20, NH25, NH35, NH45 and Gorokhova & Kyle 2002, Speekmann et al. 2007, Holmbo- NH65), the R/V Mirai from June to August 2011 at sub- rn et al. 2009). Starvation is considered to increase cope- tropical (S1) and subarctic (K2) sites in the western North pod mortality risks due to the reduction of their swimming Pacific Ocean, the T/S Nansei-Maru during May 2012 in activity (Mackas & Burns 1986). Although biochemical Kagoshima Bay (KB1 to KB15), and the T/S Kagoshima- approaches have been applied for identifying starvation in Maru during November 2012 in the Kuroshio region (KR4 naturally occurring larvae of pelagic fishes and gastropods to KR9) (Fig. 1). Temperature, salinity and chlorophyll flu- (e.g., Chícharo et al. 1998, Vidal et al. 2006) and incubated orescence was recorded from the sea surface to 100 m us- copepods of coastal species (e.g., Saiz et al. 1998, Speek- ing a CTD system. Water samples for chlorophyll measure- mann et al. 2007), we have little information on its poten- ments were collected with a plastic bucket from the sea tial use for pelagic zooplankton species in nature. surface in the eastern North Pacific or a CTD-CMS system In the present study, we investigated geographical varia- at 7 discrete depths (0, 10, 20, 30, 50, 75, 100 m) in the tions in individual contents of RNA to those of DNA for western North Pacific. These samples were filtered through copepods collected from coastal to pelagic sites across the Whatman GF/F filters and chlorophyll pigments were ex- North Pacific Ocean, in order to investigate how nucleic tracted with 90% Acetone (eastern North Pacific) or N,N- acid ratios of coastal to pelagic copepods change under dimethylformamide (western North Pacific). Chlorophyll a the ambient temperature and food supply. Moreover, we concentration was measured with a fluorometer (10-AU, compared changes in RNA/DNA ratios of several species Turner Designs, USA) by the acidified fluorometric method and developmental stages of copepods, following exposure (Holm-Hansen et al. 1965) (eastern North Pacific) or the to starvation conditions (i.e., no food supply), to determine non-acidified fluorometric method (Welschmeyer 1994) if particular geographic regions and life-history stages are (western North Pacific). Chlorophyll fluorescence was cali- more susceptible to low food concentrations. From these brated with the observed chlorophyll a concentrations. results, we discuss the potential use of RNA/DNA ratios as We collected copepods from the upper 25 m using a a proxy of starvation in copepods. Bongo net with a 2 L cod end (diameter 60 cm, mesh size 0.335 mm) (eastern North Pacific) or from the upper 100 m using a North Pacific Standard net with a 3 L cod (diam- eter 45 cm, mesh size 0.335 mm) (western North Pacific). Bongo (obliquely) and North Pacific Standard (vertically) Fig. 1. Sampling stations in the eastern (NH5–NH65) and western (K2, S1, KNOT, KR4–KR9, KB1–KB15) North Pacific Ocean. Shaded box shows Kuroshio area enlarged in the map. Current systems are superimposed. Nucleic acid ratio as a proxy for starvation in copepods 27 nets were towed at a speed of 0.5 to 1 m sec−1. After col- NGE= GGE/. AE (4) lection, the predominant copepods among the zooplankton community were identified to species and developmental Assuming 0.3 for GGE and 0.7 for AE (Omori & Ikeda stage under a dissecting microscope. For the incubation 1984), NGE is estimated to be 0.43. If copepod NGE under experiments, individual copepod was put into a 500 mL starvation exposure is lower than 0.43, they metabolize polycarbonate bottle containing filtered seawater (pore more body carbon for their respiratory requirements than size: 0.7 µm). For geographical comparisons of RNA/DNA ingested carbon. Thus, we define NGE of 0.43 of as the ratios, individual copepods were put into a 2 mL vial and starvation point. Assuming 0.4 for the carbon content (Pe- preserved at −80°C until analysis. ters & Downing 1984) and 0.7 for the respiratory quotient (lipid metabolism: Omori & Ikeda 1984), ACW and RC Incubations were estimated as per the following equations: For comparing the response of the nucleic acid ratio to ACW =×0.4 10(1.1623×− log(PL ) 1.688) (5) starvation, we incubated copepods collected from the sub- tropical and subarctic sites in the western North Pacific un- RC=××0.7 RO 12/22.4 (6) der starvation conditions. Individuals (up to 50 animals) of the dominant species and life stages were transferred into where PL is prosome length (mm) and RO is oxygen con- −1 −1 a 500 mL polycarbonate bottle containing filtered seawater sumption (µL O2 ind h ) estimated with the empirical (pore size: 0.7 µm) and incubated under dark conditions model (Ikeda et al. 2001): at 20°C for 2 d at S1 and at 5°C for 5 d at K2. Due to the RO =exp[0.124 +× 0.780 ln(ACW ) +× 0.073 ( WT )] × 24 (7) slow physiological responses to starvation exposure at low temperature for the subarctic copepods, the incubation du- Nucleic acids analyses ration at K2 was prolonged to be able to compare the meta- bolic consumption between the subarctic and subtropical RNA and DNA were measured following the micro- copepods using the empirical model of Ikeda et al.