Cowen et at: Evaluation of the In Situ Ichthyoplankton Imaging System and comparison with the bongo-net sampler 
3 
mesozooplankton), which enables the concentration of 
even relatively rare mesoplankters, such as larval fish- 
es and gelatinous zooplankton, to be quantified (Cowen 
and Guigand, 2008; McClatchie et ah, 2012). Using 
the image analysis software that we have developed 
(Tsechpenakis et al., 2007, 2008), we could essential- 
ly quantify the plankton field for every centimeter of 
our tow, and we could match these data centimeter by 
centimeter with the corresponding environmental data 
collected by the onboard sensors (pressure [depth], 
temperature, salinity, and fluorometry). Consequently, 
ISIIS can evaluate from very fine-scale (centimeters) 
to submesoscale features. ISIIS sensors for this study 
were those for temperature (SBE 3 1 Sea-Bird Electron- 
ics, Inc., Bellevue, WA) and conductivity (SBE 4) and 
a fluorometer (ECO FLRT, WET Labs, Philomath, OR). 
A 61-cm bongo sampler was used and fitted with 
505- and 333-pm mesh nets (Posgay and Marak, 1980). 
A flowmeter (General Oceanics, Miami, FL) was at- 
tached in the center of each mouth opening to quantify 
the volume of water filtered by the net. A conductivity, 
temperature, depth (CTD) instrument (SeaCAT SBE 
19) was attached to the tow wire above the bongo net. 
The CTD was used in real time to monitor the depth of 
the bongo net during deployment. 
Sampling approach 
For this study, ISIIS was towed at a speed of 2.5 m s -1 
in a tow-yo (vertically undulating) fashion between the 
surface and a target depth of 10 m above the seafloor, 
thereby following changes in seafloor depth. The ISIIS 
was towed in an undulating manner by paying cable in 
and out from the winch, and therefore continual winch 
operation was required. (Since this study, a self-undu- 
lating version of ISIIS has been designed and the need 
for continual winch operation has been eliminated). 
Each undulation (surface to depth to surface) took ~10 
min, resulting in a distance covered of 1.5 km, which 
also equates to the distance between downcasts (or up- 
casts). While being towed, ISIIS records environmental 
data (temperature, salinity, fluorescence) and imagery 
continually, sending the data up the fiber-optic cable 
for onboard recording. The continual imagery is parsed 
into single images of 13x13 cm at a rate of 17.3 images 
s -1 . Thus, ISIIS generates -64,000 images h 1 , and for 
this study, an estimated total of -478,000 images over 
-7.68 h of total recording time. 
Because the focus of this study was specifically lar- 
val fishes, processing of images specifically targeted lar- 
val fishes, thereby eliminating the need to capture and 
classify all imaged particles (e.g., copepods, larvaceans, 
medusae, and cfenophores). Consequently, all images 
were manually reviewed for larval fishes. This process 
is relatively rapid, although -3 months were required 
1 Mention of trade names or commercial companies is for 
identification purposes only and does not imply endorsement 
by the National Marine Fisheries Service, NOAA. 
to complete this task because of the large number of 
images. Future development of ISIIS will include auto- 
mated image processing; however, the current manual 
processing requires viewing each image. When a lar- 
val fish was present, that portion of the image was ex- 
tracted and saved to a file. All fish images were then 
reviewed for identification to the lowest taxonomic 
level possible and measured with ImageJ (National 
Institute of Health public domain Java-based image- 
analysis program available at http://rsbweb.nih.gov/ 
ij/). Environmental data from ISIIS were interpolated 
across each transect with a cubic interpolation function 
in Matlab (vers. 7.11.0.584 [R2010b], The MathWorks, 
Inc., Natick, MA). The depth and environmental vari- 
ables associated with each fish larva were obtained by 
matching time stamps from image and environmental 
data. 
The bongo tows were conducted in standard fashion 
by following Jossi and Marak (1983). For each tow, the 
wire was paid-out at a rate of 50 m min 1 to a depth of 
10 m above the seafloor, then the wire was retrieved 
to the surface obliquely at 20 m min -1 , while the ship 
moved at 0.75-1.0 m s -1 . At completion of each tow, 
the nets were washed down and the contents rinsed 
onto a 333-pm sieve. The sample was preserved in 5% 
buffered formalin. Samples were then sorted for larval 
fishes under a dissecting microscope and identified to 
the lowest taxonomic level following Fahay (2007). The 
333-pm mesh bongo samples were used for compari- 
sons of the bongo and ISIIS methods since this mesh 
size is the one that has been used for more than 20 
years by the Northeast Fisheries Science Center for 
ichthyoplankton surveys. 
To compare larval fish concentrations, each bongo 
tow and each ISIIS undulation were treated as rep- 
licates. There are potential statistical problems with 
this assumption, but to date, the decorrelation length 
scale in ichthyoplankton distributions in the study re- 
gion has not been calculated. This assumption will be 
examined in future studies with ISIIS. The larval fish 
concentrations were transformed by the natural log, 
and a Shapiro test was performed to test for normal- 
ity of larval fish concentrations within each gear type. 
Where the null hypothesis of normality was accepted, 
a Welch’s f-test was used to compare larval fish con- 
centrations between transects within gear and then 
between gear across both transects. Comparisons were 
made for total larvae, family-level larvae, and species- 
level larvae both within and between gears for abun- 
dance and size differences. In these tests, the nonpara- 
metric Kruskal-Wallis test was used because concen- 
trations at the family level were zero-inflated, making 
transformations to a normal distribution impossible. 
All counts per tow (or undulation) were standardized 
to volume sampled (number of fish larvae per cubic 
meter). 
All larvae collected in the bongo net were measured 
to the nearest 0.1 mm for notochord (preflexion) or 
standard length under a dissecting microscope with 
