Fish Sci (2009) 75:13771387 DOI 10.

1007/s12562-009-0172-3

ORIGINAL ARTICLE

Fisheries

Measurements of density contrast and sound-speed contrast for target strength estimation of Neocalanus copepods (Neocalanus cristatus and Neocalanus plumchrus) in the North Pacic Ocean
Ryuichi Matsukura Hiroki Yasuma Hiroto Murase Shiroh Yonezaki Tetsuichiro Funamoto Satoshi Honda Kazushi Miyashita

Received: 30 June 2009 / Accepted: 1 September 2009 / Published online: 15 October 2009 The Japanese Society of Fisheries Science 2009

Abstract The mass density and sound-speed contrasts against surrounding seawater (g and h, respectively) of Neocalanus copepods (N. cristatus and N. plumchrus) were measured in 2006 and 2007 to compute the theoretical target strength (TS). The values of g ranged from 0.997 to 1.009 in N. cristatus and from 0.995 to 1.009 in N. plumchrus. There were no correlations between prosome length (PL) and g. The values of h ranged from 1.006 to 1.021 in N. cristatus and from 1.013 to 1.025 in N. plumchrus and varied with changes in temperature. TS was estimated with the theoretical sound scattering model using the values of g and h based on the temperature, salinity, and depth of the

location where the specimens were collected. Regressions of the tilt-averaged TS versus PL were obtained at 38, 120, and 200 kHz. The averaged TS of N. cristatus and N. plumchrus at 120 kHz, which is widely used as a high frequency, ranged from -110.0 to -103.1 dB and from -121.4 to -109.7 dB, respectively. There was a positive correlation between frequency and averaged TS: the higher the frequency, the higher the value of averaged TS. The TS at 120 and 38 kHz varied from 14.8 to 16.4 dB in N. cristatus and from 17.9 to 18.7 dB in N. plumchrus, respectively; that at 200 and 120 kHz varied from 2.9 to 5.5 dB in N. cristatus and from 5.3 to 6.5 dB in N. plumchrus, respectively. Keywords Copepod Density contrast Neocalanus cristatus Neocalanus plumchrus Signal-to-noise ratio Sound-speed contrast Target strength

R. Matsukura (&) Laboratory of Marine Ecosystem Change Analysis, Graduate School of Environmental Science, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan e-mail: ryuichi_mtkr@yahoo.co.jp H. Yasuma K. Miyashita Laboratory of Marine Ecosystem Change Analysis, Field Science Center for Northern Biosphere, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan H. Murase S. Yonezaki The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan T. Funamoto Hokkaido National Fisheries Research Institute, Fisheries Research Agency, 116 Katsurakoi, Kushiro, Hokkaido 085-0802, Japan S. Honda National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan

Introduction Copepoda represents one of the most dominant groups of zooplankton communities in the ocean. Of these, Neocalanus species are the most dominant large grazing copepods found in the subarctic Pacic and its marginal seas [1]. In the Oyashio region, N. cristatus and N. plumchrus are important prey for shes and whales, forming the basis of the energy channel [2, 3] in the surface layer during their growth from spring to summer. Their life cycles are annual or biennial and include a vertical migration down to depths C500 m following their growth in the surface layer during the spring to summer [4, 5]. A classical approach to studying the vertical distribution of zooplankton is the use of a closing net to collect

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samples. For more complex studies in which the patch size and abundance in the horizontal plane are to be monitored continuously, electric plankton counters [6], laser optical plankton counters [7], and video plankton recorders [8] have been developed and used. The primary aims of these sampling methods are to collect specimens and/or to identify particles. This has lead to the proposal that an acoustic survey with a quantitative echosounder would be an easier approach for carrying out large-scale studies [9]. In acoustic survey, acoustic backscattering strength is measured and translated into abundance by the target strength (TS) of the individual target. It is therefore necessary to know the TS of the target species. For shes with a swimbladder lled with gas [10] and those with no swimbladder (e.g., Todarodes pacicus [11]), in situ or ex situ TS measurements are made with a split or dual beam echosounder [9]. However, theoretical sound scattering models are generally used to obtain the TS of small zooplankton [1215]. The mass density contrast g and soundspeed contrast h against surrounding seawater are two of the most important factors needed to compute the TS of uid like zooplankton. For example, it has been reported that the g and h for euphausiids are different from each species and that the values change seasonally within each species [1416]. In copepods, g and h data are limited. Greenlaw and Johnson [17] reported the g and h of N. plumchrus, which was preserved, and Kgeler et al. [16] reported the g and h of Calanus nmarchicus but did not specify the stage of the samples. The sound-speed as well as the body-mass density of copepods is thought to change because the body-mass density of copepods changes according to season and life stage [18]. In terms of an acoustic survey, the TS should be computed with g and h that are appropriate for the season, the location, and the life-cycle stage because changes in g and h values affect the variations in the theoretical TS. In addition, preserved specimens should be avoided, and live specimen should be used for measuring g and h because preserved samples lose their volume or weight, thereby causing errors in the estimation of TS [12, 14, 19]. In the study reported here, our aims were (1) to measure the mass density contrast g and sound-speed contrast h of live N. cristatus and N. plumchrus and (2) to estimate TS using a theoretical sound scattering model with the value range of g and h.

