The ZooCAM, a new in-flow imaging system for fast onboard counting, sizing and classification of fish eggs and metazooplankton

Highlights

• The ZooCAM is an in-flow system for on-board imaging of fish eggs and zooplankton.

• It enabled the analysis ~10,000 samples on-board in 4 years, without any failure.

• It provides staged fish eggs counts equivalent to those done with microscopes.

• It provides results comparable to the ZooScan on complex planktonic assemblages.

• The ZooCAM helps improve the spatio-temporal resolution of zooplanktonic studies.

Abstract

In this paper we present the ZooCAM, a system designed to digitize and analyse on board large volume samples of preserved and living metazooplankton (i.e. multicellular zooplankton) and fish eggs >300 µm ESD. The ZooCAM has been specifically designed to overcome the difficulties to analyse zooplankton and fish eggs in the framework of the PELGAS survey, and provide high frequency data. The ZooCAM fish eggs counts were comparable to those done with a dissecting microscope. The ZooCAM enabled the accurate prediction and fast on board validation of staged anchovy and sardine eggs in almost real time after collection. A comparison with the ZooScan, on a more complex zooplanktonic community, provided encouraging results on the agreement between the 2 instruments. ZooCAM and ZooScan enabled the identification of similar communities and produced similar total zooplankton abundances, size distributions, and size spectra slopes, when tested on the same samples. However these results need to be further refined due to the small number of samples used to compare the two instruments. The main ZooCAM drawback resides in a slight but sensible underestimation of abundances and sizes, and therefore individual and community biovolumes. The ZooCAM have been successfully deployed over 4 years, on numerous surveys without suffering any major failure. When used in line with the CUFES it provided high resolution maps of staged fish eggs and zooplanktonic functional groups. Hence the ZooCAM is an appropriate tool for the development of on board, high frequency, high spatial coverage zooplanktonic and ecosystemic studies.

Introduction

In the last decade, the European Commission encouraged member states to monitor marine ecosystems (Marine Strategy Framework Directive, MSFD: 2008/56/EC) to achieve and maintain Good Environmental Status (GES). MSFD promotes ecosystemic monitoring programs that would serve as a basis for the development of ecosystemic sustainable management. Ecosystemic monitoring implies (i) the capacity to monitor the whole spatial range of the ecosystem at appropriate frequency and spatial resolution to capture relevant ecological processes, and (ii) observe all the compartments of the ecosystem simultaneously, at the relevant scale for each compartment. The PELGAS survey has been developed since 2000 in the Bay of Biscay. The survey now provides spatialized data on numerous pelagic compartments of the Bay of Biscay ecosystem (hydrology, phytoplankton, mesozooplankton, fish and megafauna), informing fish stock and ecosystem-based management, and supporting ecosystem science (Doray et al., 2017).

Monitoring physical and biogeochemical parameters at high frequency and spatial resolution is now possible by the use of autonomous platforms such as gliders (Schofield et al., 2007, Queste et al., 2016) or autonomous profilers (de Fommervault et al., 2015). Conversely, biological variables, i.e. plankton or fish dynamics, remain hard to capture at high spatio-temporal resolution. Monitoring the biota often requires the deployment of ship based sampling devices, i.e. oceanographic bottles, plankton nets and pelagic and bottom trawls, and subsequent time consuming analyses by human examination of samples. Nevertheless, in-situ, semi-automated, high frequency phytoplankton long term observation have been achieved (Thyssen et al., 2014, Campbell et al., 2013) using imaging in-flow instruments (i.e. Olson and Sosik, 2007) in coastal sites. High spatio-temporal frequency observation of phytoplanktonic community proxies is now possible through the use of remote sensing products coupled with discrete in situ pigment measurement (Uitz et al., 2006, McClain, 2009). In a similar manner, direct and indirect observation of fishes are now routinely performed in the framework of stock assessment surveys and ecosystemic surveys such as PELGAS (Doray et al., 2017, Doray et al., 2014) by the use of acoustic techniques and midwater trawls hauls, at the regional scale. Data on birds (Certain and Bretagnolle, 2008) and top predators, essentially marine mammals, are now available thanks to dedicated monitoring programs, at the relevant scales (Mannocci et al., 2015). Despite recent efforts, the zooplankton remain a biological compartment that is still hard to analyse at the relevant spatial scales and frequencies, at a reasonable biological/taxonomical resolution, in the framework of marine ecosystemic, empirical, observation based, or modelling based, studies (Mitra et al., 2014). The Continuous plankton Recorder (CPR) program which exists since the 1930’s is an example of what can be done to monitor zooplankton efficiently (Richardson et al., 2004). Nonetheless, zooplankton analysis still represent a major bottleneck in the design of truly ecosystemic studies of marine systems.

