Environmental water flow can boost foraging success of the juvenile rapa whelk Rapana venosa (Muricidae) in aquaculture tanks with still or flowing water: Indication of chemosensory foraging

Aquaculture 513 (2019) 734392

Contents lists available at ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aquaculture

Environmental water flow can boost foraging success of the juvenile rapa whelk Rapana venosa (Muricidae) in aquaculture tanks with still or flowing water: Indication of chemosensory foraging

T

Zheng-Lin Yua,b,d, Nan Hue, Mei-Jie Yanga,b,c,d, Hao Songa,b,d, Zhi Hua,b,c,d, Xiao-Long Wangf, ⁎ Cong Zhoua,b,c,d, Zhi-Xin Zhangg, Tao Zhanga,b,d, a

CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China University of Chinese Academy of Sciences, Beijing 100049, China d Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China e Aquatic Ecology, Department of Biology, Lund University, Lund, Sweden f Marine Biology Institute of Shandong Province, Qingdao 266000, China g The ocean and fisheries bureau of rongcheng shandong, Rongcheng 264300, China b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Artificial breeding Foraging behavior Search time Velocity of water flow

Artificial breeding of Rapana venosa has been attempted in China, but the high mortality rate of rapa whelk juveniles (10–40 mm) seriously restricts the breeding success of this species in artificial cultivation and the overall aquaculture industry, and thus the scale of industrialization is far from being realized. One main factor was found to contribute to this high mortality rate: the low predation efficiency of juveniles. We studied the foraging behavior of various sized R. venosa juveniles in still, flowing, and circulating water, with the juveniles being positioned either upstream or downstream from the prey in the flowing water experiments. Our findings demonstrated that the distance between juveniles and prey in still water significantly restricted the ability of juveniles to locate food, but water flow significantly enhanced this ability. In addition, the small-sized juveniles were found to be more active predators than the larger sized juveniles. Our findings demonstrated that circulating water flow is important to improve the survival and growth rate of juveniles in R. venosa cultures. Our results broaden the understanding of chemical orientation in gastropods and can be used to develop or improve commercial breeding strategies for R. venosa.

1. Introduction Rapana venosa (Valenciennes, 1846) is an important economic species that accounts for > 20% of gastropod production in China (Song et al., 2016a). However, due to overexploitation, habitat destruction, and other threats, its natural resources are seriously declining (Song et al., 2016b). Therefore, it is necessary to conduct artificial breeding, aquaculture, and resource recovery, and as a result, artificial seedling rearing of R. venosa has attracted wide attention. The artificial breeding of Rapana venosa has not realized industrialized production, and there is no related research on the intermediate cultivation of juvenile Rapana venosa (Pan et al., 2013; Song et al., 2016a). Herbivorous plankton larvae metamorphose into carnivorous juveniles in the life cycle of R. venosa (Pan et al., 2013). After

metamorphosis, the growth rate of juvenile snails was fast and can grow to 7–10 mm in about 10 days (Production data, unpublished). Enough food supply can reduce the cannibalism rate of juvenile snails (1.5 mm – 10 mm), and the mortality of juvenile snails is low, and the cannibalism rate decreased significantly with the increase of juvenile snail size (Yu et al., 2018). When the snail grew to > 10 mm, we adopt the method of putting the juvenile snails in a culture pond, and feeding living clams. It was found that the juvenile snails with shell height 10–40 mm had a high mortality rate (30%–50%, production data, unpublished). We speculated that this had nothing to do with cannibalism, and the reason may be: (1) the predation efficiency of juvenile snail to clam was low; (2) planktonic larvae are fed on planktonic food and enough carnivorous food for benthic juvenile were not provided in time. This leads to low emergence efficiency and an undesirably small scale of

⁎ Corresponding author at: CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. E-mail address: [email protected] (T. Zhang).

https://doi.org/10.1016/j.aquaculture.2019.734392 Received 7 January 2019; Received in revised form 7 August 2019; Accepted 8 August 2019 Available online 09 August 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

