Phototropic response features for different systematic groups of mesoplankton under adverse environmental conditions

Abstract Current trends in the application of bioindication methods are related to the use of submersible tools that perform real‐time measurements directly in the studied aquatic environment. The methods based on the registration of changes in the behavioral responses of zooplankton, in particular Crustaceans, which make up the vast majority of the biomass in water areas, seem quite promising. However, the multispecies composition of natural planktonic biocenoses poses the need to consider the potential difference in the sensitivity of organisms to pollutants. This paper describes laboratory studies of the phototropic response of plankton to attracting light. The studies were carried out on a model natural community that in equal amounts includes Daphnia magna, Daphnia pulex, and Cyclops vicinus, as well as on the monoculture groups of these species. The phototropic response was initiated by the attracting light with a wavelength of 532 nm close to the local maximum of the reflection spectrum of chlorella microalgae. Standard potassium bichromate was used as the model pollutant. The largest phototropic response value is registered in the assemblage. The concentration growth rate of crustaceans in the illuminated volume was 4.5 ± 0.3 ind (L min)−1. Of the studied species, the phototropic response was mostly expressed in Daphnia magna (3.7 ± 0.4 ind (L min)−1), while in Daphnia pulex, it was reduced to 2.4 ± 0.2 ind (L min)−1, and in Cyclops vicinus, it was very small—0.16 ± 0.02 ind (L min)−1. This is caused by peculiar trophic behavior of phyto‐ and zoophages. The addition of a pollutant, namely potassium bichromate, caused a decrease in the concentration rate of crustaceans in the attracting light zone, while a dose‐dependent change in phototropic responses was observed in a group of species and the Daphnia magna assemblage. The results of laboratory studies showed high potential of using the phototropic response of zooplankton to monitor the quality of its habitat thus ensuring the early diagnostics of water pollution. Besides, the paper shows the possibility of quantifying the phototropic response of zooplankton using submersible digital holographic cameras (DHC).