(499 ton) within the framework of the project Research on the Assessment of Fisheries Stocks in the Waters around Japan. Collections were also made in August 2007 in the Western North Pacic (4456.90 N, 16554.70 E) using a NORPAC net (mouth diameter 45 cm, mesh size 0.35 mm) and an Isaacs-Kidd midwater trawl (mesh size 13 mm) aboard the R/V Kaikou Maru (860 ton) during a cruise of the Japanese Whale Research Program under Special Permit issued within the framework of the Western North Pacic-Phase II (JARPN II) study (Fig. 1). The net tows were conducted to target a strong scattering layer observed on the echograms. Before or after each net tow at each sampling point, oceanographic observations were conducted with a conductivity-temperature-depth proler (CTD; model SBE19, Sea-Bird, Bellevue, WA) or with an expendable conductivity temperature depth probe (XCTD; model XCTD-1, Tsurumi-Seiki, Yokohama, Japan) to obtain the temperature and salinity. The following measurements were made with live copepods on board immediately after the nets were reeled in. Body-mass density was measured using a density-bottle method in which body-mass density is determined by evaluating the buoyancy of each specimen [12, 14, 21]. A series of solutions was prepared in which mixed seawater was mixed with glycerol in steps of 0.002 g/cm3. The zooplankton was anesthetized (FA100, 4-allyl-2-methoxyphenol) before measurement, and a specimen was placed in each solution in turn. The density of the specimen was determined as the mean density of the last sinking solution and rst oating solution. The measurement methodology followed that of Mikami et al. [17] and Yasuma et al. [21].

55N

50N

45N 2007 40N 2006

Materials and methods Specimens were collected in June 2006 offshore Hiroo, Hokkaido, Japan (428.30 N, 14337.70 E) using a framed midwater sampling trawl (FMT) with a 2 9 2-m opening (mesh size 8 mm) [20] aboard the R/V Kaiyou Maru No.7

35N

Japan

Pacific Ocean

140E

150E

160E

170E

Fig. 1 Sampling location of Neocalanus cristatus and N. plumchrus. The crosses correspond to sampled areas in 2006 and 2007

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1379

The mass density of seawater (qsw) was calculated as a function of the mean temperature, salinity, and pressure [22] observed at depth range where the net tows were conducted. Density contrast g was given by q g animal qsw 1

fbs

Z
rpos

  2 ksw ac 1 h2 2 4kanimal gh2  J1 2kanimal ac cos btilt  drpos  cos btilt 4

e2ikanimal rpos

where qanimal is the mass density of the specimen. After the measurement, each specimen was preserved in individual microtubes containing buffered formalin with seawater (10% v/v). The speed of sound through the animal body was estimated using a time-average approach that is based on the measurement of the time-of-ight method [23]. The measurements of sound-speed were performed using an acrylic T-tube [23] with two transducers mounted at both ends of the horizontal tube. The 5-ls sinus pulse at 400 kHz was transmitted from one of the transducers and received the other. The transmitting time of the pulse between transducers was measured with a digital oscilloscope (2006: model 9314AM; LeCroy, Chestnut Ridge, NY; 2007: model DSO6012A; Agilent Technologies, Santa Clara, CA). We measured the transmitting time of the seawater copepod mixture (Ttotal), of only seawater (Tsw), and of the volume fraction of copepod in the mixture (V). These relations are given as Ttotal 1 V Tsw VTanimal 2

where fbs is the complex backscattering amplitude, the relation to rbs is given by rbs = |fbs|2, rpos is the position along the axis of the deformed cylinder, and k is the acoustic wavenumber given by k = 2p/k, where k is the acoustic wavelength. The subscript sw refers to the surrounding seawater, while the subscript animal refers to zooplankton, J1 is a Bessel function of the rst kind of order 1, ac is the cross-section radius of the cylinder, and btilt is the local angle between the cylinder and incident wave. We digitized and obtained at least 79 sets of rpos and ac from each image of specimens on which the body-mass density or sound-speed was measured. In this study, the swimming angle of copepods was assumed to be random. TS (dB) is dened as TS = 10 log10(rbs), and the averaged TS was dened as the mean TS of each 1.

Results Once we had obtained the acoustical parameters of the seawater (i.e. sound-speed and mass density), we measured the temperature and salinity using the CTD or XCTD. The acoustical parameters were calculated from the temperature, salinity, and depth data where the net tows were conducted (Table 1). The specimens collected in 2006 were distributed in layers that were deeper and colder than that those in 2007. The differences in Csw and the mass density of seawater between 2006 and 2007 were negligible. In 2006 and 2007, both the body-mass densities of N. cristatus and N. plumchrus were measured by the density-bottle method, and then the PL was measured on individual specimens (Table 2). We calculated the qsw with each parameter listed in Table 1. All of the specimens were at copepodite stage 5 (C5) [26]. In 2006, the PL of N. cristatus and N. plumchrus ranged from 6.94 to 7.95 mm (mean 7.38 mm) and from 3.68 to 4.61 mm (mean 4.09 mm), respectively. In 2007, the PL of N. cristatus and N. plumchrus ranged from 6.30 to 7.41 mm (mean 6.92 mm) and from 3.77 to 4.35 mm (mean 4.03 mm), respectively. There was a signicant difference between the PL of N. cristatus in 2006 and 2007 (P \ 0.05, Mann Whitney U test), but there was no remarkable difference between the PL of N. plumchrus in 2006 and 2007 (P [ 0.05). In 2006, the body-mass density of N. cristatus and N. plumchrus ranged from 1.026 to 1.037 g/cm3 (mean 1.030; g = 1.003) and from 1.022 to 1.036 g/cm3 (mean