industrialization, which seriously restricts the development of its aquaculture industry. Water flow is one of the most important environmental driving factors affecting feeding behavior of aquatic animals (Pan et al., 2015). Animals usually use chemical cues released by prey and current conditions to prey (Kamio and Derby, 2017). According to many freshwater studies, increased water flow can negatively affect the ability of predators to locate prey and can result in unsuccessfully capture (Powers and Kittinger, 2002). Similarly, extensive studies conducted on crustaceans in different flow conditions, revealed the same findings (Weissburg and Zimmer-Faust, 1993, 1994). Powers and Kittinger (2002) demonstrated that blue crabs (Callinectes sapidus) had diminished search success in increased turbulent water flow. However, Moore and Grills (1999) highlighted that crayfish can track the source of chemical cues in different turbulent flow structures, thus increasing their predation efficiency. And the hydrodynamics of different environment can influence the chemical orientation strategies of the crayfish Orconectes virilis (Moore et al., 2015). Moore et al. (1991) found that the lobster, Homarus americanus, can make initial directional choices in turbulent flow by lateral antennules and use chemical signals to locate food Drolet and Himmelman (2004) demonstrated that sea star Asterias vulgaris has the ability that is dependent on hydrodynamic conditions to locate prey by chemodetection. Other studies have demonstrated that gastropods can also accurately detect and locate prey positions by using chemical cues in water flow (Ferner and Weissburg, 2005; Kamio and Derby, 2017; Murphy, 2001). In contrast to crustaceans, gastropods are slow-moving animals that search for similar prey in similar habitats but use completely different sensors and behavioral strategies (Ferner and Weissburg, 2005) slow-moving whelks use temporal sampling, while fast-moving crustaceans use spatial sampling to foraging in water flow (Powers and Kittinger, 2002). Thus, it is important to understand how juveniles find food to be able to identify ways to improve their feeding efficiency, their survival rate, and their growth rate in intermediate cultures. Therefore, the present study investigated the foraging behavior of R. venosa juveniles in still and flowing water. 2. Material and methods 2.1. Larval culture and holding methods Adult Rapana venosa broodstock were collected from Laizhou Bay (37°17′7″N, 119°35′10″E) in Shandong Province. Culturing of parental whelks, as well as mating, spawning, hatching, and larval rearing were carried out based on Yang et al. (2007). After settling and undergoing metamorphosis, the planktonic larvae grew into juveniles that reached 40 mm (shell length, SL) after an additional 70 ± 2 days of culturing. All experimental juveniles were cultured in laboratory conditions (water temperature: 24–26 °C, salinity: 29.8–31.7‰ with constant aeration) and fed Ruditapes philippinarum bivalves (15 ± 0.5 mm, SL) for 5 days prior to experimentation. To standardize their hunger level, juveniles were starved for 96 h before the experiments were conducted.

Fig. 1. The experimental set up (tank) used in still and flow through water system (A. perspective drawing, B. top view) and circulating water system (C. perspective drawing, D top view).

upper end, the middle and the lower end of the experimental water flow with a flow meter (INFINITY-EM AEM-USB, JFEHoldings Inc., Japan). The behavior of the juveniles was recorded at 5-s intervals using time lapse cameras (Brinno TLC-200). 2.3. Experimental design 2.3.1. Still and flowing water Foraging behavior was examined in three different water environments, namely: still water, flow through water system (Fig. 1 A, B), and circulating water system (Fig. 1 C, D). In the experimental setups of the still water and flowing water environments (outflow water did not come back to inflow), the juvenile predators and prey were separated by five different distances (S): 0.5, 1, 2, 3, and 4 m. Select the active juvenile snails for the experiment, such as the moving or attached juveniles. A total of 30 R. philippinarum (15 ± 0.5 mm, SL) were introduced as prey, after which 30 (20 ± 2 mm, SL) R. venosa juveniles were introduced into each distance group. The prey was fixed with thin lines to prevent its movement affecting the result (Wrap a round fence

2.2. Experimental facilities The experiments of still water and flowing water were conducted in a straight tank (6 m × 0.3 m × 0.3 m, Fig. 1 A, B). The experiments of circulating water were conducted in an annular tank (5 m × 0.3 m × 0.3 m, Fig. 1 C, D). Two baffles with mesh were set in up and down stream part of the experimental area. No other substrates were placed at the bottom of the tank. Seawater enters and leaves through 20 holes in the head and rear of the tank, respectively, by water pumps. The experimental water depth was 5 cm. Frequency converters were connected to pumps to adjust water velocity at 0.8, 4.5, 9.5 and 14.5 cm s−1. The average water flow velocity was measured at the 2

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

successful foraging behavior and the foraging time as valid. The recording began after juvenile snails and prey were placed in the tank. In this study, the duration of foraging behavior was defined as the total time taken from beginning of recording by camera until a prey item was encountered and captured by R. venosa (search time). In this process, the juveniles show two states: static immobility and walking. Therefore, we divide the search time into residence time (Total time without walking) and activity time (Total of walking time). If a juvenile moves along a wall and finds its prey when passes through, it was not a successful foraging behavior. When the predator avoided the prey, the recording terminated and used as a sequence of behavior. The number of successful foraging juveniles and their search time were counted to calculate the ratio of successful foraging and average search time, as follows:

with three lines, diameter 10 cm, high 0.5 cm). In addition, the experimental setup of the flow through water system included a set of upstream and downstream predator experiment in each distance group. The flow rate was maintained at 4.5 ± 0.5 cm s−1. Upstream predator experiment: a. initial position of juveniles, b. initial position of prey; Downstream predator experiment: a. initial position of prey, b. initial position of juveniles (Fig. 1 B). To check the influence of flow (without prey) on foraging behavior of juveniles in the flow through water system, controls were setup that involved the same experimental groups and conditions without the addition of prey (Fig.1 A, B). Based on the position of juvenile snails, the experimental area was divided into upstream and downstream regions. Each treatment was conducted for 12 h, i.e., a sufficient amount of time for juveniles to locate prey. Treatments were replicated three times and different juveniles were used in all experiments. The experimental tank was cleaned every day. Due to circulating water carried back molecules released from the prey sometime ago with dilution in the moving water, circulating water system was setup to study the foraging behavior of R. venosa juveniles in circulating water. The flow through water system and circulating water system involved a distance of 1 m between the juvenile predators and prey, and a set of downstream predator experiment. The flow rate was maintained at 4.5 ± 0.5 cm s−1. Controls were setup that involved the same experimental groups and conditions without the addition of prey. The experimental area was divided into four areas: A = water inflow area, B = prey (Downstream predator), or blank (control), C = predator, D = water outflow area (Fig. 1 D).

