Aquatic Toxicology 104 (2011) 5660

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Effects of the pyrethroid fenvalerate on the alarm response and on the vulnerability of the mosquito larva Culex pipiens molestus to the predator Notonecta glauca
Sebastin Reynaldi a,b,c, , Maximilian Meiser a , Matthias Liess a
a

Department System Ecotoxicology, Helmholtz Centre for Environmental Research UFZ, Permoserstrae 15, D-04318 Leipzig, Germany Department Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research UFZ, Permoserstrae 15, D-04318 Leipzig, Germany c Department of Agronomical Science, National University of Colombia, Calle 59a 20-63, Medelln, Colombia
b

a r t i c l e

i n f o

a b s t r a c t
Previous studies have shown that mosquito larvae retreat from the surface of water as an alarm response to sudden changes in light intensity. We investigated the effects of the insecticide fenvalerate on this alarm response in larvae of the mosquito Culix pipiens molestus and on the related vulnerability of these larvae to the predator Notonecta glauca. For the alarm response, after 1 h of exposure to fenvalerate, no immediate effects were observed. However, after a 5-h postexposure period following 1 h of exposure, the proportion of larvae that showed the alarm response decreased with increasing fenvalerate concentration. The median effective concentration (EC50) was 0.1 g/L. After 6 h of continuous exposure, the EC50 decreased to 0.06 g/L. In addition, vulnerability to the predator N. glauca increased after 6 h of continuous exposure. The median time needed for N. glauca to prey 50% of the larvae (PT50) decreased from 5 h 48 min in the control to 3 h 8 min at 0.3 g/L fenvalerate. No mortality occurred after 48 h when larvae were exposed for 6 h in the absence of N. glauca. The median lethal concentration (LC50) was 0.3 g/L after 48 h of continuous exposure. The decrease in the PT50 was related strongly to the increase in the proportion of larvae that did not exhibit an alarm response. These results show that the alarm response can be inhibited by low levels of fenvalerate, and this inhibition seems to increase larval mortality due to predation. 2011 Elsevier B.V. All rights reserved.

Article history: Received 9 February 2011 Received in revised form 21 March 2011 Accepted 27 March 2011 Keywords: Insecticide Mosquito larvae survival Alarm response Vulnerability to predation Short-term exposure

1. Introduction The use of pyrethroid insecticides has increased markedly in recent years (Amweg et al., 2005). Pyrethroids are a good alternative to organophosphate insecticides because they are over 10 times more effective against pests than organophosphates, while at the same time less toxic to mammals, including human beings (Amweg et al., 2005). In particular, the nonagricultural use of pyrethroids, for example to control mosquitoes, has been promoted by these characteristics. Pyrethroids are hydrophobic compounds that can adsorb rapidly onto sediments and vegetation in aquatic environments. This characteristic might decrease their bioavailability to aquatic organisms (Adelsbach and Tjeerdema, 2003). The concentration of fenvalerate in lake enclosures was found to decrease more than 50%

Corresponding author at: Department Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research - UFZ, Permoserstrae 15, D-04318 Leipzig, Germany. Tel.: +49 341 235 1515. E-mail address: sreynaldi@unal.edu.co (S. Reynaldi). 0166-445X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.03.017

after the rst 24 h following a single surface spray (Day et al., 1987). For this reason, it is expected that, when pyrethroids enter aquatic ecosystems, organisms are only exposed to them for a short period. However, short-term exposure to pyrethroids such as fenvalerate can cause long-term effects in aquatic organisms (Liess and Schulz, 1996; Schulz and Liess, 2000). Rapid uptake of fenvalerate can be expected because, in general, pyrethroids are very hydrophobic: the log ko/w for fenvalerate is 6.4 (Adelsbach and Tjeerdema, 2003). Exposure for 1 h was sufcient to cause long-term effects in Limnephilus lunatus: the emergence pattern was affected more by 1 h of exposure to 1 g/L fenvalerate than by 10 h of exposure to 0.1 g/L fenvalerate (Schulz and Liess, 2000). This shows that biological responses might vary more with increasing concentration of fenvalerate than with increasing duration of exposure. However, the median lethal concentration (LC50) of permethrin was 4.67, 1.15 and 0.45 g/L after 1, 4 and 24 h exposure, respectively (Parsons and Surgeoner, 1991). This shows that lethal effects caused by pyrethroids can also increase with duration of exposure in mosquito larvae. Mosquito larvae breathe atmospheric air through tubes with siphonal valves that are located at their tails (Corbet et al., 2000).