where Tanimal is the passing time through that part of copepods body in the T-tube. The sound-speed ratio h is given by h Tsw Canimal Tanimal Csw 3

where Csw and Canimal are the speed of sound through seawater and body of copepods, respectively. Because Csw is known from Tsw, the measurement distance (200 mm), Canimal can be obtained. The sound-speed measurements were performed with the T-tube bathed in a temperaturecontrolled tank, in which the temperature was varied by about 0.5C per 10 min. After each measurement, the copepods volume V was measured by the volume displaced by submerging the specimens in a graduated cylinder. At the end of the experiment, specimens were preserved in buffered formalin with seawater (10% v/v). The scaled prosome length (PL) of specimens used for measuring the body-mass density and sound-speed was measured under a microscope. The sound scattering from zooplankton was estimated using the distorted wave born approximation (DWBA [24, 25]) model. A modication of the algorithm of Matlab ver. 6.1.0.450 (MathWorks, Natick, MA) was used to estimate the TS of the deformed cylinder [13] and given as

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Table 1 Mean oceanographic conditions and acoustical parameters of seawater where the net tows were conducted Targeted specimen 2006 2007 Neocalanus cristatus Neocalanus plumchrus Neocalanus cristatus Neocalanus plumchrus Temperature (C) 2.9 2.9 7.5 9.1 Salinity (PSU) 33.3 33.3 32.9 32.9 Depth (m) 152.5 161.0 40.0 25.0 Csw (m/s) 1462.0 1462.3 1478.3 1484.3 qsw (g/cm3) 1.027 1.027 1.026 1.026

Csw, the speed of sound through seawater; qsw, mass density of seawater

Table 2 Prosome length and body-mass density of Neocalanus cristatus and N. plumchrus Targeted specimen na PL (mm) Range 2006 2007 Neocalanus cristatus Neocalanus plumchrus Neocalanus cristatus Neocalanus plumchrus 37 49 52 37 6.947.95 3.684.61 6.307.41 3.774.35 Mean SD 7.38 0.23 4.09 0.19 6.92 0.22 4.03 0.12 Body-mass density (g/cm3) Range 1.0261.037 1.0221.036 1.0231.035 1.0231.032 Mean SD 1.030 0.003 1.026 0.003 1.026 0.004 1.024 0.002 1.003 0.998 1.000 0.998 g

PL Prosome length, SD standard deviation, g mass density contrast Density contrasts g is calculated from the qsw listed in Table 1
a

Number of specimens

1.026, g = 0.998), respectively. In 2007, the body-mass density of N. cristatus and N. plumchrus ranged from 1.023 to 1.035 g/cm3 (mean 1.026, g = 1.000) and from 1.023 to 1.032 g/cm3 (mean 1.024, g = 0.998). The body-mass density were signicantly different between species and year (P \ 0.05). There was no signicant correlation between the body-mass density and PL in 2006 and 2007. The sound-speed measurements were conducted by a time-averaged approach using the T-tube. The measurements of the sound-speed through N. cristatus (Ccristatus), N. plumchrus (Cplumchrus), and seawater (Csw) were made at temperatures ranging from about 1 to 10C (Table 3). All of the specimens were at C5. In 2006, the PL (mean standard deviation, SD) of N. cristatus and N. plumchrus were 7.29 0.27 and 4.01 0.16 mm, respectively; in 2007, these were 6.90 0.26 and 3.96 0.14 mm, respectively. The PL of the specimens varied between species and year (P \ 0.05). The value of Ccristatus, Cplumchrus, and Csw relatively increased with temperature. In 2006, the sound-speed contrast h of N. cristatus and N. plumchrus ranged from 1.006 to 1.011 and from 1.013 to 1.025, respectively; in 2007, these ranged from 1.014 to 1.021 and from 1.018 to 1.024, respectively. The values of sound-speed contrast h determined for the nearest known temperature where the nets were towed are highlighted in bold in Table 3. Approximating to the deformed cylinder, we digitized the shape of N. cristatus and of N. plumchrus in 2006 and 2007. The TS was estimated using DWBA model with the

values for g and h given in Tables 2 and 3. Typical examples of the relationship between variations in TS and body tilt angle at 38, 120, and 200 kHz computed with the value of g and h in 2006 are shown in Fig. 2. The variation in TS versus tilt angle showed peaks around 0 (dorsal) and 180 (abdominal) at a broadside incidence at 120 and 200 kHz. In N. cristatus, the TS as a function of tilt angle was about 12 and 19 dB at 120 and 200 kHz, respectively. In N. plumchrus, peaks also appeared at 120 and 200 kHz, with the TS being about 3 and 10 dB, respectively. The values of TS versus tilt angle at 38 kHz made a smooth curve without obvious peaks. The values of TS at 38 kHz were about -119 and -131 dB in N. cristatus and N. plumchrus, respectively. The relationship between PL and the tilt-averaged TS are shown in Fig. 3, and the equations of logarithmic regression are shown in Table 4. The PL range shown in Fig. 3 is as follows: 6.607.95 mm, N. cristatus in 2006 (Fig. 3a); 3.684.61 mm, N. plumchrus in 2006 (Fig. 3b); 6.267.69 mm, N. cristatus in 2007 (Fig. 3c); 3.54 4.26 mm, N. plumchrus in 2007 (Fig. 3d). The higher the frequency was, the higher the value of averaged TS. The averaged TS values at 38 and 120 kHz (DTS12038 = TS120 - TS38) among the N. cristatus speciments tested ranged from 14.8 to 16.3 dB; those among N. plumchrus ranged from 17.9 to 18.6 dB. The values of DTS200-120 among all of the N. cristatus and N. plumchrus specimens ranged from 2.9 to 5.5 dB and from 5.3 to 6.5 dB, respectively.