M=

n × 100% N

where M is the ratio of successful foraging, n is the total number of successful foraging, and N is the total number of juveniles. ∑n ti

T = ni (i = 1, 2, 3, …, n)where T is the average search time, n is the total number of successful foraging, i is the number of successful foraging for prey juveniles, and ti is the search time of the juvenile snail i. 2.5. Statistical analysis The data were tested for normality (Shapiro-Wilk test) and homogeneity (Levene's test). The direction of movement of juveniles in the still and flowing water environments was analyzed using an Independent Samples t-Test. Data related to the ratio of successful foraging in different distance and water flow groups was analyzed using a two-way analysis of variance (ANOVA). When the interaction was significant, one-way ANOVA was used to compare the distance effect at each flow, and the flow effect at each distance. Difference in search time among flow through water system, and difference in ratio of distributions in different areas among circulating water system, and difference in ratio of successful foraging and search time among various flow velocity groups, and among various juvenile size groups were analyzed using one-way ANOVA, respectively.; Tukey's post-hoc test was performed to identify significant differences among groups. All statistical computations were conducted using SPSS v. 16.0 software (SPSS Inc., Chicago, IL) and α-values = 0.05 were considered to be statistically significant.

2.3.2. Flow velocity To assess the effect of flow velocity in the flow through water system (The tank was Fig.1 A, B), four different flow velocities were used: 0.8 ± 0.5 cm s−1, 4.5 ± 0.5 cm s−1, 9.5 ± 0.5 cm s−1, and 14.5 ± 0.5 cm s−1. Experiments were conducted as previously described downstream predator condition with R. venosa juveniles (n = 30) and R. philippinarum prey (n = 30) that were introduced 1 m apart. Successful foraging rate and search time were recorded for each experiment. 2.3.3. Size of juveniles To assess the effect of the size of the juveniles in the still water environment, three different sized juveniles were used: 10 ± 2 mm, 20 ± 2 mm, and 40 ± 2 mm. Experiments were conducted as previously described with R. venosa juveniles (n = 30) and R. philippinarum prey (n = 30) that were introduced 1 m apart. Successful foraging rate and search time were recorded for each experiment.

3. Results

2.4. Collection of behavioral data

3.1. Still and flowing water

Successful foraging behavior comprises a variety of different behaviors, namely searching for food, encountering, capturing and testing the food if it is fine to eat (Kamio and Derby, 2017), and consuming prey (Wong and Barbeau, 2003). Rapana venosa has been shown to either avoid or capture prey after they encounter a prey item (Hu et al., 2016). In the present study, when a prey item was avoided, we deemed it a failed foraging behavior. When a prey item was captured, we deem it a

3.1.1. The effect of flow on behavior of the predator in absence of prey Presence and absence of water flow did not affect movement behavior of juveniles in absence of prey. The direction of movement of the juveniles in the still and flowing water environment when prey was not provided was shown in Table. 1. There was no significant difference in the average ratio of R. venosa juveniles in the upstream region (t = 0.72, P = .51), downstream region (t = 0.33, P = .76), and initial position (t = 0.67, P = .54) between the still and flowing water

Table 1 The average ratio and movement distance of Rapana venosa juveniles in the still and flowing water environment at different regions when prey was not supplied. Still water

Upstream region Downstream region Initial position

Flow water

Average ratio

Movement distance/m

Average ratio

Movement distance/m

44.44% 40.00% 15.56%

1.47 1.58 0

40.00% 42.22% 17.78%

1.41 1.37 0

3

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

Fig. 2. Ratio of successful foraging in different distance groups in the still and flowing water environments. In the flowing water experiments, Rapana venosa juveniles were positioned either upstream or downstream from the prey. Bars represent mean values. Error bars indicate standard errors. Fig. 3. The search time, residence time and activity time of Rapana venosa juveniles spent foraging for prey in still and flowing water environments (i.e., the juveniles were positioned either upstream or downstream from the prey and the juveniles and prey were introduced at 3 m from each other). Bars represent mean values. Error bars indicate standard errors. The different lowercase letters (a, ab, b, c) indicate significant differences between the mean values (P < .05).