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For this reason, mosquito larvae spend a substantial part of their time just under the surface of the water with their head hung downwards. However, sudden changes in light intensity induce the larvae to retreat from the surface; they dive towards the bottom and return to the surface some minutes later (Corbet et al., 2000). This behavior is considered to be an alarm response because it results from the perception of environmental changes (Mellanby, 1958). The frequency of this response can depend on temperature (Mellanby, 1958) or availability of food (Olsson and Klowden, 1998). The alarm response was shown to be related to the avoidance of predation by larvae of the mosquito Anopheles gambiae because the absence of this response increased the vulnerability of these larvae to the wolf spider Pardosa messingerae (Futami et al., 2008). Larvae of the mosquito Culex pipiens showed a specic antipredator response to the backswimmer Notonecta glauca (Sih, 1986). Fenvalerate is a neurotoxic pesticide that affects the ion channels of neurons (Adelsbach and Tjeerdema, 2003), which can affect the behavior of the exposed organisms (Wendt-Rasch et al., 1998). Thus, we hypothesized that the pyrethroid fenvalerate would affect the alarm response and the vulnerability of larvae of the mosquito Culex pipiens molestus to the predator N. glauca. 2. Materials and methods 2.1. Test organism Larvae of C. pipiens molestus were bred in glass cylinders, 10 cm high and 10 cm in diameter. These cylinders contained 100 larvae in 400 ml of Elendt M4 medium (Elendt and Bias, 1990). The cylinders were placed in an temperature-controlled room at 20 1 C in a light: dark regime of 16:8 h, with light intensity of approximately 15 mmol/m2 /s. First-stage larvae were fed daily with three drops of the invertebrate food Liquidzell (Dohse Aquaristik, GrafschaftGelsdorf, Germany). From the second stage onwards, larvae were fed daily with 1.5 cm3 of a 1:1 mixture of dried and powdered leaves of Urtica dioica and ground dog food (Ssniff Spezialitten, Soest, Germany). 2.2. Fenvalerate exposure To investigate the effects of fenvalerate on the alarm response, fourth-stage larvae were exposed to 0, 0.01, 0.03, 0.06, 0.1, 0.3, and 0.6 g/L fenvalerate (CAS: 51630-58-1) in the following schemas: (1) 1 h of exposure; (2) 1 h of exposure followed by 5 h in the absence of toxicant; (3) 6 h of exposure; and (4) 6 h of exposure followed by 18 h in the absence of toxicant. Four groups of 10 larvae were exposed at each concentration as replicates. To investigate the effects of fenvalerate on vulnerability to N. glauca, groups of 100 larvae in the fourth stage were exposed to 0, 0.01, 0.06, and 0.3 g/L fenvalerate for 6 h. Fenvalerate (99.9%) was obtained from Riedel-de Han (Seelze, Germany). A stock solution of 40 mg/L was prepared in dimethylsulfoxide (DMSO; 99.8%; Merck , Darmstadt, Germany). The maximum concentration of DMSO in the test medium was 0.0003% (v/v). The solutions to which the larvae were exposed were analyzed by solid-phase extraction of 1-L volumes with C18 columns (Baker, Phillipsburg, NJ, USA), followed by gas chromatographyelectron-capture detection (CP-3800 Saturn 2200, Varian, Walnut Creek, USA). The actual concentration of fenvalerate was 93.1% 16.5 (mean standard error) of the nominal concentration for the range 0.061 g/L. 2.3. Measurement of effects of fenvalerate on the alarm response After exposure to fenvalerate, the groups of larvae were placed in test tubes (3 cm in diameter, 6 cm high) that contained 40 ml of

Elendt M4 medium (Elendt and Bias, 1990). These test tubes were initially kept in the dark for approximately 5 min. Subsequently, a light source was placed above the test tubes. The intensity of the light source was 20 klux (Olsson and Klowden, 1998). After all the larvae had surfaced and remained motionless, the test tube was covered for a short period of time. The proportion of larvae that dived when the illumination was interrupted was recorded and expressed as a percentage. 2.4. Measurement of effects of fenvalerate on larval survival The effects of the pesticide on larval survival were tested by exposing groups of 10 larvae continuously to 0 (control), 0.01, 0.06, 0.3, and 1 g/L fenvalerate for 6 h or 48 h. In both exposure schemas, three groups of 10 larvae were tested per concentration and the survival was recorded after 48 h. Exposure for 6 h was tested in order to ensure the absence of mortality during the vulnerability experiment due to direct effects of the pesticide. 2.5. Measurement of effects of fenvalerate on vulnerability to N. glauca After exposure to fenvalerate, 100 larvae per concentration were placed in plastic containers (30 cm long, 20 cm wide and 8 cm depth) that were lled to a depth of 5 cm with M4 medium. Three individuals of N. glauca were added per container. These individuals were adults (length: 1516 mm) and had been starved for two days before the experiment. The plastic containers were divided diagonally from the bottom left side to the top right by sheets of plastic (20 cm long, 15 cm high, and 0.5 cm thick) that contained 1.5-cm holes. Although both N. glauca and mosquito larvae could pass through these holes, this setup was intended to mimic natural obstacles such as rocks and vegetation. The density of N. glauca individuals was chosen in accordance with Sih (1986) in order to avoid effects of predator density on the predation process. Every hour for 5 h, the number of living larvae was recorded and N. glauca individuals were exchanged randomly among the containers. 2.6. Statistical analysis The median concentration of fenvalerate that reduced the number of larvae that exhibited the alarm response to 50% (EC50), the median time that N. glauca needed to prey 50% of the larvae (PT50), and the median lethal concentration (LC50) were estimated with the Hill model (1). f (x) = 100 xnH xnH + anH 100 (1)