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Table 3 Sound-speed through Neocalanus cristatus, N. plumchrus, and seawater measured by the T-tube (2006) Neocalanus plumchrus V = 16% PL = 4.01 0.16 mm h Temperature (oC) 10.0 9.5 9.0 8.5 8.0 7.5 7.7 7.0 6.7 6.0 5.6 5.0 4.5 4.0 3.5 3.0 2.5 1496.0 1497.2 1495.4 1496.1 1492.0 1490.6 1491.3 1489.3 1496.3 1502.9 1502.1 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 1486.6 1449.7 1485.9 1482.5 1453.8 1451.7 1489.2 1455.8 1.023 1.022 1.021 1.025 1485.1 1457.9 1.019 1495.9 1460.0 1.025 1491.8 1462.0 1.020 1495.2 1464.1 1.021 1491.0 1466.2 1.017 1501.9 1468.3 1.023 1497.8 1470.4 1.019 1501.2 1472.5 1.019 1504.6 1474.6 1.020 1508.0 1476.7 1.021 1477.9 1475.2 1474.1 1471.4 1469.9 1467.6 1465.7 1463.8 1461.9 1460.1 1458.2 1503.8 1478.8 1.017 8.0 1502.5 1479.0 1507.2 1480.9 1.018 8.5 1504.9 1481.0 1.016 1.016 1.016 1.019 1.015 1.017 1.019 1.019 1.021 1.019 1.020 1.021 1.021 1506.4 1503.0 1485.2 1483.1 1.014 1.013 9.5 9.0 1507.0 1503.9 1484.8 1482.9 1.015 1.014 1509.8 1487.3 1.015 10.0 1511.9 1486.8 1.017 10.1 9.7 9.3 8.5 7.9 7.5 7.0 6.6 6.1 5.7 5.1 4.5 4.0 3.7 3.0 2.1 1.5 Cplumchrus (m/s) Csw (m/s) h Temperature (oC) Ccristatus (m/s) Csw (m/s) Temperature (oC) PL = 6.90 0.26 mm V = 34% V = 34% PL = 3.96 0.14 mm Cplumchrus (m/s) 1514.6 1516.6 1510.3 1510.0 1511.3 1509.4 1507.3 1504.3 1506.5 1501.3 1498.8 1500.1 1496.3 1491.0 1487.7 1484.4 1482.0 Csw (m/s) 1487.1 1485.6 1484.0 1481.0 1478.6 1477.1 1475.2 1473.7 1471.8 1470.2 1468.0 1465.7 1463.8 1462.7 1460.1 1456.7 1454.5 h (2007) Neocalanus cristatus (2007) Neocalanus plumchrus

(2006) Neocalanus cristatus

Fish Sci (2009) 75:13771387

V = 35%

PL = 7.29 0.27 mm

Temperature (oC) 1.006 1.008 1.008 1.008 1.008 1.008 1.007 1.009 1.009 1.009 1.009 1.009 1.009 1.011 1.011 1.010 1.010 1.010

Ccristatus (m/s)

Csw (m/s)

10.0

1496.4

1487.3

1.018 1.021 1.018 1.020 1.022 1.022 1.022 1.021 1.024 1.021 1.021 1.023 1.022 1.019 1.019 1.019 1.019

9.5 9.0

1497.3 1494.9

1485.2 1483.1

8.5

1492.6

1480.9

8.0

1490.2

1478.8

7.5

1487.8

1476.7

7.0

1485.4

1474.6

6.5

1486.4

1472.5

6.0

1484.1

1470.4

5.5

1481.7

1468.3

5.0

1479.4

1466.2

4.5

1477.0

1464.1

4.0

1474.7

1462.0

3.5

1475.6

1460.0

3.0

1473.3

1457.9

2.5

1471.0

1455.8

2.0 1.5

1468.7 1466.4

1453.8 1451.7

Canimal, speed of sound through the body of the specic copepod; V, copepods volume; h, sound-speed contrasts of seawater

The values of h determined for the temperature nearest where the nets were towed are highlighted in bold

1381

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-90 -100 -110 -120 -130 -180 -120 -60 0 60 120 180
38kHz 120kHz 200kHz

Fish Sci (2009) 75:13771387

-100 -110 -120 -130 -140 -180 -120 -60 0 60 120 180
38kHz 120kHz 200kHz

Target strength (dB)

(PL: 7.38 mm)

Target strength (dB)

(PL: 4.09 mm)

Tilt angle (deg)

Tilt angle (deg)