environments. There was no significant difference of movement distance in the upstream region (t = 0.80, P = .38), downstream region (t = 1.37, P = .25) between the still and flowing water environments. 3.1.2. Distance Ratio of successful foraging differed significantly among the different distances (F 4, 30 = 37.60, P < .001) and among the different types of water flow (still, downstream, and upstream) (F 2, 30 = 878.60, P < .001) (Fig. 2). Distance and flow had significant interactive effects on the ratio of successful foraging (F 8, 30 = 4.44, P < .01). In the still water environment, the foraging behavior was mainly affected by the distance between the juvenile predators and prey (F 4, 10 = 22.73, P < .05, Fig. 2). The ratio of successful foraging decreased as the distance increased. The ratio of successful foraging of the 0.5 m group was higher than that of the 1 m (P < .05), 2 m (P < .05), 3 m (P < .05), and 4 m (P < .05) groups. The ratio of successful foraging of the 1 m group was higher than that of the 3 m (P < .05), and 4 m (P < .05) groups, and there was no significant difference when the distance was > 2 m (P > .05). Most juveniles (84.43% - 91.11%) did not locate food at distance 2-4 m. In the flowing water environment, the direction of water flow significantly affected the foraging behavior of the juveniles (Fig. 2). When downstream predator experiment, there was no significant difference in ratio of successful foraging (F 4, 10 = 3.18, P > .05) in different distanc groups.. The downstream flow significantly enhanced the foraging success of the juveniles. Even when the distance between the juveniles and prey was 4 m, the ratio of successful foraging was still as high as 78.89%, which was higher (P < .05) than that of the 0.5 m group in the still water environment (51.11%). The ratio of successful foraging decreased as the distance increased in the upstream predator experiments (F 4, 10 = 19.45, P < .05). The ratio of successful foraging of the 0.5 m group was higher than that of the 1 m (P < .01), 2 m (P < .01),3 m (P < .01), and 4 m (P < .01) groups. The ratio of successful foraging of the 1 m group was higher than that of the 4 m (P < .05) groups, and there was no significant difference when the distance was > 2 m (P > .05). When the distance was < 3 m, only a few juveniles (3.33%–25.55%) found and captured their prey by walking for a long time. None of the juveniles found and captured prey when the distance was 4 m. When at the same distance groups, ratio of successful foraging differed significantly (0.5 m: F 2, 6 = 48.25, P < .05; 1 m: F 2, 6 = 235.24, P < .05; 2 m: F 2, 6 = 150.03, P < .05; 3 m: F 2, 6 = 2494, P < .05;

4 m: F 2, 6 = 553.88, P < .05;)in different types of water flow (still, downstream, and upstream). The result of posthoc test were: The ratio of successful foraging of the downstream groups was higher than that of the still (P < .05) and upstream (P < .05) in 0.5 m, 1 m, 2 m, 3 m, and 4 m groups, respectively. The ratio of successful foraging of the still groups was higher than that of the upstream (P < .05) in 0.5 m, 1 m, 3 m, and 4 m groups, respectively. 3.1.3. Search time The search time of successful foraging differed significantly in different types of water flow (still, downstream, and upstream) (F 2, 6 = 42.22, P < .001) at 3 m distances between the juvenile predators and their prey (Fig. 3 A). The search time was significantly shorter in downstream predator experiment than that in still (P < .05) and upstream predator experiment (P < .05). And the search time was significantly shorter in still water experiment than that in upstream predator experiment (P < .05). The residence time (Fig.3 B) and activity time (Fig.3 C) of juveniles differed significantly among the different types of water flow (F 2, 6 = 50.78, P < .001; and F 2, 6 = 5.59, P < .05, respectively). The residence time was significantly shorter in downstream predator experiment than that in still (P < .05) and upstream predator experiment (P < .05). And the residence time was significantly shorter in still water experiment than that in upstream predator experiment (P < .05). The activity time was significantly shorter in downstream predator experiment than that in still water experiment (P < .05). The shortest residence (19.53 min) and activity time (16.44 min) were recorded in the downstream predator experiment. The longest residence time (620.33 min) was recorded in the upstream predator experimentand the longest activity time (82.66 min) was recorded in the still water experiments. 3.1.4. Motion path The motion paths of successful foraging behavior in different types of water flow (still, downstream, and upstream) at 4 m distances between the juvenile predators and their prey are shown in Fig. 4. In the 4

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

Fig. 4. The motion paths of the foraging success of Rapana venosa juveniles in different types of water flow (still - A, upstream - B, and downstream - C) at 4 m distances between the juvenile predators and their prey. The juveniles were positioned either upstream or downstream from the prey. The different colored fine lines represent the different foraging success of each repeating group. The big black dots on the right and left indicate the initial position of R. venosa and prey, respectively. The time between the small dots on the fine lines is 5 min. The numbers > 5 indicate the residence time of R. venosa and the numbers < 5 indicate the activity time between the last two dots (i.e., the big black dot on the left and the last dot on the fine line).

still water environment, the motion paths overlapped and was complicated and confused (i.e., the first half of the moving path was overlapped and the latter half was more linear). The motion characteristics of the juveniles are repeating stop and walk cycle. Some R. venosa were observed to pass the prey and then return to capture it. Different from the still water environment, the motion paths in the upstream predator environment demonstrated more linearity. The motion characteristics of the juveniles are that they stop for a long time and walk less. In contrast, the motion paths in the downstream predator environment were obviously linear and directed towards the prey. The motion characteristics of the juveniles are that they just stop for a period of time at the beginning of the experiment, then walks towards the prey without stopping. 3.2. Circulating water Fig. 5. Ratio of Rapana venosa juveniles in different regions of different water flow system. Bars represent mean values. Error bars indicate standard errors. The different lowercase letters (a, b, c, cd, d) indicate significant differences between the mean values (P < .05).

We found that the juveniles moved to the direction where the prey is (either downstream or via the inflow, Fig. 5). When downstream predator experiment in the circulating water, the ratio of juveniles were able to locate their prey successfully was 74.44%, while the ratio of juveniles moved to the water inflow area was 25.56% (i.e., the inflow of water that carried the prey odor). No juveniles were found in the water 5

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

Fig. 6. Ratio of successful foraging (A) and search time (B) of Rapana venosa juveniles under different flow velocities (means ± velocity range). The predators and prey were introduced 1 m apart. Bars represent mean values. Error bars indicate standard errors. The lowercase letter (a) indicates that the means are not significantly different (P > .05).

