Adopting cannibalism substantially affects individual fitness, and recognizing the presence of other cannibals provides additional benefits such as the opportunity to prepare for hunting or defense. This recognition can be facilitated by perceiving conspecific chemical cues. Their role in cannibalistic interactions is less studied than in interspecific predation and it is unclear whether these cues inform individuals of danger or of food availability. Interpretation of these cues is crucial to balance the costs and benefits of anti-predator and feeding strategies, which can directly influence individual fitness. In this study we aimed to test whether damselfly larvae shift towards bolder and more exploratory (cannibalistic) behavior, or become more careful to avoid potential cannibals (as prey) in response to such cues. We conducted behavioral and respiratory experiments with Ischnura elegans larvae to investigate their response to chemical cues from older and larger conspecific larvae. We found that I. elegans larvae decrease their activity and shift their respiratory-related behavior, indicating activation of anti-predator defense mechanisms in response to conspecific chemical cues. Our findings indicate that individuals exposed to conspecific chemical cues balance catching prey with staying safe.
Cannibalism is a widespread phenomenon[
Cannibalistic individuals may enhance their fitness through nutritional benefits, yet are exposed to additional risks. Well-fed cannibals increase their own survival, developmental rate, and fecundity[
The effectiveness of strategies employed by prey to evade predators relies on the accessibility and precision of sensory data. While vision theoretically allows for immediate perception of predator's location, approach trajectory, and identity, prey confronted with active predators must react so early that visual cues become unreliable indicators of real danger[
Therefore, in predator–prey interactions, recognizing each other's presence via chemical cues is one of the most effective and reliable mechanisms of assessing either risk or prey availability. Such cues may be of one of three types: alarm signal, diet cues, or kairomones. Infochemicals released by injured individuals form alarm signal which serves as cue of danger to other potential prey. Diet cues, released by hunted prey and produced during prey consumption and expulsion by predators, provide information to other predators about food availability. Lastly, kairomones are involuntarily released by both prey and predator and betray each other's presence without the necessity of direct interaction[
In interspecific predation, predator's reaction to the chemically-detected presence of prey is often limited to behavioral changes in foraging strategy[
The role of these chemicals in intraspecific, cannibalistic interactions is more complex and far less understood. Most of the hitherto conducted studies on cannibalistic interactions concern the effect of conspecific alarm signal, while kairomones have been studied much less in this respect. For example, blue crabs (Callinectes sapidus) avoid areas of intense conflicts in response to odors released from freshly injured conspecifics. Yet, alarm signal can also function as diet cues in this intraspecific interaction[
Odonate larvae are a suitable model for investigating the importance of chemical cues in cannibalistic interactions. Larval cannibalism has been found in different odonate species[
Behavioral changes are a common anti-predator strategy in zygopteran larvae and alterations to foraging are expected as the response to conspecific cues. For example, a typical anti-predator defense response in damselfly larvae is reduction in foraging intensity (decreasing the number of captured prey) and prolonging immobility[
Predator pressure can also lead to changes in respiratory-related behavior, thus causing changes in the oxygen uptake pattern, which we call here oxygen consumption variability. Stressed zygopteran larvae modify the pattern of rectal breathing by increasing the number of inhalations, which shortens the pause between them and intensifies exhalation[
Here, we conducted behavioral (mobility and feeding) and respiratory experiments with larvae of damselfly Ischnura elegans to test if they: (a) change their behavioral strategy in response to conspecific chemical cues by reducing mobility and staying in refuge (i.e. adopt prey strategy), (b) decrease oxygen consumption and change oxygen consumption pattern by increasing its consumption variability in response to those signals (i.e. adopt prey strategy), and if (c) the strength of the individual reaction to conspecific cues depends on their type and concentration.
Damselfly larvae Ischnura elegans were collected from Czerniakowskie lake in Warsaw—a natural urban lake inhabited by both fish and invertebrate predators—in July 2020. Larvae were photographed and measured using NIS-Elements software, and assigned to instars according to parameters proposed by Thompson: body length and head width[
All experimental animals were synchronized to the 10th instar. Knowing the date of the larva's last molt allowed us to choose the time of the experiment to exclude unexpected molting during exposition to kairomones. Individuals in the 9th instar were kept in specially-designed containers (material: colorless filament ASA 1.75 mm, dimensions: 50 × 70 × 40 mm) created with 3D printing technology that guaranteed the physical separation of individuals. The aquarium with the containers was filled with 10 L of water. There were 10 individuals per aquarium. The culturing setup consisted of a series of interconnected chambers that were separated by a mesh, enabling a continuous flow of fresh, oxygenated water through each individual chamber. The aquarium contained a designated vegetation zone (to prevent the accumulation of excess nitrogen compounds), wherein a pump was located. The pump functioned to expel water from the vegetation zone to the opposite end of the aquarium, thereby maintaining a consistent flow of oxygenated water throughout the entirety of the system.