Fig. 2 Typical variations in the target strength (TS) of N. cristatus (left) and N. plumchrus (right) as a function of tilt angle, estimated by the distorted wave born approximation (DWBA) model at 38 (dotted Fig. 3 Correlation between prosome length and averaged TS estimated by the DWBA model of N. cristatus and N. plumchrus in 2006 and 2007. The TS and logarithmic regression are indicated as follows; triangle and solid line, 38 kHz, square and broken line 120 kHz, diamond and dotted line, 200 kHz. Equations of the regression line are given in Table 4. a N. cristatus in 2006, b N. plumchrus in 2006, c N. cristatus in 2007, d N. plumchrus in 2007

line), 120 (dashed line), and 200 kHz (solid line). The positive value of the tilt angle denotes head up and the negative value denotes head down

-95 -100

(a)
Target strength (dB)

-100 -105 -110 -115 -120 -125 -130 -135 -140 6.5 7.0 7.5 8.0 -145 3.3

(b)

Target strength (dB)

-105 -110 -115 -120 -125 -130 6.0

38 kHz 120 kHz 200 kHz

38 kHz 120 kHz 200 kHz

3.6

3.9

4.2

4.5

4.8

Prosome length (mm)

-95 -100

Prosome length (mm)

-100 -105

(c)
Target strength (dB)

(d)

Target strength (dB)

-110 -115 -120 -125 -130 -135 -140

38 kHz 120 kHz 200 kHz

-105 -110 -115 -120 -125 -130 6.0 6.5 7.0 7.5 8.0
38 kHz 120 kHz 200 kHz

-145 3.3

3.6

3.9

4.2

4.5

4.8

Prosome length (mm)

Prosome length (mm)

Discussion The focus of this study was to estimate the TS of N. cristatus and N. plumchrus. The value of TS was affected by changes in the value of g, h, and the tilt angle of the body at an invariable frequency. In particular, we have discussed the inuence of g and h on TS. Averaged TS at 200 kHz with changing g was estimated by h (1.0001.060 by steps of 0.010) and is shown in Fig. 4. The same specimens as in Fig. 2 were used. For each h, the averaged TS increased when the value of g increased, and for each g, averaged TS increased when the value of h increased. Areas in Fig. 4

enclosed with a dotted gray line correspond to the range of averaged TS estimated by the whole value range of g and h measured in this study. Sound scattering occurred at the boundary between different elements of the acoustic impedance, but it did not occur when the product of g and h was one, because there was no difference of acoustic impedance between specimens and seawater. Therefore, the values of TS around the enclosed area varied dynamically with changes in g and/or h. The value of the averaged TS indicates that these areas differed by a maximum of about 20 dB in N. cristatus and about 12 dB in N. plumchrus, even if the same digitized shape was used. The TS

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Fish Sci (2009) 75:13771387 Table 4 Logarithmic regression of prosome length and target strength of N. cristatus and N. plumchrus given in Fig. 3
Targeted specimen n 38 kHz Approximate expression 79 90 146 136 TS = 50.5 log(PL) - 164.3 TS = 49.0 log(PL) - 162.9 TS = 39.8 log(PL) - 157.3 TS = 47.7 log(PL) - 163.3 R
2

1383

120 kHz SE Approximate expression TS = 35.2 log(PL) - 135.8 TS = 41.8 log(PL) - 140.2 TS = 26.2 log(PL) - 130.0 TS = 40.9 log(PL) - 140.9 R
2

200 kHz SE Approximate expression TS = 28.8 log(PL) - 126.3 TS = 32.4 log(PL) - 128.5 TS = 24.4 log(PL) - 123.9 TS = 32.7 log(PL) - 129.8 R2 SE

2006

Neocalanus cristatus Neocalanus plumchrus

0.38 0.38 0.44 0.43

1.07 1.09 0.70 0.82

0.25 0.30 0.25 0.35

1.00 1.15 0.72 0.84

0.36 0.22 0.38 0.25

0.63 1.06 0.49 0.84

2007

Neocalanus cristatus Neocalanus plumchrus

TS Target strength

-80 -85 -90 6 4 -95 5 7 -100 1 2 -105 -110 -115 -120 0.990 1.010

3 8 9

1.030

1.050

-90 -95 -100 6 -105 7 1 -110 -115 -120 -125 -130 0.990

Averaged TS (dB)

Averaged TS (dB)

3 8 4 5 2 9

1.010

1.030

1.050

Density contrast g
Fig. 4 Relationship between density contrast g and averaged TS at 200 kHz estimated in the same specimens as in Fig. 2 (left N. cristatus, right N. plumchrus). The values of sound-speed contrast h are given from 1.000 to 1.060 in steps of 0.010. Areas enclosed with a dotted gray line are the range of averaged TS estimated by g and h measured in this study. The lled circles, numbered 1 and 2 in both gures correspond to TS estimated by the values of g and h obtained in 2006 and 2007, respectively. Open circles are the referenced values