Fig. 7. Ratio of successful foraging (A) and search time (B) of different sized Rapana venosa juvenile groups (mean ± size range) in the still water experiments. The predators and prey were introduced 1 m apart. Bars represent mean values. Error bars indicate standard errors. The lowercase letters (a, b, c) indicate that the means are significantly different (P < .05).

outflow and predator areas. When flow through water system, the ratio of juveniles were able to locate their prey successfully was 88.89%, which was not significantly different from that of circulating water system (P = .11), while the ratio of juveniles moved to the water inflow area was 5.55%, which was significantly lower than that of circulating water system (P < .05). In contrast to the above two situations, the ratio of the juveniles in the still water control (i.e., without food) stayed in the predator area for a long time was 43.33%, which was significantly higher than that of circulating water system (0%, P < .05) and flow through water system (1.11%, P < .05), and these juveniles showed less activity while the others showed a short period of movement followed by apparent inactivity. There was no significant difference in the distribution ratio of juvenile snails in the other three areas (water inflow = 20.00%, water outflow = 17.18%, prey = 18.89%, P < .05).

successful locating prey (F 3, 8 = 0.73, P = .56, Fig. 6 A) and the search time (F 3, 8 = 0.17, P = .91, Fig. 6 B) did not differ significantly among the different flow velocities. Even in the fast water flow, juveniles were still able to accurately locate their prey. The tracking behavior did not vary greatly with the acceleration of the flow rate. 3.4. Size Ratio of successful foraging differed among the different size groups in the still water environment (Fig. 7 A). As the size of the individuals increased, the ratio of successful foraging decreased (F 2, 6 = 63.00, P < .01) and the search time increased (Fig. 7 B, F 2, 6 = 10.45, P < .05). The ratio of successful foraging in 10 mm group was higher than that in 20 mm (P < .05) and 40 mm (P < .01) group. The ratio of successful foraging in 20 mm group was higher than that in 40 mm (P < .01) group. The search time in 10 mm group was shorter than that in 20 mm (P < .05) and 40 mm (P < .05) group. Small-sized juveniles were more active, often moving before feeding, and remained inactive for a short period of time, while the large-sized juveniles spent a longer

3.3. Velocity of flow Successful foraging was independent of flow speed as ratio of 6

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

consumes more energy. However, it is possible that given a longer search time, some of the other juveniles may also have found food. This suggests that the closer the juveniles were to their prey, the higher the locating prey efficiency and when the distance increased to 2 m, the locating prey efficiency was very low.

period of time searching, and most of the juveniles remained near the initial position of the experiment for a longer period of time. 4. Discussion In this study, R. venosa juveniles showed active foraging behavior only when the prey was located at upstream and this is indicating that they used chemical cue to find the prey. The juveniles might use visual and acoustic cues but we assume that they use chemical cues combining with rheological flow direction to locate prey as reported in other marine snails and invertebrates (Ferner and Weissburg, 2005; Kamio and Derby, 2017; Powers and Kittinger, 2002). Distance should work in the same way for auditory and visual tracking. Their ability to sense does not change. What was changed is the information that they can use and the change affected their foraging success. Many aquatic mobile predators rely on chemical cues when foraging for food (Weissburg et al., 2002). The ability to sense chemical cues and to locate the position of prey determines whether the predation is successful (Ferner and Weissburg, 2005), and water flow in aquatic systems can affect these advantages (Benfield and Minello, 1996; Breitburg, 1994; Ferner and Weissburg, 2005; Moore et al., 1991).

4.2. Flowing water To track odor successfully, organisms need to perceive their position relative to the source, e.g., their prey (Wilson and Weissburg, 2012). However, chemical concentration cannot provide dependable information regarding the distance of the source; therefore, organisms use additional information about the hydrodynamics to track the source of the odor (Brönmark and Hansson, 2012; Kamio and Derby, 2017). The Nudibranch Mollusc Tritonia diomedea can use odors and water flow to locate prey, predators, and conspecifics, and rhinophores mediate orientation to flow (Wyeth and Willows, 2006; Wyeth et al., 2006). Fluid environments may provide mechanical stimuli combined with odor cues to provide some measure of the direction of flow (Vickers, 2000) and different organisms adopt different navigation mechanisms (Powers and Kittinger, 2002). For example, male moths are able to track female moths or synthetic sex pheromone sources and use visual cues to assist in their behavior (Vickers, 2000). Salmon use their olfactory system to detect their native streams, and their activities are closely related to the obvious stratification of water (Døving and Stabell, 2003). Marine invertebrates use two main mechanisms to locate chemical sources. Fast-moving and large organisms use spatial sampling strategies, and slow-moving and small organisms use temporal sampling strategies (Weissburg, 2000). As a typical fast-moving animal that uses chemical cues to locate prey, the blue crab has been widely studied under quantitative flow conditions (Powers and Kittinger, 2002; Weissburg and Zimmer-Faust, 1993, 1994). Studies have shown that increased water velocity and turbulence restrain the ability of blue crabs to track odor plumes because they rely on spatial sampling of odor plumes to perceive and locate prey. Due to the increase in turbulent mixing, chemical odors propagated in water at high speed rapidly mix and become diluted which makes it difficult for the crabs to perceive and locate the odorproducing prey (Powers and Kittinger, 2002). In contrast, slow-moving animals, such as whelks, rely on temporal sampling and their slow movement leads to a high degree of temporal integration of odor plumes. Compared to blue crabs, whelks collect temporal averages of chemical concentrations over a period of time to locate prey (Kamio and Derby, 2017; Wilson and Weissburg, 2012). In our study, we thought that juveniles may sense and locate prey using chemical cues in flowing water and that water flow can significantly enhance the ability of the juveniles to perceive and locate food. In the downstream predator experiments, the distance between the predator and prey have little effect on the ability of the predators to locate food. Their movement was characterized by a straight line towards the prey without a large turning angle (Fig. 4 C). They moved quickly towards their prey, featuring a short search time and no stopping. This feature indicated that the juveniles had successfully perceived and located the prey. Compared with 8.89%–51.11% in the still water experiment, the ratio of successful foraging was over 78.89% in the downstream predator experiments. However, in the upstream predator experiments, juveniles exhibited completely different behavioral characteristics. The movement of the juveniles was more random, with no fixed direction, and most of them remained near the initial position for a long time and rarely moved. When the predators were close to the prey, they moved randomly to the vicinity or downstream of the prey. When the distance was far, few juveniles were able to successfully locate their prey (Fig. 4 B). This movement characteristic indicated that the upstream environments made it difficult for R. venosa to successfully perceive and locate prey. Compared with the residence time of juveniles in the still water environment, the residence time of the