| INTRODUC TI ON
The contamination of water bodies with small concentrations of pollutants at first may not have a visible toxic effect. Moreover, the violation of biological well-being may not be detected during a single examination. But in case of chronic impact on the biota, this may lead to a shift in the ratio between species. This will inevitably cause changes in the quality of the ecosystem and potential disastrous reduction in the number of aboriginal species. Therefore, today there is an urgent need for prompt early control of pollution of natural water areas by microconcentration of pollutants (Sukharenko et al., 2017).
Besides, early detection of pollutants is critical in hazardous areas such as nuclear stations, oil platforms, and gas pipelines.
Over the past two decades, quite a few studies have been devoted to water biotesting techniques based on the analysis of various features of aquatic organisms, namely survival registration (OECD, 2013(OECD, , 2019, reproducibility, offspring quality, morphological parameter changes (OECD, 2013), physiological functions, and behavioral responses (Lechelt et al., 2000;Morgalev et al., 2015;Nikitin, 2014;Wang et al., 2019). The most promising are the methods of water biomonitoring using behavioral responses of local species of hydrobionts, and primarily bivalves (Sukharenko et al., 2017), branchiopoda (Carreño-León et al., 2014) and copepoda (Lechelt et al., 2000;Pan et al., 2015Pan et al., , 2017Ren et al., 2017), and fish (Ren et al., 2016). Daphnia are particularly interesting with regard to these methods. They filter a large amount of water by feeding on bacteria and algae contained in it, as a result of which the presence of harmful substances even at low concentrations causes significant changes in their state.
Behavioral response recording techniques are more sensitive than the methods that register mortality or growth and developmental inhibition. There are automated methods of continuous biological control that can generate an alarm signal based on recording physiological parameters and behavioral responses, such as speed and trajectory of swimming, frequency of swimming movements, etc. (Dodson et al., 1997;ISO, 2012;Lechelt et al., 2000). However, these methods are implemented by stationary flow devices, so the analyzed water samples shall be delivered to them, which significantly reduces the dynamics of monitoring. Besides, they use special laboratory types of daphnia aligned by sensitivity to model toxicants.
Such disadvantages, including a limited set of test species, are typical for other devices of this type.
The current trend in world ocean monitoring is the use of submersible tools that perform real-time measurements directly in the studied aquatic environment. The same approach should be applied to study the responses of biological species thus ensuring high representativeness of sampling and more reliable bioindication. The second important advantage here is the recording of responses of autochthonous organisms adapted to local changes in environmental factors. Therefore, the development of methods that record behavioral responses directly in the habitat is justified and promising.
Phototropism is particularly interesting among other behavioral responses. The phenomenon of phototaxis in hydrobionts has long been known. It is most clearly observed in diel vertical migration along the water column (Cousyn et al., 2001)-the largest biomass migration. Light-dependent movement of microzooplankton reduces the biomass of phytoplankton at the surface (Moeller et al., 2019).
With the illumination change, the aggregation of daphniae may be changed by toxicants, for example, by titanium oxide nanoparticles (Noss et al., 2013). On the one hand, the variability of diel vertical migration indicates the participation of the nervous system in the phototropic response: In environments where the influence of predators and other factors change over time, normal migration (nocturnal ascent) may be replaced by reverse migration (nocturnal descent) (Ohman, 1990). On the other hand, phototactic responses change when such psychotropic substances as diazepam, fluoxetine, and carbamazepine are introduced (Rivetti et al., 2016). Therefore, it is possible to expect a significant change in the phototropic response when the nervous system of planktonic organisms changes as a result of pollutants in their habitat.
At the same time, there are contradictory data regarding the phototropic response: Thus, according to Simão et al. (2019), when faced with a sudden increase in light intensity, Daphnia magna show a photomotor reaction, period of hyperlocomotion, when animals try to escape from light to avoid predatory fish. At the same time, De Meester (1991) describes both swimming toward (positive phototaxis) and away from light (negative phototaxis).
Besides, most of such studies do not focus on the wavelength of stimulating light being only limited to the term "daylight." Another significant disadvantage of most trajectory-tracking studies is the small number of individuals: from 1 to 10 in different studies.
The laboratory studies on a large number of individuals, but not on single representatives of zooplankton, make it possible, firstly, to vary the spectrum and concentration of pollutants and, secondly, to obtain a statistically significant response assessment of hydrobionts.
Besides, the use of natural autochthonous organisms (and not only their laboratory analogues) allows extrapolating the resulting patterns to subsequent field studies. the phototropic response of zooplankton using submersible digital holographic cameras (DHC).

K E Y W O R D S
bioindication, early diagnostics of ecosystem pollution, environmental monitoring, mesoplankton, phototropic response, submersible digital holographic camera This requires special technical means that allow detecting and classifying indicator organisms in the monitoring mode, as well as monitoring the dynamics of their behavioral responses.
The operational oceanology utilizes a large number of devices used to study the properties of plankton using fluorimetric, nephelometric, and turbidimetric measurements ("SBE 19plus V2 SeaCAT Profiler CTD | Sea-Bird Scientific-Overview | Sea-Bird", 2020). However, the registration using such equipment does not make it possible to perform differential bioindication, including the study of behavioral responses. There are several commercial submersible holographic cameras on the market that provide measurements of individual particles, in particular, LISST-Holo ("Environmental Archives-Sequoia ScientificSequoia Scientific", 2020; Ouillon, 2018) and Submersible Microscope ("HoloSea: Submersible Holographic Microscope-4Deep", 2020 ;Rotermund & Samson, 2015). On the contrary, compared to photographic cameras (Cowen & Guigand, 2008;Lertvilai, 2020;Ohman et al., 2012) holographic cameras record information on all particles in volume per one exposure, which allows obtaining a focused image of each particle in the recorded volume from one hologram, identifying geometric parameters and classifying the type of each of the particles in the recorded volume, and using the time sequence of holograms to build a trajectory and study the motion pattern of each particle (Bochdansky et al., 2013;Graham et al., 2012;Nayak et al., 2018;Owen & Zozulya, 2000;Pfitsch et al., 2007;Sun et al., 2008;Talapatra et al., 2013). The studies Giering et al., 2020;Nayak et al., 2021) show the comparison of such cameras.
The equipment created at Tomsk State University (TSU) (digital holographic cameras and hydrobiological probes based on them) Dyomin, Gribenyukov, Davydova, et al., 2019;Dyomin et al., 2018) also provides measurements of individual particles, but differs from known analogues in the possibility of photostimulation with attracting radiation causing a phototropic response of zooplankton (Dyomin, Davydova, Morgalev, Olshukov, et al., 2019). The advantages include both a large size of the controlled volume and thus obtained representativeness of data.
The purpose of this study was to assess the contribution of crustaceans of different species represented in natural freshwater populations to changing the phototropic responses of a group of hydrobionts under the action of pollutants.