Density contrast g
of other zooplanktons: 3 Euphausia pacica caught in June (g = 1.046, h = 1.043) [15], 4, 5 Calanus nmarchicus caught in winter (g = 1.000, h = 1.029) and in May (g = 0.999, h = 1.021) [16], respectively, 6, 7 Calanus hyperboreus caught in winter (g = 0.999, h = 1.029) and in May (g = 0.997, h = 1.021) [16], respectively, 8, 9 both N. plumchrus caught in summer (g = 1.047, h = 1.012 and g = 1.047, h = 1.006) [17]

that differed by 20 dB caused an overestimation (more than 102 times) or an underestimation (less than 10-2 times) in terms of the translation from the acoustic backscattering strength to abundance. Measuring the local g and h and estimating the practical TS with the theoretical model are important for uid-like zooplankton. However, prior to this study, the g and h values of zooplankton had not been described in the Oyashio region, only those for E. pacica [14, 15]. If the averaged TS of Neocalanus copepods were to be estimated using the g and h values for E. pacica instead of their own value, the former (open circles, numbered 3) would be about 15 dB higher than the latter (lled circles, numbered 1 and 2) in Fig. 4. Estimations of TS using g and h may be used to distinguished E. pacica values from those of Neocalanus copepods even if they inhabit the same Oyashio region [27]. TS estimated using g and h values for C. nmarchicus (open circles, numbered 4 and 5) and C. hyperboreus (open circles, numbered 6 and 7) [16] were 16 dB higher than those of this study. One explanation for this difference may be that different genera have different values of g and h, even if all specimens are

live copepod. The TS estimated using g and h values for N. plumchrus (open circles, numbered 8 and 9) preserved in formalin have been reported by Greenlaw and Johnson [17] and are about 1012 dB higher than those in this study. The body-mass density value differs between live and preserved specimens because the composition and condition of the body changes with emersion in formalin [12, 14, 19]. Live specimens should be used for measurements. Estimating the theoretical TS therefore requires adequate values of g and h derived from live specimens. Although the swimming angles of Neocalanus copepods were assumed randomly, the differences in TS as a function of tilt angle at 120 and 200 kHz (Fig. 2) were not negligible. Thus, the validity of the hypothesis should be discussed. The values of g were almost one (Table 2); in other words, the Neocalanus copepods were neutrally buoyant in the layer where they were distributed. Therefore, they need not swim upward or downward to maintain their position against negative or positive buoyancy. In contrast, E. pacica swim upward with a mean of 30.4 and a standard deviation of 19.9 [28] because their body-mass density is

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Fish Sci (2009) 75:13771387 Table 5 Parameters for calculating detectable depth using the model EK60 echosounder (Simrad, Horten, Norway) Frequency, f (kHz) Electro-acoustic efciency, g Maximum electrical power, W (W) Radius of transducer, at (m) Band width, Df (Hz) Absorption coefcient, a (dB/m) Individual TS of Neocalanus cristatus (dB) Individual TS of Neocalanus plumchrus (dB) 38 0.7 2000 0.212 2425 0.010 -119.5 -133.1 120 0.75 1000 0.076 3026 0.028 -104.2 -114.8 200 0.75 1000 0.050 3088 0.041 -100.2 -108.7

higher than that of seawater [15]. Neocalanus copepods are known to carry out little or no dial vertical migration during growth in the surface layer from copepodite stage 1 to stage 5 [4, 5]. As they are plankton, they do not move like sh, so they would not exhibit a constant distribution of swimming angle. Hence, we assumed the swimming angle of Neocalanus copepods to be distributed randomly. However, we did apply three sets of swimming angle distribution (mean SD), i.e., -5 10 (head down, like sh [29]), 0 10 (horizontal position), and 30.4 19.9 (head up, like E. pacica [28]), to N. cristatus, whose difference of TS as a function of tilt angle at 200 kHz was the largest (Fig. 2). The values of averaged TS obtained using these three sets of swimming angles were -94.5 (-5 10), -94.3 (0 10), and -98.8 dB (30.4 19.9) although the averaged TS (random) was -100.1 dB. Consequently, the swimming angle distribution did have an effect on our estimation of TS, thereby emphasizing the need for direct observations to obtain information on the swimming angle in future work. N. cristatus and N. plumchrus are known to migrate from layers near the sea surface to the bathypelagic zone [4, 5] where they are undetectable by echosounders at high frequencies (C120 kHz. Although the acoustic survey of zooplankton required high frequencies, the higher the frequency, the larger the absorption loss of echo intensity, and the detectable distance decreases. Thus, the relationship between the detectable range derived from the signal-tonoise ratio (SNR) [30] and minimum number of N. cristatus and of N. plumchrus per cubic meter need to be discussed. The SNR was expressed by Furusawa et al. [30] as h i 4p3 qsw gWa4 f 5:8 exp 2pat f =Csw 2 h2 100:2ar TS t SN 2 3 Csw r 4 NP0 Df 5 where SN is the linear variable of SNR [i.e., SNR = 10 log(SN)], qsw is the mass density (kg/m3 seawater only in Eq. 5), g is electro-acoustic efciency, W is the maximum electrical power, at is the radius of transducer, f is the frequency, h is the the angle measured from the beam axis, a is the absorption coefcient, r is the detectable depth that corresponds to distance between the transducer and target species, TS is the product of the number of target species and the linear scale of the averaged TS, NP0 is the noise spectrum extrapolated to 1 Hz, and Df is the bandwidth of the transducer. When the beam axis where the SNR is the largest was restricted, h was substituted as zero. The averaged TS in 2006 shown in Fig. 4 were used. NP0 is an eigenvalue which was assumed to be 145 dB [31]. The parameters used for computing the detectable depth with the echosounder, which was a Simrad