Every second day, the odonates were fed with live adult Daphnia pulex. After reaching the 10th instar, the larvae were transferred to a thermostatic chamber and each was kept in a separate container (to prevent cannibalistic interaction and exposition to conspecific cues), at 4 °C, the low temperature preventing them from molting. The cannibals (11th and 12th instar larvae) and prey (7th and 8th instar larvae) were also maintained at 4 °C. Additionally, as an injury may affect larvae behaviors and respiratory activities, all experimental animals were checked for signs of mechanical damage (such as loss of lamellae or legs). All larvae were cultivated in conditioned (filtered through 1 µm mesh and aerated at least for two weeks) lake water.
Before the experiment, all cannibals were acclimatized to 20 °C for three days. These damselflies were not fed prior to the experiment 1) to prevent contamination of their kairomones with food particles that could serve as potential alarm signal (AS) for prey, and 2) to increase their propensity for cannibalism. A day before the experiment, the predators were transferred to separate containers—to prevent competitive or cannibalistic interactions—where they were exposed in conditioned lake water for 24 h to produce kairomones.
The experimental media were as follows: 1) Control medium—kairomone free medium (C); 2) Medium with kairomones in concentration of 2 I. elegans × L
To prepare experimental media, the replicated cannibal-exposed water from all containers used in the particular treatment was poured into a single one just before the experiment and filtered through a GFC filter.
The experiment was performed in a specially designed chamber (white box of dimensions 120 × 106 × 56 cm) which guaranteed stable internal conditions and silenced off external noises. The box was equipped with two lamps covered with a light diffuser to ensure uniform light conditions. It was placed in the temperature-controlled experimental room and special openings provided gravity ventilation, thus allowing it to maintain the constant 20 °C inside. The camera was inserted through a hole in the center of the box's ceiling. The front wall was removable, allowing an easy access to the animals.
Six days before the experiment, experimental animals were transferred from 4 to 20 °C for acclimatization. Each of them was placed in a separate container to prevent exposure to conspecific chemical cues and cannibalistic interactions. The next day after the transfer, the animals were moved into the box in their separate containers. These were round, non-transparent plastic vessels (diameter 10 cm, height 3 cm) filled with 200 ml of clean medium and had a centrally located refuge zone (an artificial plant on a silicone suction cup of 2 cm in diameter,—Fig. 1). The circular shape of the containers minimized the shaded area inside and thus increased the clarity of the recorded movies. Additionally, it prevented the aggregation of live food in the corners and enforced its random dispersion. Each experimental animal was fed with 10 D. pulex at the pre-reproductive instar. Live food was introduced simultaneously into the containers with I. elegans larvae with the use of a special feeding device (Fig. 1A) that consisted of a movable rack to which containers with live Daphnia were attached. When the rack was lifted, the Daphnia were released into the containers with I. elegans. Three days before the experiment, the medium was exchanged and damselflies were fed as before. Then, the larvae were starved for two days prior to the experiment.
Graph: Figure 1Experimental setup. (A) Feeding device with cylindrical bottomless chambers holding Daphnia while resting on the bottom of the experimental containers, four containers in sight. The chambers were tied to hooks on the rack (gray) and lifted during feeding, (B) An individual experimental container with arena zone and refuge zone (artificial plant on suction cup) with Daphnia released.
On the experimental day the containers were filled with 200 ml of one of the experimental media. The control medium (C) and the medium with high kairomone concentration with an alarm signal (D5 + AS) were replicated six times. The medium with low kairomone concentration (D2) and the medium with high kairomone concentration (D5) were replicated five times (this unequal number of replications resulted from problems with collecting a sufficient number of synchronized experimental individuals). The larvae were then left to acclimatize for 30 min to reduce the impact of the stress from transferring to experimental containers on mobility and feeding behaviors. After acclimatization, the feeding procedure was performed. After closing the chamber, the recording of feeding behavior was started and lasted for 63 min.