4.1. Still water In still water, molecules that leak from live organisms and fresh carrion form a concentration gradient (via molecular diffusion) that is highest closest to the source and lower further away(Webster and Weissburg, 2009; Wyeth, 2019). In such cases, molecular diffusion is the main distributing force of chemical cues and, therefore, the chemical cues and their spatiotemporal gradient are the only cues available to organisms (Vickers, 2000). Slowly evolving and smooth concentration gradients are the two main features of cue structure in still water (Webster and Weissburg, 2009). Under these circumstances, organisms using spatial sampling strategies can determine the direction of a concentration gradient (What they can detect on their chemosensory organ is frequency and intensity of chemical stimuli) to move towards or away from the odor source (Wyeth, 2019). When a concentration gradient exists in still water, gastropods will use either klinotaxis or tropotaxis to orient towards food (Croll, 1983). As distance from the source increases, the odor concentration decreases and it becomes more difficult for gastropods to locate prey. In the present study, we found that juveniles were able to capture prey from 4 m away in still water, which indicated that they have a strong ability for chemosensory perception. In contrast, sea stars (Asterias vulgaris) failed to orient towards prey even at a distance of only a few centimeters away from their prey (Drolet and Himmelman, 2004). In the present study, the motion paths of R. venosa juveniles became more linear towards the source as they approached the odor source. The overlapping of motion paths indicated that R. venosa was searching for prey, while the linearity towards prey indicated that R. venosa had determined the location of the prey. (Fig. 4 A). This phenomenon was consistent with the orientation behavior of sea stars (Asterias forbesi), which decreased their heading angle relative to the odor source as they approached the odor source (Moore and Lepper, 1997). However, it has been shown that when juveniles are searching for prey, the direction of their movement can deviate from the direction of the odor source (Drolet and Himmelman, 2004; Moore and Lepper, 1997). When searching for food, juveniles of R. venosa move their siphon back and forth to adjust their direction of movement. A large heading angle indicates that juveniles are sampling odors to help improve their predation success (Wilson and Weissburg, 2012). When the R. venosa juveniles were < 1 m away from the prey, the ratio of successful foraging was as high as 15.57%–51.11%. However, when the distance increased to 2 m or more, the ratio of successful foraging was as low as 8.89%–15.57% despite the juveniles having spent much more time searching (Fig. 3 A) and being active (Fig. 3C) in pursuit of prey, which 7

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

food types and ability to escape from cannibalism (Larger snails escape better). 4.4. Methods to improve predation efficiency According to the above results, a method to improve the predation efficiency of juvenile snail was proposed, that was, a water pump (JVA202A, SENSEN, CHINA) was placed in two corners of the culture pool, respectively, to produce circulating water flow at the bottom of the pool (Fig.8). The advantages of this type of water pump are: low power (15–24 W), low cost and easy installation. Fig. 8. A water pump used to make circulating water (A) and the position of the water pumps in the culture pool (top view).

4.5. Conclusion

juveniles in the upstream environment was very long (Fig. 3 B), and most of them remained in a static state, possibly to conserve energy. Similarly, when the juveniles detected odor cues in the circulating environment, they followed the odor upstream. This behavior is common and different organisms adopt different navigation mechanisms (Kamio and Derby, 2017). For this slow-moving gastropod, temporal sampling can effectively improve its efficiency in perceiving and locating food. In the downstream predator experiment, circulating water experiment, most of the juveniles located the prey while the others continue to move upstream towards the odor to look for other prey. This indicates that in circulating water, juvenile snails choose prey in different locations rather than concentrated in one place. This behavior was in response to the interaction between the individual prey odor plumes which are more uniform and concentrated (Wilson and Weissburg, 2012), as opposed to the odor that was recycled in the system. Juveniles are able to use temporal sampling to gather information about their position relative to their prey as the odor concentrations change (Webster and Weissburg, 2001). Ferner and Weissburg (2005) found that the strategy of temporal sampling enables whelks to track chemical cues successfully even at high water velocities and increased turbulence. Increased turbulence in water has been shown to improve the odor-tracking abilities of gastropods (Ferner et al., 2009), and the findings of our study were the same as this previous study. As for R. venosa juveniles, their predation efficiency was not affected by different flow velocities which was the result of their temporal sampling strategy. These findings were greatly different from the perceptual behavior of blue crabs (Weissburg and Zimmer-Faust, 1993) but similar to those of sea stars (Drolet and Himmelman, 2004).