In the past decade, imaging has become an operational solution to acquire meso- and macro-zooplankton data at fine spatial scale and/or high frequency (e.g. Romagnan et al., 2015) by the development of numerous dedicated instruments (Davis et al., 2005, Benfield et al., 2007, Gorsky et al., 2010, Picheral et al., 2010, Cowen and Guigand, 2008), and dedicated analytical methods (Faillettaz et al., 2016, Hirata et al., 2016, González et al., 2016). Compared to other methods to analyse zooplankton such as microscopic examination or optical methods (e.g. Optical Plankton Counter, OPC & Laser-OPC) imaging enable a good but coarse taxonomic identification coupled with the precise size measurement of numerous objects in a short time. Yet, sampling and analysing zooplankton with imaging remain challenging because the sampled volume (imaged volume) and image resolution do not always enable quantitative estimates of key zooplankton variables (see Romagnan et al., 2016 for a review addressing the quantitative analysis of zooplankton using imaging techniques). The combination of net sampling with bench-top imaging is an efficient approach because it enables the quantitative sampling (up to tens of cubic meters) and an accurate control of the image generation and resolution (Gorsky et al., 2010) compared to in-situ imaging. But these advantages are minored by the coarse spatial resolution achieved by net sampling.

The ZooScan is one of the main commercially available bench-top imaging instrument dedicated to the analysis of zooplankton (Gorsky et al., 2010). It enables the analysis of ∼3000 objects, corresponding to 2–3 samples per day (Romagnan et al., 2016). However, it has to be stable during the scanning process and therefore cannot be used onboard a research vessel. Other scanners or photographic camera have been tested (e.g. Bachiller et al., 2012) but suffer the same drawback. The FlowCAM which takes microscope images of particles in a water flow (Sieracki et al., 1998) is an alternative to the ZooScan. It is routinely used for phytoplankton analysis and can be used on board (Jenkins et al., 2016, Zarauz et al., 2009). It has also been used to analyze metazooplankton on a limited size range (<1 mm) that do not encompass the whole metazooplanktonic community (Le Bourg et al., 2015). Yet, FlowCAM analyses duration may become excessively long for concentrated samples as they require subsampling and large dilution to limit overlapping organisms and the maximum FlowCAM flow rate does not exceed 20 mL/min (Fluid Imaging documentation).

In this paper we will present the ZooCAM, a system designed to image and analyze on board large volume samples of living metazooplanktonic organisms and fish eggs larger than 300 µm ESD. The ZooCAM has been designed to overcome the difficulties to analyse zooplankton and fish eggs in the framework of the PELGAS survey. PELGAS provides data for DEPM (Daily Egg Production Method, Lasker, 1985, Stratoudakis et al., 2006, Bernal et al., 2011). DEPM is based on age-staged fish eggs data. One essential objective for the ZooCAM development was the fast counting and accurate identification of staged fish eggs. The development specifications were then: on board use and time effectiveness in analysing large volume of water to enable high frequency quantitative analysis of zooplankton and fish eggs, and coupling to an efficient classification software, for a complete analysis and sorting of fish eggs at the end of the survey. We will present the hardware, the software, and how the ZooCAM can provide data on fish eggs and zooplankton sampled during PELGAS surveys. The ZooScan is a good reference when considering benchtop imaging of zooplankton. A comparison of ZooCAM and ZooScan would therefore be an adequate benchmark to estimate the ZooCAM efficiency to provide sufficiently qualified data from in-flow imaged zooplankton samples. We will compare the results obtained with the ZooCAM and the ZooScan on key ecological variables i.e. individual and community abundances distributions, size distributions and biovolumes distributions, community size spectra slopes, and community functional groups composition.