| Experimental equipment and software
We used a digital holographic camera (DHC) developed by us to record the behavioral responses of zooplankton assemblage. The DHC allows registering a digital hologram of the entire studied volume of water with plankton per one exposure (laser pulse) and then restoring the image of this volume in a layers-by-layers mode. The software-based DHC technology for digital hologram recording and processing allows automatically restoring the spatial distribution of particles in the studied volume (3D coordinates of each particle), determining the size, shape, speed, and direction of movement of each particle, and recognizing them Dyomin, Davydova, Morgalev, Olshukov, et al., 2019;Dyomin, Gribenyukov, Davydova, et al., 2019;Dyomin et al., 2018;.
Besides, it registers individuals with the minimum size of 100 μm at the resolution sufficient enough to identify plankton individuals. It is evident that in order to study the particle motion parameters, it is necessary to record a time sequence of digital volume holograms and reconstruct the video based on holographic data (Dyomin & Olshukov, 2012).
Thus, compared to existing analogues, the DHC provides the ability not only to measure dimensions and coordinates in the monitoring mode but also to classify plankton by species and assess the motor activity of plankton.
The device can be used in field studies up to a depth of 600 m (the depth of the information layer from the point of view of plankton presence). A hologram of 1-L volume is recorded per one exposure. In the accumulation mode (e.g., when water passes through the measuring channel while moving the DHC), the studied volume may be increased to 15 L per second. The DHC base is composed of lighting (3) and recording (2) units located in sealed cases.

F I G U R E 1
Laboratory setup: 1-DHC, 2-DHC recording unit, 3-DHC lighting unit, 4-laboratory water tank, 5-studied (working) volume formed by recording (red) and attracting (green) light beams, 6-mirror-prism system to form the working volume, 7-semiconductor laser diode (λ = 650 nm), 8-semiconductor laser diode (λ = 532 nm), 9-fiber-optic multiplexer (mixer), 10-beam expander, 11-portholes, 12-selective filter, 13-receiving lens, 14-CMOS camera Light from the source 7 (λ = 650 nm) is formed by the beam expander (10) into a parallel beam with a diameter of 35 mm. Then, radiation passes through a porthole (11), the analyzed volume (5) with analyzed particles optically formed by the recording (lighting) beam and prisms (7) and gets into the recording unit (2) through a porthole (11). As a result, an interference pattern of the reference wave (part of the radiation that passed by the particles) and the object wave (part of the radiation scattered on the particles) is formed. The optical system (13) in the recording unit matches the size of the incoming beam with size of a CMOS camera (14). The camera (14) registers the mentioned interference pattern (which is then a registered digital hologram of the studied volume) and transmits it to the computer memory via a digital cable. Mathematical processing by computational algorithms (Dyomin, Gribenyukov, Davydova, et al., 2019) allows reconstructing the image of each particle (plankton individual) from one digital hologram, ensuring the spatial distribution of particles in the studied volume (3D coordinates of each particle), determining the size, shape, speed, and direction of movement of each particle, and recognizing them. Additional attracting radiation from the source (8) also passes through the volume with studied particles and is used for the photostimulation of zooplankton behavioral activity. A fiber-optic multiplexer (9) is used to input this photostimulating green radiation of a semiconductor laser (λ = 532 nm) into the same DHC optical channel. In order to prevent the damage of the camera (14) matrix, radiation at a certain wavelength (λ = 532 nm) is absorbed by a selective filter (12).
This series of experiments utilized the recording red light (wavelength-650 nm, radiation power-20.3 mW) and attracting light (wavelength-532 nm, close to the local maximum of chlorella microalgae reflection spectrum, radiation power-9.6 mW). The radiation power is indicated at the output of the porthole.
In laboratory experiments for the phototropic response of plankton, the optical part of the DHC (1) was placed in a 90-L laboratory water tank (4) filled with water containing plankton ( Figure 1).