Here, nH is the Hill number and a was the EC50, PT50, or LC50. The PT50 was related to the mean proportion of the larvae (%) without an alarm response by a linear model. The models were applied on the software SigmaPlot (Systat Software, USA). 3. Results 3.1. Effect of fenvalerate on the alarm response The proportion of larvae that showed an alarm response did not decrease after 1 h of exposure to fenvalerate at any concentration. However, for 1 h of exposure followed by a 5-h postexposure period, the proportion of larvae that exhibited the alarm response began to decrease at a concentration of 0.03 g/L and decreased further at higher concentrations (Fig. 1). In this exposure schema, the median effective concentration (EC50) was 0.1 g/L (Table 1). After 6 h of exposure without a postexposure period, the pro-

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1-h exposure 1h exp

100 80 100 80

6h exp 6-h exposure

Larvae (%)

60 40 20 0

Larvae (%)
Control 0.001
0.01 0.1 1

60 40 20 0

Control 0.001

0.01

0.1

Fenvalerate (g/L)

Fenvalerate (g/L)

1-h exposure + 5-h postexposure 1h exp. & 5h recovery

100 80

6-h exposure + 18-h postexposure 6h exp. & 18h recovery

100 80

Larvae (%)

60 40 20 0 0.001 Control 0.01 0.1 1

Larvae (%)

60 40 20 0 0.001 Control 0.01 0.1 1

Fenvalerate (g/L)

Fenvalerate (g/L)

Fig. 1. Proportion of mosquito larvae that exhibited the alarm response with increasing concentrations of fenvalerate under the following exposure schemas: 1 h of exposure; 1 h of exposure followed by 5 h in the absence of toxicant; 6 h of exposure; and 6 h of exposure followed by 18 h in the absence of toxicant.

Table 1 The EC50 and the Hill number (H), their probabilities of type I error (p), and the coefcient of determination (r2 ) of the logistic regression, which were estimated by applying the Hill equation (1) to the data obtained in the alarm response experiment. Exposure schemas 1-h exp. 6-h exp. 1-h exp. and 5-h post-exp. recovery 6-h exp. and 18-h post-exp. recovery EC50 0.05 0.08 0.33 p 0.0001 0.0001 0.0001 nH 1.53 1.08 0.62 p 0.0001 0.0001 0.0001 r2 0.96 0.89 0.87

portion of larvae that showed the alarm response also started to decrease at a concentration of 0.03 g/L (Fig. 1), but the EC50 was 0.06 g/L (Table 1). The EC50 increased to 0.37 g/L with 6 h of exposure followed by an 18-h postexposure period (Fig. 1 and Table 1). No mortality was observed during this experiment.

3.3. Effect of fenvalerate on larval vulnerability to N. glauca The median time that N. glauca needed to prey 50% of the larvae (PT50) in the control groups was 5 h 48 min. The PT50 decreased to 5 h 36 min and 4 h 27 min when the larvae were exposed to 0.01 g/L and 0.06 g/L fenvalerate, respectively. Exposure to 0.3 g/L fenvalerate decreased the PT50 to 3 h 8 min (Fig. 3 and Table 2). 3.4. Relationship between the alarm response and larval vulnerability The PT50 was signicantly (p < 0.01) related to the proportion of larvae that failed to exhibit the alarm response by the following linear model: y = 0.031x + 5.96. The PT50 and the proportion of larvae that exhibited the alarm

3.2. Effects of fenvalerate on larval survival No mortality had occurred after 48 h when larvae were exposed to fenvalerate for 6 h in the absence of N. glauca. One dead larva was observed after 48 h of continuous exposure to 0.01 g/L fenvalerate, and the median lethal concentration (LC50) was 0.3 g/L (nH = 1.35, p < 0.0001, and r2 = 0.93) (Fig. 2).

