Claus-Peter Stelzer. Automated system for sampling, counting, and biological analysis of rotifer populations.Limnol. Oceanogr.: Methods 7, 2009, 856–864

Zooplankton organisms with short generation times, such as rotifers, are ideal models to study general ecological and evolutionary questions on the population level, because meaningful experiments can often be completed within a couple of weeks. Yet biological analysis of such populations is often extremely time consuming, owing to abundance estimation by counting, measuring body size, or determining the investment into sexual versus asexual reproduction. An automated system for sampling and analyzing experimental rotifer populations is described. It relies on image analysis of digital photographs taken from subsamples of the culture. The system works completely autonomously for up to several weeks and can sample up to 12 cultures at time intervals down to a few hours. It allows quantitative analysis of female population density at a precision equivalent to that of conventional methods (i.e., manual counts of samples fixed in Lugol solution), and it can also recognize males, which allows detecting temporal variation of sexual reproduction in such cultures. Another parameter that can be automatically measured with the image analysis system is female body size. This feature may be useful for studies of population productivity and/or in competition experiments with clones of different body size. In this article, I describe the basic setup of the system and tests on the efficiency of data collection, and show some example data sets on the population dynamics of different strains of the rotifer Brachionus calyciflorus. *Corresponding author: E-mail: [email protected] Acknowledgments I thank Johanna Schmidt and Anneliese Wiedlroither for technical assistance in the maintenance of chemostats and in microscopic analyses. Hannes Höllerer crafted several of the custom-made parts of the image analysis system, most notably the optical chamber. Kurt Mayerhofer provided advice on electrical issues. Financial support was provided by FWF grant P20735-B17. Limnol. Oceanogr.: Methods 7, 2009, 856–864 © 2009, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS Stelzer Automated analysis of rotifer populations 857 culture sampling has to be done manually, which is time consuming and presents a continuing risk of contamination; (2) operation of an EPC may be faster than “manual counting,” but it is still time consuming, e.g., loading the samples, reading data, cleaning, and routine maintenance of the counting device; (3) the sample has to be diluted in a special electrolyte buffer, which lowers the detection limit if only few rotifers are present; (4) the data output of EPCs usually cannot be directly transferred to other computer programs (e.g., Excel); and (5) EPCs are expensive to buy and maintain. Recent technological advances in digital imaging sensors have opened alternatives for automatic analysis of rotifer populations. Briefly, in digital image analysis, samples are drawn from a culture vessel into a flat optical chamber (similar to a flow-through cuvette). Digital pictures are taken by a camera and sent/analyzed automatically in a PC. Live individuals, which are continuously swimming, can be detected by subtraction of two digital images that have been taken in short temporal sequence. This principle of “background subtraction” is commonly used to detect moving objects, e.g., estimating urban traffic in cities (Cucchiara et al. 2003), but has also been exploited in biological context, e.g., in population size estimation of bacteria in microchemostats (Balagadde et al. 2005). The proof-of-principle paper with rotifers was presented by Alver et al. (2007), who developed an automated system to determine the density of rotifers in first feeding tanks in fish aquaculture. They showed that image analysis can give good estimates of the rotifer concentration in such tanks. Here I describe a sampling and image analysis system that was designed for studies of aquatic ecology, i.e., where rotifers are used as model organisms to investigate general ecological questions. The image analysis system allows in-depth analysis of rotifer population, including abundance estimation, detection of males (i.e., episodes of sexual reproduction), and measurement of body size distributions. The sampling system includes several new features to efficiently work with a large number of replicates and prevents cross-contamination between measurements of different cultures. It works completely autonomously and can sample up to 12 independent rotifer populations in short time intervals (down to a few hours) and over several weeks. In this article, I describe the basic setup of the system and tests on the efficiency of data collection, and show some example data sets on the population dynamics of different strains of the rotifer Brachionus calyciflorus. Materials and procedures General setup—The main components of the automated sampling and image analysis system are shown in Fig. 1. The Fig. 1. Schematic drawing of the sampling and image analysis system. For simplicity, only two rotifer cultures are displayed (the system