| Studied organisms
The studies were carried out on the groups of individuals-Cladocera The wild Daphnia pulex species were introduced into the laboratory culture according to the recommendations (Grigoriev & Shashkova, 2011). To introduce the Cyclops vicinus wild species into the laboratory culture, an introduction procedure was developed, including a feeding regime. A cocktail consisting of a decoction of lettuce leaves, a concentrate of single-cell chlorella algae, and a suspension of Paramecium caudata infusories was used for feeding in the following proportion: 50 ml of "cocktail" containing 35 ml of lettuce leaves decoction at the concentration of 1 g/L, 10 ml of chlorella concentrate with an optical density of D = 0.450-0.500, and 5 ml of infusoria suspension at the concentration of 150 pcs/ml were added to 1 L of the cultivation medium.
To verify the stability of the culture prior to holographic registration, the sensitivity to the standard toxicant was assessed in accordance with (ISO, 2012).
The individuals of the same age (3 days) were used in the study.
In the experiments with monoculture, 270 small crustaceans were placed in a 90-L water tank, which corresponded to the concentration of 3,000 individuals per 1 m 3 . In case of mixed culture, 90 small crustaceans of each of the three considered species were used.

| Experimental procedures
The experimental procedure for each zooplankton species or their mixture included the following sequence of operations (Table 1).
The example of a 2D display of a holographic image obtained from a digital hologram recorded by a digital holographic camera is shown in Figure 2. A set of data obtained after digital hologram processing, on the basis of which the results of this study are presented, is available at Zenodo digital repository-http://dx.doi.org/10.5281/ zenodo.4308667 .
Statistical data analysis was performed using Statistica 10.
Since the distribution of variables did not always correspond to the normal law (Shapiro-Wilk's W test), and each experiment used a new assemblage of crustaceans, the assessment of differences and statistical significance of the result (p-value) was carried out using a nonparametric Mann-Whitney U test.

| Phototropic response of crustacean
The preliminary series of experiments studied the time characteristics of the phototropic response of the Daphnia magna assemblage to attracting light. The experiment is partially described by us (Dyomin, Davydova, Morgalev, Olshukov, et al., 2019). According to hologra- Based on these data, the following scheme for recording the measuring hologram set was chosen for further studies: Immediately after the attracting light is turned on, a pair of holograms is recorded with a time interval of 41.6 ms between them for subsequent calculation of the average speed of the zooplankton assemblage. This time interval is chosen based on the average speed of movement and the size of crustaceans-during this time, the displacement of each crustacean does not exceed the size of its body, that is, the images of the same particle reconstructed from adjacent holograms partially  The concentration growth rate in the "concentration-lighting time" coordinates was 4.49 ± 0.29 ind (L min) −1 . The pronounced phototropic response is associated with the prevalence of Daphniidae phytotrophes in the assemblage (180 of 270 individuals).
An attempt to isolate the mobility characteristics of different species from the group of species was not quite successful. First, as we have previously noted , the accuracy of taxonomic affiliation of objects reconstructed from a hologram is not high: Copepoda-86 ± 9% and Cladocera-77 ± 2%.
Second, the conjugation of the recognition algorithm with the hologram reconstruction algorithm requires quite large computational resources and did not allow obtaining behavioral response characteristics in dynamics in our experiment. Therefore, the contribution of crustaceans of different species to general reactivity was studied in the experiments with monocultures.
It should be noted that the response of zooplankton assemblage formed from crustaceans of older age (5-7 days) may vary quantitatively, but the overall responsiveness ratio remains the same: Concentration growth rates are maximum for the mixed group of species (5.95 ± 0.38 ind (L min) −1 ) and decrease for Daphnia magna and Cyclops vicinus (3.54 ± 0.33 ind (L min) −1 and 0.05 ± 0.02 ind (L min) −1 , respectively). Toxicity is generally estimated to be LC 50 (50% lethal concentration), which is the amount of a substance dissolved in water required to kill 50% of test animals during a predetermined observation period. EC 50 (half-maximum effective concentration) is used to assess nonlethal effects on physiological and behavioral functions. This is the concentration of a substance causing an effect equal to half the maximum possible for a given substance after a certain period of time.