Before the analysis, the initial three minutes were cut from the raw recording (starting from the moment of closing the box), as in that time larvae might have shown altered behavior caused by retraction of the feeding device and movements associated with the closing of the box. An hour-long recording was used for the analysis. To compare the behavior of odonate larvae at the beginning (immediate response to cue—we assume that in this time the effect of kairomones is most detectable) and at the end of this period, the movie's first and last 15 min were analyzed. Each larva was analyzed independently. Nine types of behaviors were observed and coded with three letters. The first letter denotes where the behavior takes place, in refuge (R) or in arena (A). The second letter denotes whether the animal is immobile (I) or mobile (M). The third letter after the hyphen specifies further either the location of the activity: on plant (-P) or on cup (-C), or the type of mobility: leaning (-L), treading (-T), gentle movements (-G) or swimming (-S). The behaviors were: 1) RI-P: Immobility on plant—most camouflaging behavior; 2) RI-C: Immobility on suction cup—observation of prey; 3) RM-P: Mobility on plant (vertical movement)—stalking of prey; 4) RM-C: Mobility on suction cup (horizontal fast walking)—stalking of prey; 5) RM-L: Leaning out in search of prey—stalking of prey; 6) AI: Immobility within arena—observation of prey; 7) AM-G: Gentle movements within arena (turning the head behind the prey, slow initial walking)—initial stalking of prey; 8) AM-T: Treading within arena—(decisive, quick following of the prey) main stalking of prey; 9) AM-S: Swimming—quick escape. Behavior timeline was created separately for each individual's first and last 15 min of the recording.
From the timeline of individuals assigned to each treatment (i.e. different infochemicals), the first and last 15 min of experiment were taken and two parameters were determined for each treatment for each period: i) time spent immobile (sum of times spent on RI-P, RI-C, AI behaviors), ii) time spent in refuge (sum of times spent on RI-P, RI-C, RM-P, RM-C, RM-L behaviors). We chose these parameters expecting that movement and switching between different types of behavior may attract predator. Thus, time spent in refuge, and time spent immobile should be a good measure of fear of a cannibal predator. We analyzed the data for the first and last 15-min intervals separately using TIBCO Statistica 13.3 and Kruskal–Wallis test (results of Leven's and Shapiro–Wilk test showed that data failed to meet the criteria for parametric analysis) and a nonparametric two-tailed post-hoc test with Bonferroni correction described by Castellan, with significance level at p < 0.05[
Also, the total number of attacks on prey made in the first and last 15 min of the experiment was counted. We counted all attack attempts, including those that were not successful. An attack was considered an ejection of the mask in the direction of the prey. Simultaneously, we also recorded the success of the attack, which involved mask ejection leading to the capture of the prey. We analyzed the data for the first and last 15-min intervals separately. Additionally, at the end of the experiment the number of Daphnia left in each experimental container was counted to obtain the number of prey consumed during the whole experiment. All three parameters: 1) total number of attacks, 2) number of successful attacks, and 3) number of consumed Daphnia until the end of the exposure were analyzed using TIBCO Statistica 13.3. and Kruskal–Wallis test (data failed to meet the criteria for parametric analysis) and nonparametric two-tailed nonparametric post-hoc test with Bonferroni correction described by Castellan with significance level at p < 0.05[
Second, to analyze the overall effects of different chemical cues on time spent in different behaviors, but also on time in immobility and in refuge, we fitted linear models using generalized least squares by maximizing the restricted log-likelihood (nlme R package[
We visualized behavioral patterns for the first and last 15 min in each treatment as directed graphs. Each of the nine observed behavioral types was represented on a graph as an ellipse (bubble) corresponding in area to the total time spent on this behavior by all individuals from the particular treatment. The ellipse area was calculated as log (time) * 10. The number of direction-specific transitions between behaviors for all individuals from each treatment was visualized by arrow thickness, which was calculated as log (number of transitions*2) * 1.5. The graphs were created in CorelDRAW2020.
The media used in respiratory experiments were prepared as described above, except that a 0.2 μm antibacterial filter was used for media filtration before the proper experiment, and next, the proper medium was aerated for 15 min.