model EK60 (Simrad, Horten, Norway) on the R/V Kaiyou Maru No.7, are listed in Table 5. R/V Kaikou Maru, which was used for the survey in 2007, carried an EK500 echosounder (Simrad) that was not equipped 200-kHz splitbeam transducer. Therefore, we used the EK60 apparatus for estimating the detectable depth (Fig. 5). The detectable depth increased with increasing frequency. The solid gray vertical line numbered 1 in Fig. 5 is the number of N. cristatus in C5 corresponding to the 2 ind./m3 reported by Kobari et al. [32]. In this case, the detectable depth was spread to 10 m at 38 kHz, 34 m at 120 kHz, and 52 m at 200 kHz. For Nagasawa et al. [33] (solid gray vertical line, numbered 2), the number of N. cristatus in C5 was 11 ind./ m3, and the detectable depth was 15, 50, and 73 m; for Barraclough et al. [34] (gray solid vertical line, numbered 3), the number of N. cristatus in C5 was 93 ind./m3, and the detectable depth was 26, 79, and 106 m; for Kobari et al. [32] (solid gray vertical line, numbered 4), the number of N. plumchrus in C5 is 50 ind./m3, and the detectable depth was 10, 41, and 67 m; for Kawamura and Hirano [35] (solid gray vertical line, numbered 5), the number of N. plumchrus in C5 was 745 ind./m3, and the detectable depth was 20, 7, and 108 m, respectively. N. cristatus and N. plumchrus are distributed patchily on a horizontal plane near the sea surface during the foraging period. Areas inside and outside of these patches show remarkable differences in abundance [34, 35]. If we assume that the size of patches was smaller than the resolution of the net tow, the number of copepods per cubic meter obtained from the net tow was smaller than that in the patches because the number of copepods caught with net was divided by the volume ltered by net towing, regardless of the presence of the patches. This may be one of the reasons why the values reported by Nagasawa et al. [33] and Kobari et al. [32] are smaller than those reported by Barraclough et al. [34] and Kawamura and Hirano [33]. The former estimated the abundance of copepods by vertical net tow, while the latter used a LonghurstHardy plankton recorder [6] or horizontal towing of the net.

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Fish Sci (2009) 75:13771387

Number of Neocalanus cristatus (ind./m3)
0 1 10 102 103 104 105

1385
Number of Neocalanus plumchrus (ind./m3)
0 1 10 102 103 104 105

Detectable depth (m)

Detectable depth (m)

38 kHz

30 60 90 120 150
1 2 3

120 kHz 200 kHz

30 60 90 120 150
38 kHz 120 kHz 200 kHz

Fig. 5 Relationship between detectable depth and number of copepods per cubic meter when the sound-to-noise ratio (SNR) = 10 dB in N. cristatus (left) and N. plumchrus (right). The black lines (dotted line 38 kHz, dashed line 120 kHz, solid line 200 kHz) correspond to the minimum number of copepods that can be detected by the EK60
Number of Neocalanus cristatus (ind./m3)
0 1 10 10
2

echosounder at each depth. The solid gray vertical lines numbered 15 are the number of copepods (ind./m3) referenced as: 1 2 ind./m3 [32], 2 11 ind./m3 [33], 3 93 ind./m3 [34], 4 50 ind./m3 [32], 5 745 ind./m3 [35]

Number of Neocalanus plumchrus (ind./m3)

0 1 10 102 103 104 105

10

10

10

Detectable depth (m)

30 60 90 120 150

Detectable depth (m)

Minimum TS Typical TS Maximum TS

Minimum TS

30 60 90 120 150

Typical TS Maximum TS

Fig. 6 Relationship between detectable depth and number of copepods per cubic meter when the SNR = 10 dB in N. cristatus (left) and N. plumchrus (right) at 200 kHz. The solid black lines are typical TS corresponding to the detectable depth at 200 kHz shown in Fig. 5, long-dashed lines and dash-dotted lines correspond to the detectable

depth calculated with the maximum and the minimum TS within the area enclosed area by the gray dotted line in Fig. 4. Solid gray vertical lines are the number of copepods per cubic meter, assumed to be 93 ind./m3 in N. cristatus and 745 ind./m3 in N. plumchrus

However, there were fewer N. cristatus and N. plumchrus per cubic meter obtained by the horizontal sampling mentioned above than other copepods. For example, Kawamura [36] reported from 3.3 9 102 to 2.4 9 104 ind./m3 for N. tonsus, and Wiborg [37] reported 4 9 107 ind./m3 for C. nmarchicus. Although the number of N. cristatus and N. plumchrus per cubic meter in their patch might be more than that reported by Barraclough et al. [34] and Kawamura and Hirano [35], we took them to be 93 and 745 ind./m3, respectively. The inuence of TS on detectable depth also needs to be discussed. Figure 6 shows the difference between detectable depth calculated by the maximum and the minimum TS at 200 kHz where area enclosed by the dotted gray line in Fig. 4. In N. cristatus, there was a 83-m difference between the detectable depth calculated from the maximum TS, which indicated 131 m, and the minimum TS, which indicated 47 m. In N. plumchrus, there was 56-m difference between the maximum TS, which indicated 123 m, and the minimum TS, which indicated 67 m. The difference between detectable depths could not be neglected in

the calculations using typical TS and minimum TS or maximum TS. Therefore, an acoustical survey is needed to determine the adequate TS of the target species inhabiting the survey area. As a result, the EK60 echosounder was able to detect the patches of N. cristatus and of N. plumchrus within 79 and 73 m at 120 kHz, and between 106 and 108 m at 200 kHz. N. cristatus and N. plumchrus are distributed near the sea surface during the foraging period and during our study, if their patches were distributed at depths of less than 106 m, they could be detected at 200 kHz with an echosounder attached to the bottom of the ship. Moreover, N. cristatus and N. plumchrus can be identied using the two-frequency method [38] within 73 m from the transducer, except in an acoustical dead zone near the sea surface. However, the detectable depth varies according to the TS of target species, components in the patches (e.g., other copepodite stages except C5, other zooplankton), noise level, and marine environments. The value of g and h inuenced the TS of copepods remarkably, much as it does other uid-like zooplankton.