The present study investigated the foraging behavior of juvenile R. venosa gastropods in still and flowing water and demonstrated that the predation efficiency of juveniles in still water was low when the distance between the predators and the prey was high, however, water flow enhanced their predation efficiency. Small-sized juveniles were found to be more active in feeding. Accordingly, our study lead us to recommend culture practices that can increase the survival rate and growth rate of juveniles. The recommendation is to provide circulating water flow in the aquaculture pools by placing several water pumps in the corners. Resulting statistics from the actual production data through this method revealed that the survival rate of juveniles can be increased from 30 to 50% to > 90%, and can grow to 3–5 cm (compared to 1–3 cm, without using this method) in 3 months after settlement and metamorphosis (Production data, unpublished). In summary, our findings broaden the understanding of foraging behavior in gastropods and can be used to develop or improve commercial breeding strategies for R. venosa. Acknowledgments This study was supported by the National Natural Science Foundation of China [grant number 31572636], Natural Science Foundation of Shandong Province (Grant No. ZR2019BD003), China Postdoctoral Science Foundation (Grant No. 2019M652498), the earmarked fund for Modern Agro-industry Technology Research System (CARS-49), the Special Funds for Talent Project of Taishan Industry Leader, the ‘Double Hundred’ Blue Industry Leader Team of Yantai. This study was financially supported by the Creative Team Project of the Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology (no. LMEESCTSP-2018-1). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

4.3. Size Smaller juveniles were found to be more active and have high ratio (40.00%) of successful foraging and short search times (Fig. 7). Is the perception ability of the smaller-sized juveniles better than that of the larger-sized juveniles? Larger organisms are advantageous because they can move receptors over larger distances and move faster, and can thus increase their ability to sample gradients across space or time domains (Weissburg, 2000). The reason for the apparent difference in foraging may be that the different stages of aquatic species have different tolerances to hunger; whereby larger individuals are more tolerant of hunger, while smaller individuals need to eat more and regularly to survive (Bougrier et al., 1995; Mehner and Wieser, 1994). In addition, even in the presence of prey, a few small-sized (1.5–10 mm) juveniles may be cannibalized by other juveniles (The cannibalism behavior mainly occurred in the absence of food, and with the increase of juvenile snail size, the cannibalism rate decreased significantly), but this rarely happens and had no important effect on the whole seedling raising process. (Yu et al., 2018). In this study, no cannibalism behavior was found in juvenile snails (10 mm–40 mm). This may be related to the development of their olfactory organs, their ability to identify certain

Author contributions TZ conceived and designed the experiments. ZY and NH conducted the experiments. ZY analyzed the data. MY, HS, ZH, XW, CZ and ZZ contributed the reagents, materials, and analytical tools. ZY wrote the manuscript. Declarations of interest None. Compliance with ethical standards All applicable international, national, and institutional guidelines for the care and use of animals were followed. 8

Aquaculture 513 (2019) 734392

Z.-L. Yu, et al.

References

52–59. Powers, S.P., Kittinger, J.N., 2002. Hydrodynamic mediation of predator–prey interactions: differential patterns of prey susceptibility and predator success explained by variation in water flow. J. Exp. Mar. Biol. Ecol. 273, 171–187. Song, H., Yu, Z.L., Sun, L.N., Gao, Y., Zhang, T., Wang, H.Y., 2016a. De novo transcriptome sequencing and analysis of Rapana venosa from six different developmental stages using Hi-seq 2500. Comp. Biochem. Physiol. Part D 17, 48–57. Song, J.P., Fang, J.B., Song, H., Zhang, T., Ban, S.J., Pan, Y., Li, Z.X., 2016b. Effect of feeding preferences, temperature, and size on food intake of Rapana venosa. Mar. Sci. 40, 48–53. Vickers, N.J., 2000. Mechanisms of animal navigation in odor plumes. Biol. Bull. 198, 203–212. Webster, D., Weissburg, M., 2009. The hydrodynamics of chemical cues among aquatic organisms. Annu. Rev. Fluid Mech. 41, 73–90. Webster, D.R., Weissburg, M.J., 2001. Chemosensory guidance cues in a turbulent chemical odor plume. Limnol. Oceanogr. 46, 1034–1047. Weissburg, M.J., 2000. The fluid dynamical context of chemosensory behavior. Biol. Bull. 198, 188–202. Weissburg, M.J., Zimmer-Faust, R.K., 1993. Life and death in moving fluids: hydrodynamic effects on chemosensory-mediated predation. Ecology. 74, 1428–1443. Weissburg, M.J., Zimmer-Faust, R.K., 1994. Odor plumes and how blue crabs use them in finding prey. J. Exp. Biol. 197, 349–375. Weissburg, M.J., Ferner, M.C., Pisut, D.P., Smee, D.L., 2002. Ecological consequences of chemically mediated prey perception. J. Chem. Ecol. 28, 1953–1970. Wilson, M.L., Weissburg, M.J., 2012. Temporal and spatial sampling strategies maintain tracking success of whelks to prey patches of differing distributions. Anim. Behav. 84, 1323–1330. Wong, M.C., Barbeau, M.A., 2003. Effects of substrate on interactions between juvenile sea scallops (Placopecten magellanicus Gmelin) and predatory sea stars (Asterias vulgaris Verrill) and rock crabs (Cancer irroratus say). J. Exp. Mar. Biol. Ecol. 287, 155–178. Wyeth, R.C., 2019. Olfactory navigation in aquatic gastropods. J. Exp. Biol. 222, jeb185843. Wyeth, R.C., Willows, A.D., 2006. Odours detected by rhinophores mediate orientation to flow in the nudibranch mollusc, Tritonia diomedea. J. Exp. Biol. 209, 1441–1453. Wyeth, R.C., Woodward, O.M., Willows, A.D., 2006. Orientation and navigation relative to water flow, prey, conspecifics, and predators by the nudibranch mollusc Tritonia diomedea. Biol. Bull. 210, 97–108. Yang, D.Z., Zhou, Y.B., Guan, Z.C., Zhao, F.F., 2007. Technique for industrial breeding in Rapana venosa valenciennes. Fish. Sci. 26, 237–239. Yu, Z.L., Wang, H., Song, H., Bai, Y.C., Sun, J.C., Qian, Y.S., Hu, N., Yang, M.J., Zhang, T., 2018. Cannibalism by the juveniles of the gastropod Rapana venosa (Muricidae) reared under laboratory conditions. J. Molluscan Stud. 84, 303–309.