On each day of the experiment, only one replication of one of the four experimental treatments was carried out (one individual per day). This makes a total of twelve experimental days, with three repetitions per treatment throughout the entire experiment. The selection of a day for each treatment tested was random. Two days before testing, one experimental animal was transferred from 4 to 20 °C. On the day before testing, an I. elegans larva was placed in 200 ml of conditioned and filtered (GFC) lake water. During that day, the larva was starved to empty its digestive tract. The exchange of water was conducted two hours before the experiment.
The oxygen content was measured with a UNICENSE MicroRespiratory System. Two breathing chambers were filled with 40 ml experimental medium (one with the animal and one blank), filtered with a 0.2 μm filter. In the next stage, the filled breathing chambers were placed in a stand located in a water bath set at 20 °C. The blank chamber was measured to account for unexpected oxygen consumption. The oxygen content in each chamber containing an animal was measured continuously for 12 h (with 1 s intervals between measurements) starting from the acclimatization period of 40 min after closing. Oxygen loss at that time ranged between 5 and 27%, not causing the alerting behavior of I. elegans associated with oxygen deficiency, which could disturb the hunting process. The volume of each chamber and the animals' body mass was measured after the experiment.
We analyzed the effect of experimental treatment on damselfly respiration separately for four consecutive three-hour intervals. Since the linear regression parameters changed over time, dividing the analysis into four time intervals increased the precision of deviations from the trendline analysis, see below. To obtain the initial and final oxygen concentration for each of the periods, the measurements for the first and last 15 min were averaged (to neutralize the effect of short-term fluctuations in measurements) and converted per unit mass, by taking into account the body mass of the particular animal, and time. In the blank chambers the measurements did not indicate any unexpected oxygen consumption, hence blank chambers were not included in the analysis.
The variability of oxygen consumption derived from calculating the average deviation from the regression curve calculated separately for each animal. Raw data were log transformed and analyzed by ANOVA and LSD post-hoc test.
In the first 15 min of observation, conspecific kairomones had a significant effect on time the experimental individuals spent immobile (Kruskal–Wallis test: H (
Graph: Figure 2Time spent immobile (A, B) and time spent in refuge (C, D) by I. elegans exposed to different concentrations of kairomones (C, D2, D5) or kairomones with alarm signal (D5 + AS); Median (circle), 1st and 3rd quartiles (box), min/max (whiskers), *denotes treatments significantly different from the control treatment, C.
In the first 15 min, conspecific kairomones with alarm signal had a significant effect on time spent in refuge (Kruskal–Wallis test: H (
In the first 15 min, there was a significant effect of conspecific kairomones with alarm signal on the total number of attacks (Kruskal–Wallis test: H (
Graph: Figure 3Total number of attacks on Daphnia by I. elegans exposed to different concentrations of kairomones (C, D2, D5) or kairomones with alarm signal (D5 + AS) in the first 15 min (A), and the last 15 min (B) of the experiment; Median (circle), 1st and 3rd quartiles (box), min/max (whiskers); Percentages of successful attacks are given over the whiskers; *denotes treatments significantly different from the control treatment (C).
The number of consumed Daphnia was not significantly different between treatments (Kruskal–Wallis test: H (
Graph: Figure 4Number of Daphnia consumed by I. elegans exposed to different concentrations of kairomones (C, D2, D5) or kairomones with alarm signal (D5 + AS); Median (circle), 1st and 3rd quartiles (box), min/max (whiskers).
The presence of conspecific chemical cues had a significant effect on time spent in immobility on suction cup (RI-C), on immobility, slow movements and treading within arena (AI, AM-M, AM-T), and on total time spent immobile and in refuge (Anova on GLS results: Chi
Table 1 Analysis of deviance table (type II tests) for linear models fitted using generalized least squares (GLS) and p values for pairwise comparisons with control treatment using Wilcoxon rank sum test with Holm p value adjustment for time I. elegans exposed to different concentrations of kairomones (C, D2, D5) or kairomones with alarm signal (D5 + AS) spent in different behaviors, immobility, and refuge during the whole analyzed time.