can handle up to 12 cultures). The tubing connecting to these cultures was 120 cm in length, measured from the outlet of the culture to the dedicated simple magnetic valve (not true to scale in Fig. 1). All electrical parts, i.e., magnetic valves, air pump, peristaltic pump, camera, and illumination, can be controlled by a PC (PC and wiring not shown). Simple magnetic valves are either opened or closed (default: closed); coupled magnetic valves can switch between the states: valve 1 open/valve 2 closed and vice versa. Stelzer Automated analysis of rotifer populations 858 system is composed of parts dedicated to the automated sampling (silicone tubing, magnetic valves, air pump, peristaltic pump) and parts associated with image acquisition (optical unit in Fig. 1: camera, optical chamber, illumination). All electrical parts were controlled by a PC: The digital camera was controlled through a Firewire connection, the peristaltic pump through a serial RS232 connection, and some parts were indirectly controlled through a Quancom® USBREL64 relay interface (magnetic valves, LED illumination unit). For clarity, the computer and the connecting cables were omitted from Fig. 1. The heart of the image acquisition system is the optical unit, consisting of optical chamber, ring illumination, and digital camera (Fig. 1). The optical chamber consists of two glass plates separated by a frame to form a chamber with the inner dimensions of 18 × 24 × 3 mm (i.e., approximately 1.2 mL volume). Two stainless-steel tubes attached at the lower left and upper right end of the chamber provide inand outflow of the culture medium. The chamber is fixed in a metal frame, which itself is mounted onto a commercially available LED darkfield illumination unit (CCS LDR2-74SW2-LA). The images are taken with an industrial monochrome digital camera with a resolution of 6 megapixels (PixeLINK® PL-B781F). As optics, I use a SchneiderKreuznach APO-Componon 45mm lens with two 10-mm macro distance rings. One pixel corresponds to a square with approximately 9 μm length. All optical parts, chamber, illumination unit, and camera are mounted on a metal support so that the field of view of the camera exactly matches the size of the chamber (not visible in Fig. 1). Sampling system—Up to 12 rotifer cultures are connected via silicone tubing with the optical chamber (Fig. 1). The length of these connections is 120 cm, measured from the outlet of the culture to the dedicated simple magnetic valve (not true to scale in Fig. 1). The magnetic valves (FluidConcept S104) of each culture only open during sampling events (controlled by the PC). Individual cultures are sampled using the following sequence of events: (1) Filling the chamber. The magnetic valve connecting to the focal rotifer culture is opened and the peristaltic pump is switched on. This creates suction in the silicone tubing, which causes the culture medium to flow into the optical chamber. When the chamber is filled with the rotifer culture to be sampled, the pump stops and the digital camera takes two pictures and sends them to the PC, where they are digitally subtracted, stored, and analyzed. (2) Emptying the chamber. Used samples are expelled from the optical chamber with the membrane air pump: the two magnetic valves directly flanking the chamber are switched and the air pump is turned on. Note that these two magnetic valves are coupled (one channel is opened while the other is closed and vice versa). This allows changing the direction of flow so that the used culture medium is expelled in reverse direction into a waste reservoir (Fig. 1). The change in flow direction is essential, because otherwise the chamber cannot be emptied completely. (3) Rinsing the chamber. Between samplings from two different rotifer cultures, or at the end of sampling, the optical chamber is rinsed with deionized (DI) water from a separate reservoir (Fig. 1) to prevent cross-contamination. To initiate the rinsing cycle, the magnetic valve connecting to the DI reservoir is opened (connecting the DI water bottle but disconnecting the rotifer cultures) and the peristaltic pump sucks DI water into the chamber. The DI water is expelled in the same way as described in step 2. With this sequence of events, each rotifer culture can be sampled, and afterward the system can be paused by filling the chamber with DI water until the next sampling event (to prevent fouling within the chamber). To avoid dilution with residual DI water, the optical chamber is emptied before each sampling event with the membrane air pump (step 2). Additionally, old culture medium and DI water in the tubing are removed by filling the chamber once before each measurement (step 1) and immediately emptying it without taking pictures (step 2). The complete sampling procedure for 12 rotifer cultures and three samplings per culture, including all rinsing cycles, takes less than 90 min. All events of the sampling procedure are controlled by a program written in Labview® 8.5 and NI Vision (National Instruments). Figure 2 displays a schematic depiction of the program’s general structure. The actual Labview code is available from the author on request. The program represents a simple “state machine” that switches between two states: a waiting state and a sampling state (Fig. 2). Within the waiting state, the program reads the current time and compares it to the preset sampling times, which may be one or several different time points during a day. When the time of a sampling event is reached, the program switches to the sampling state, which causes all cultures to be sampled in sequential order. After sampling is completed and image analysis is done, the program switches back to the waiting state. Due to its modular structure, the program can be adjusted quickly to accommodate a wide range of variation in experimental designs: number of cultures sampled, number of samplings per day, number of samples per culture (within one sampling). Image analysis—Images are automatically processed using NI Vision, running within the Labview programming environment. Briefly, the two pictures from each sample are loaded into the memory of the PC and one image is digitally subtracted from the other. After subtraction, the thresholding algorithm of NI Vision generates a binary image (i.e., an image consisting of black and white pixels only). Shape recognition algorithms are used on the binary image to detect rotifers and classify them. The detection criteria for female and male rotifers are summarized in Table 1. These criteria are empirical estimates and are optimized for the lightning conditions provided by the light ring and settings of the camera (brightness, gain, exposure time). I found that a relatively long exposure time of 100 ms and a waiting time of 1 s between the two pictures gave best Stelzer Automated analysis of rotifer populations 859 results for detecting both female and male rotifers: Individual females look like rice grains, whereas the fast-swimming but much smaller males leave more narrow traces, reminding of eyelashes (Fig. 3). These two shapes are clearly distinct and can be reliably separated by the exclusion criteria listed in Table 1. Note that due to the long exposure time of 100 ms, rotifers tend look a bit more elongated than their natural shape, because of the distance they swim during exposure. Rotifer cultures—The performance of the sampling and image analysis system was assessed using laboratory cultures of the rotifer Brachionus calyciflorus. All Brachionus lines used in this study were descendants of two strains from Florida and Georgia, respectively, and were originally provided by J.J. Gilbert (Dartmouth College, Hanover, NH, USA). They have been maintained in my laboratory since autumn 2006. Clonal lines of these strains differed with respect to body size and Fig. 2. Simplified structure of the program that controls sampling, image acquisition, and image analysis (Nassi-Shneidermann diagram). Note that the program switches between two fundamental states, a waiting state (for the times between two samplings) and a sampling state. The switch between these two states would continue indefinitely, unless the program is interrupted or terminated by the user. Table 1. Detection criteria of females and males in the image analysis program. Command/detection criteria Parameter range Female preprocessing 1. Erode picture (removes random noise) 2. Set thresholda 50–255 3. Fill holes (of particles) Female detection Particle area 80–2000 pixels Elongation factorb 1–4.5 Equivalent ellipse minor axis 8–30 pixels Male preprocessing 1. Erode picture 2. Set threshold 50–150 3. Exclude particles with holes >50 pixels 4. Fill holes (of remaining particles) Male detection Elongation factor >6 Particle area 80–200 pixels Equivalent ellipse major axis 20–40 pixels Equivalent ellipse minor axis 4–7 pixels aThresholding transforms a grayscale picture to a binary picture: All pixels meeting the inclusion criteria are set to white; all remaining pixels are set to black. After thresholding, particles can be identified as a group of white pixels directly adjacent to each other. bElongation factor was defined as a particle’s largest intercept divided by the mean perpendicular intercept. Stelzer Automated analysis of rotifer populations 860 their ability to induce sexual reproduction: Some of them were cyclical parthenogens and others were obligate parthenogens that had lost the ability to reproduce sexually (Stelzer 2008). The rotifers were cultured in COMBO medium (Kilham et al. 1998) with the unicellular algae Chlamydomonas reinhardii as food source (strain SAG11-32b, Sammlung fuer Algenkulturen). The rotifer populations used in this study were cultured in chemostats, i.e., flow-through cultures that were continuously diluted with food suspension harvested from a separate algal culture. Algae in this algal chemostat grew at a dilution rate of 0.89 d–1 and had entered a steady state with a relatively constant biovolume of 1.2 × 108 femtoliters mL–1 (approximately 750,000 cells mL–1). This algal suspension was diluted 1:1 with fresh culture medium before being distributed among the rotifer chemostats. In the experiments, I used chemostats with 380 mL volume and a dilution rate of 0.56 d–1. All experiments were done in a temperature controlled room at 24°C. For further details on culturing methods, see Stelzer (2008).