| Phototropic response of crustacean in the presence of potassium bichromate
The earlier studies showed that the toxic effect on the behavioral responses of Daphnia magna consisting in a change in the nature of movement was observed from the concentration of 0.011 ± 0.001 mg/L, and a 50% response change (EC 50 ) was observed at a pollutant concentration of 0.15 ± 0.02 mg/L (Dyomin, Gribenyukov, Davydova, et al., 2019).
The sensitivity of Cyclops vicinus to potassium bichromate is significantly lower. The study showed that the LC 50, therefore, was 29.6 ± 9.6 mg/L. This major difference in sensitivity is caused by the fact that Cyclops are not filtration organisms. Similar data are obtained by other researchers (Noskov, 2011).

| Behavioral response of the mixed group of crustacean species
The At a higher bichromate concentration of 0.12 mg/L, there was no reliable change in the concentration growth rate compared to the control situation without a pollutant-from 3.54 ± 0.33 ind (L min) −1 to 3. 9 ± 0.26 ind (L min) −1 (p > .05).

| Behavioral response of Daphnia pulex
There was a paradoxical response of Daphnia pulex-a critical increase in the phototropic response of this group of species (Figure 4c): The concentration growth rate increased from 2.42 ± 0.19 ind (L min) −1 to 5.17 ± 0.46 ind (L min) −1 (p = .0001).
A slight decrease in the phototropic response was observed at the concentration of 0.12 mg/L: The concentration growth rate decreased from 8.14 ± 0.80 ind (L min) −1 to 7.86 ± 0.69 ind (L min) −1 (p = .40). The tendency to inhibit the phototropic response at a higher toxicant concentration than that of Daphnia magna may be caused by the fact that the size of the body, and therefore, the amount of fluid to be filtered is smaller for Daphnia pulex.

| Possible reasons for differences in responses with a toxicant
The most pronounced pattern of zooplankton response to a toxicant is inhibition of phototropic response-concentration of crustaceans in the attracting light zone. One mechanism for such changes may be the direct effect of the toxicant on the nervous system of crustaceans thus leading to a change in their behavior (Dallakyan et al., 2017;Kikuchi et al., 2017).  (Selye, 1946). These factors have been repeatedly confirmed both for certain different organisms and entire populations (Patin, 2004).
The most rapid and relatively easily recorded effects occur at physiological, biochemical, and organizational levels. Daphnia pulex due to their reduced sensitivity. This is due to the fact that the size of the body, and therefore the amount of fluid to be filtered, is less.
The increase of the sensitivity of Daphnia as age increases, and accordingly body sizes, is described in Traudt et al. (2017) and studied by us in the survival test (LC 50 ) of Daphnia pulex ( Figure 6). As the age of crustaceans increases, the toxicant concentration leading to the death of 50% individuals (LC 50 ) decreases from 1.84 mg/L for 1day individuals to 0.13 mg/L for 9-day individuals, that is, the sensitivity to this toxicant increases by more than an order of magnitude.
Other reasons may explain the differences in Cyclops vicinus responses. Cyclops are mainly zootrophes and feed on protozoa, rotifers, small crustaceans. In this regard, light with a wavelength corresponding to the reflection spectrum of chlorophyll does not seem to be a signal of food for them and, accordingly, an attractor.
The phototropic response of Cyclops vicinus increasing with an increase in toxicant concentration may be associated with the avoidance response at high toxicity of the medium and active movement to a "safe" zone with chlorophyll-containing microorganisms.

| CON CLUS ION
The phototropic response of the model Crustacea assemblage

ACK N OWLED G M ENTS
The study was carried out with the support of the grant of the Russian Science Foundation .

CO N FLI C T O F I NTE R E S T
The authors claim no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data set is available at the digital repository Zenodo with https:// doi.org/10.5281/zenodo.4308667 .