Behavior df χ2 D2 D5 D5 + AS RI-P 3 3.74 0.291 0.300 1.000 0.420 RI-C 0.126 0.079 RM-L 3 1.77 0.622 0.650 1.000 0.710 RM-P 3 2.18 0.535 0.680 0.110 0.110 RM-C 3 2.50 0.476 1.000 0.820 1.000 AI AM-G 0.001 0.106 AM-T 0.127 0.070 AM-S 3 1.85 0.605 1.000 0.920 1.000 Immobility Refuge 0.054
Designation of letters in behavioral abbreviations: R Refuge, A Arena, I Immobility, M Mobility, P Plant, C Suction cup, L Leaning, T Treading, G Gentle, S Swimming. Immobility and Refuge—total time spend immobile or in refuge. Significant values are in bold.
In the first 15 min, control individuals displayed all behaviors quite evenly (Fig. 5A). They mostly stayed immobile or gently moved through the arena (AI and AM-M), and least often leaned out in search of prey (RM-L) or trod on suction cup (RM-T) in refuge. Behavioral transitions were frequent and occurred almost between all types of behaviors, yet often between immobility and moving forward. The arena-refuge boundary was often crossed. In the last 15 min (Fig. 5B), the diversity of the behavioral transitions decreased and the arena-refuge boundary was crossed only sporadically.
Graph: Figure 5Behavioral patterns of experimental I. elegans larvae in each of the four treatments: C (A, B), D2 (C, D), D5 (E, F) and D5 + AS (G, H). The size of the bubble is proportional to the time spent on the behavior. The thickness of the arrow is proportional to the number of transitions between behaviors. Green colors indicate behaviors considered safe (in refuge), and red-yellow colors are for ones considered dangerous (in the open arena) for the odonates.
The behavioral pattern of larvae exposed to information on low population density of conspecifics (D2, Fig. 5C) was similar to the control pattern, though it was less movement-intense and the differences between first and last 15 min were less pronounced. The larvae transitioned between many different behaviors, yet they spent more time immobile in refuge (RI-C, RI-P) than control ones. The arena-refuge boundary was crossed a few times. In the last 15 min (Fig. 5D), D2 individuals spent less time in the arena than in the first 15 min.
The behavioral pattern of larvae exposed to information on high population density (D5), in the first 15 min (Fig. 5E) appeared to be simplified compared to the control. Stalking behaviors (RM-L and RM-T) were not observed. The behavioral pattern was mainly limited to transitions between immobility on cup and mobility in plant in refuge (RI-C and RM-P) and between immobility and gentle movements in the arena (AI and AM-G). The former were more frequent and the latter less frequent compared to control. The arena-refuge boundary was crossed sporadically. In the last 15 min (Fig. 5F), D5 individuals stayed in refuge and their behavioral pattern was almost exclusively limited to the above refuge transitions (between RI-C and RM-P).
The larvae exposed to information on high population density with alarm signal (D5 + AS), in the first 15 min (Fig. 5G) spent most of the time immobile in refuge. As above, most transitions occurred between immobility on cup and mobility in plant (RI-C and RM-P), but transitions between mobility on cup and leaning out were also included (RM-C and RM-L). The refuge-arena boundary was crossed only at times. In the last 15 min (Fig. 5H), mobility within refuge increased. Also, the use of the arena increased. There, the transitions took place between immobility and gentle movements or treading (AI and AM-G or AM-T). The refuge-arena boundary was crossed a few times.
Oxygen consumption of experimental larvae was not significantly different between treatments in any of the four analyzed time intervals (ANOVA time: F (
Graph: Figure 6Mean oxygen consumption (O 2 μmol/ individual mass (mg)) (A), and mean oxygen consumption variability (B) of I. elegans exposed to different concentrations of kairomones (C, D2, D5) or kairomones with alarm signal (D5 + AS). I–IV consecutive time intervals lasting three hours each. The bars indicate standard deviation. * denotes treatments significantly different from the control treatment (C) according to LSD (p < 0.05).