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Fish Sci (2009) 75:13771387 14. Mikami H, Mukai T, Iida K (2000) Measurements of density and sound speed contrasts for estimating krill target strength using theoretical models. Nippon Suisan Gakkaishi 66:682689 (in Japanese with English abstract) 15. Matsukura R, Mukai T, Ando Y, Iida K (2009) The variation of density and sound speed contrasts and theoretical target strength estimation of Euphausia pacica. Nippon Suisan Gakkaishi 75:3844 (in Japanese with English abstract) 16. Kgeler JW, Falk-Petersen S, Kristensen A, Pettersen F, Dalen D (1987) Density- and sound speed contrasts in sub-Arctic zooplankton. Polar Biol 7:231235 17. Greenlaw CF, Johnson RK (1982) Physical and acoustical properties of zooplankton. J Acoust Soc Am 72:17061710 18. Knutsen T, Melle W, Calise L (2001) Determining the mass density of marine copepods and their eggs with a critical focus on some of the previously used methods. J Plankton Res 23: 859873 19. Richter KE (1985) Acoustic scattering at 1.2 MHz from individual zooplankters and copepod populations. Deep Sea Res 32:149161 20. Itaya K, Fujimori Y, Shimizu S, Komatsu T, Miura T (2007) Effect of towing speed and net mouth size on catch efciency in framed midwater trawls. Fish Sci 73:10071016 21. Yasuma H, Takao Y, Sawada K, Miyashita K, Aoki I (2006) Target strength of the lanternsh, Stenobrachius leucopsarus (family Myctophidae), a sh without an airbladder, measured in the Bering Sea. ICES J Mar Sci 63:683692 22. Mellor GL (1996) Introduction to physical oceanography. American Institute of Physics, New York 23. Foote KG (1990) Speed of sound in Euphausia superba. J Acoust Soc Am 87:14051408 24. Chu D, Foote KG, Stanton TK (1993) Further analysis of target strength measurements of Antarctic krill at 38 and 120 kHz: Comparison with deformed cylinder model and inference of orientation distribution. J Acoust Soc Am 93:29852988 25. Stanton TK, Chu D, Wiebe PH, Clay CS (1993) Average echoes from randomly oriented random-length nite cylinders: Zooplankton models. J Acoust Soc Am 94:34633472 26. Chihara M, Murano M (1977) An illustrated guide to marine plankton in Japan. Tokai University press, Tokyo (in Japanese) 27. Yamamura O, Honda S, Shida O, Hamatsu T (2002) Diets of walleye pollock Theragra chalcogramma in the Doto area, northern Japan: ontogenetic and seasonal variations. Mar Ecol Prog Ser 238:187198 28. Miyashita K, Aoki I, Inagaki T (1996) Swimming behaviour and target strength of isada krill (Euphausia pacica). ICES J Mar Sci 53:303308 29. Miyanohana Y, Ishii K, Furusawa M (1990) Measurements and analysis of dorsal-aspect target strength of six species of sh at four frequencies. Rapp Pv Reun Cons Int Explor Mer 189: 317324 30. Furusawa M, Asami T, Hamada E (1999) Detection range of echo sounders. In: The 3rd JSPS International Seminar Sustainable Fishing Technology in Asia towards the 21st Century, pp 207213 31. Nishimura M (1969) Study on the optimum frequency of echo sounders (in Japanese). PhD thesis. Tohoku University, Miyagi 32. Kobari T, Sternberg DK, Ueda A, Tsuda A, Silver MW, Kitamura M (2008) Impacts of ontogenetically migrating copepods on downward carbon ux in the western subarctic Pacic Ocean. Deep Sea Res II 55:16481660 33. Nagasawa K, Ohtsuka S, Saeki S, Ohtani S, Zhu G, Shiomoto A (2001) Abundance and in situ feeding habitats of Neocalanus cristatus (Copepoda: Calanoida) in the central and Western North Pacic Ocean in summer and winter. Bull Natl Res Inst Far Seas Fish 38:3752

The local values of g and h should be measured for theoretical TS estimation, which is the scale factor of acoustic surveys, because the values of g and h do vary according to species and life stage. In addition, in evaluating the abundance of weak sound-scattering organisms, such as copepods, we should consider the detectable depth derived from the SNR of the echosounder. In this study, the detectable depth was restricted to the beam axis. Knowledge of the number of copepods that can be detected in the entire acoustical beam of an echosounder will be required in future studies.
Acknowledgments We thank the captains and crew of R/V Kaiyou Maru No.7 and R/V Kaikou Maru for their cooperation in collecting specimens. This study was supported in part by the Fisheries Agency of Japan under the project Research on the Assessment of Fisheries Stocks in the Waters around Japan. JARPNII survey was supported by Fisheries Agency of Japan, Fisheries Research Agency of Japan, and the Institute of Cetacean Research. We thank to these institutions for their support.

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