Benfield, M.C., Minello, T.J., 1996. Relative effects of turbidity and light intensity on reactive distance and feeding of an estuarine fish. Environ. Biol. Fish 46, 211–216. Bougrier, S., Geairon, P., Deslouspaoli, J.M., Bacher, C., Jonquières, G., 1995. Allometric relationships and effects of temperature on clearance and oxygen consumption rates of Crassostrea gigas (Thunberg). Aquaculture. 134, 143–154. Breitburg, D., 1994. Effects of low oxygen on predation on estuarine fish larvae. Marine Ecology Progress. 104, 235–246. Brönmark, C., Hansson, L.A., 2012. Chemical Ecology in Aquatic Systems. Oxford University Press, New York. Croll, R.P., 1983. Gastropod chemoreception. Biol. Rev. 58, 293–319. Døving, K.B., Stabell, O.B., R, F.R., 2003. Trails in open waters: sensory cues in salmon migration. In: T, B., J, A., AN, P., WN, T. (Eds.), Sensory Processing in Aquatic Environments. Springer, New York, pp. 39–52. Drolet, D., Himmelman, J.H., 2004. Role of current and prey odour in the displacement behaviour of the sea star Asterias vulgaris. Can. J. Zool. 82, 1547–1553. Ferner, M.C., Weissburg, M.J., 2005. Slow-moving predatory gastropods track prey odors in fast and turbulent flow. J. Exp. Biol. 208, 809–819. Ferner, M.C., Smee, D.L., Weissburg, M.J., 2009. Habitat complexity alters lethal and nonlethal olfactory interactions between predators and prey. Mar. Ecol. Prog. Ser. 374, 13–22. Hu, N., Wang, F., Zhang, T., Song, H., Yu, Z.L., Liu, D.P., 2016. Prey selection and foraging behavior of the whelk Rapana venosa. Mar. Biol. 163, 233. Kamio, M., Derby, C.D., 2017. Finding food: how marine invertebrates use chemical cues to track and select food. Nat. Prod. Rep. 34, 514–528. Mehner, T., Wieser, W., 1994. Energetics and metabolic correlates of starvation in juvenile perch (Perca fluviatilis). J. Fish Biol. 45, 325–333. Moore, P.A., Grills, J.L., 1999. Chemical orientation to food by the crayfish Orconectes rusticus: influence of hydrodynamics. Anim. Behav. 58, 953–963. Moore, P.A., Lepper, D.M.E., 1997. Role of chemical signals in the orientation behavior of the sea star Asterias forbesi. Biol. Bull. 192, 410–417. Moore, P.A., Scholz, N., Atema, J., 1991. Chemical orientation of lobsters, Homarus americanus, in turbulent odor plumes. J. Chem. Ecol. 17, 1293–1307. Moore, P.A., Ferrante, P.A., Bergner, J.L., 2015. Chemical orientation strategies of the crayfish are influenced by the hydrodynamics of their native environment. Am. Midl. Nat. 173, 17–30. Murphy, A.D., 2001. The neuronal basis of feeding in the snail, Helisoma , with comparisons to selected gastropods. Prog. Neurobiol. 63, 383–408. Pan, Y., Qiu, T., Zhang, T., Wang, P., Ban, S., 2013. Morphological studies on the early development of Rapana venosa. J. Fish. China 37, 1503–1510. Pan, Y., Zhang, L., Lin, C., Sun, J., Kan, R., Yang, H.J.P., 2015. Influence of flow velocity on motor behavior of sea cucumber Apostichopus japonicus. Physiol. Behav. 144,

9