A significant effect of time and conspecific cue type on oxygen consumption variability was observed (ANOVA time: F (
The role of chemical cues in cannibalistic interactions is more complex than in interspecific predation, yet, it is less understood. Most studies focused on the effects of conspecific alarm signal (AS, i.e. compounds released by injured individuals[
We approached this gap and demonstrated that I. elegans odonate larvae decrease their activity in response to conspecific chemical cues—both kairomones and kairomones with alarm signal (Fig. 2A). This indicates recognizing those cues as danger and triggering anti-predator defense, since decreased activity was observed previously in the presence of interspecific predators[
The behavioral experiment demonstrated that I. elegans larvae can recognize conspecific kairomones, as indicated by increased time spent immobile and increased refuge use (Table 1) in kairomones-exposed individuals compared to control. The reaction to kairomones was significant immediately after their application, i.e., within the first 15 min of exposure. (Fig. 2). Reduced foraging activity (Fig. 3) and increased refuge use lower the risk of being detected and thus can serve as predator avoidance strategy[
Such a reaction to conspecific chemical cues (kairomones and AS) can be related to the odonate larvae's ambush-hunting strategy. They usually remain immobile on habitat structures while waiting for an apparent visual or mechanical stimulus (i.e. rapid movement) from potential prey, after which they either begin to slowly approach the prey or wait for it to come closer by itself[
Alternatively, the change in foraging strategy in response to conspecific chemical cues may also result from the expected increase in competition. For example, damselfly larvae, during an encounter with a competitor, exhibit aggressive postures and strike each other with their labium until one individual swims away. Such aggressive behavior incurs metabolic costs, which can subsequently affect growth rates[
The risk of cannibalistic interaction increases with population density[
The behaviors of individuals exposed to low density of conspecifics (D2), indicating low risk of dying due to cannibalistic predation, were similar to behaviors in control. Yet, these individuals appeared to stay more in refuge and immobile (Table 1). The frequent transitions from immobility to gentle movements or treading in both control and D2 individuals indicate a 'stop-and-go strategy' (actively following the prey)[
The behaviors of individuals exposed to high kairomone concentration (D5), indicating high probability of death due to cannibalism, differed from the control. D5 individuals balanced between hunting in refuge and arena, however, towards more time spent immobile in refuge and less time mobile in arena compared to control group (Table 1). Our results suggest that D5 individuals also simplified their behavioral pattern (Fig. 5E). The perceived predation pressure here was strong enough to make the more efficient arena-hunting less profitable than in C, well visible especially shortly after receiving the cue. Also the percentage of successful attacks seemed highest in this experimental group (78% vs. ≤ 13% in other treatments, though not statistically significant).This, combined with observations of the behaviors, indicates that these larvae attacked almost only when catching the prey was most likely successful (Fig. 3A). Still, they consumed fewer Daphnia during the experiment than control individuals (Fig. 4).
D5 + AS individuals, i.e. exposed to cues of high mortality risk indicated not only by high population density but also by actively foraging cannibals, did not balance between arena and refuge behaviors and chose only safe low-mobility foraging in refuge. They spent more time in refuge and immobile, than the control group. (Table 1), which was evident in the first 15 min of the experiment (Fig. 5G), when the larvae did not attack even once (Fig. 3A). Similarly to I. elegans responses observed here, feeding and respiratory changes of Daphnia magna studied as prey were also strong when they were exposed to a mixture of kairomones from predators and alarm cues from conspecifics[
The results of the respiratory experiment showed no significant effect of conspecific chemical cues on I. elegans oxygen consumption (Fig. 6A). Also Kolar found no increased respiration rate under predator pressure in Ischnura larvae[
In summary, we showed that 1) I. elegans larvae recognized conspecific chemical cues as a signal about danger or expected increase in competition; 2) Conspecific alarm signal applied with kairomones increased the intensity of refuge use and immobility in I. elegans.; 3) Individuals exposed to conspecific chemical cues balanced food intake and risk avoidance compared to non-exposed individuals, which was observed in behavioral changes.; 4) Oxygen consumption of I. elegans did not change in response to conspecific chemical cues. However, I. elegans changed their respiratory-related behavior by –increasing the variability of oxygen consumption, which may indicate compensation for the inability to perform respiratory movements other than rectal ones in the face of danger.
This study was supported by a grant from the National Science Centre, Poland, project no: 2018/31/N/NZ8/03800
M.S.: designed and implemented the experiments, wrote the first version of the manuscript, managed the project, and acquired funding. B.P.: conducted the data and statistical analysis, reviewed and edited the manuscript, and helped write the final version. M.K.: implemented the behavioral experiment. A.B.: helped develop the experimental protocol and implement the respiratory experiment, and reviewed and edited the manuscript. A.M.: designed the experiments, conducted the data and statistical analysis, and reviewed and edited the manuscript.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request (contact: ma.sysiak@student.uw.edu.pl).
The authors declare no competing interests.
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By Monika Sysiak; Barbara Pietrzak; Matylda Kubiak; Anna Bednarska and Andrzej Mikulski
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