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100

Survival after 48 h (%)

80

PT50 (h)

60 40 20 0 0,001 Control 0,01 0,1 1 10

4
y=-0.031x+5.96 r2=0.96 p<0.001

6 h-pulse Continuous

0 0 20 40 60 80 100

Fenvalerate (g/L)
Fig. 2. Number of larvae that avoided predation by N. glauca over time, which was expressed as the number of living larvae. The mosquito larvae were exposed to 0, 0.01, 0.06, and 0.3 g/L fenvalerate for 6 h. Subsequently, the larvae were placed in fresh medium and N. glauca individuals were introduced. The number of living larvae was monitored every hour for 5 h for each concentration.

Laevae (%)
Fig. 4. Proportion of larvae that survived at 48 h after a continuous and a 6-h-pulse exposure to fenvalerate in the absence of N. glauca individuals. For the continuous exposure, larval survival was signicantly (p < 0.001) related to the concentration of fenvalerate by applying the Hill equation (1); the estimated LC50 (48 h) was 0.3 g/L.

100

80

60 Control 0.01 g/L 0.06 g/L 0.3 g/L

40

20 0 1 2 3 4 5

Time (hours)
Fig. 3. Relationship between the proportion of larvae that failed to exhibit an alarm response and the median time that N. glauca needed to prey 50% of the larvae (PT50). The larvae were exposed for 6 h to 0.01, 0.06, and 0.3 g/L fenvalerate. Table 2 The PT50 and the Hill number (H), their probabilities of type I error (p), and the coefcient of determination (r2 ) of the logistic regression, which were estimated by applying the Hill equation (1) to the data obtained in the larval vulnerability experiment. Exposure schemas Control 0.01 g/L 0.06 g/L 0.3 g/L PT50 5 h 48 min 5 h 36 min 4 h 27 min 3 h 8 min p 0.0003 0.0004 0.0001 0.0001 nH 3.8 2.5 2.2 1.9 p 0.0252 0.0122 0.0081 0.0006 r2 0.90 0.93 0.95 0.99

response decreased with increasing fenvalerate concentration (Fig. 4). 4. Discussion Mosquito larvae retreat from the surface of the water upon sudden changes in light intensity, and this was dened as an alarm response (Mellanby, 1958). The results of the present study conrm the existence of this response, and also show that exposure to fenvalerate can affect it. The proportion of larvae that showed an alarm response decreased with increasing concentration of fenvalerate. However, this decrease depended on the exposure schema used (Fig. 1). No effects on the alarm response were observed after

1 h of exposure without a postexposure period. However, after the 1-h exposure followed by a 5-h postexposure period, the proportion of larvae that failed to exhibit the alarm response increased with increasing fenvalerate concentration (Fig. 1). The results of a previous study showed that 1 h of exposure to 1 g/L fenvalerate can be more toxic than 10 h of exposure to a concentration of 0.1 g/L (Schulz and Liess, 2000). Fast uptake can be expected for very hydrophobic pesticides, such as fenvalerate (Adelsbach and Tjeerdema, 2003). However, in the present work, the EC50 was higher for 1 h of exposure followed by a 5-h postexposure period than for 6 h of continuous exposure (Table 1), which suggests that fenvalerate might be taken up for more than 1 h. Nevertheless, the alarm response can also recover; the EC50 was six times higher when the 6-h exposure period was followed by an 18-h postexposure period. Given that the period when the larvae are more vulnerable might correspond to the time between the onset of the effects of fenvalerate and the recovery of the alarm response, vulnerability to N. glauca was tested for 5 h after the larvae had been exposed to fenvalerate for 6 h. The median time that N. glauca needed to prey 50% of the larvae (PT50) decreased with increasing fenvalerate concentration. The PT50 decreased by more than 1 and 2 h when the larvae were exposed to 0.06 and 0.3 g/L fenvalerate, respectively (Table 2), which demonstrated a strong increase in the rate of predation of N. glauca on mosquito larvae. Exposure to fenvalerate was also shown previously to increase the predation of Baetis mayy nymphs by the sh Galaxias zebratus. The activity of the mayies increased after exposure to fenvalerate, which probably made them more visible to the predator (Schulz and Dabrowski, 2001). In the present study, the decrease in PT50 was directly proportional to the proportion of larvae that failed to exhibit the alarm response (Fig. 4), which suggests that exposure to fenvalerate increases the vulnerability of mosquito larvae to N. glauca by affecting their avoidance behavior. Avoidance behavior in response to N. glauca has been observed previously in C. pipiens, and it was suggested that this behavior evolved to reduce the likelihood of capture by N. glauca because C. pipiens and N. glauca co-exist in many aquatic habitats (Sih, 1986). Moreover, this avoidance behavior seems to be genetically determined because larvae of C. pipiens that carried genes for insecticide resistance were found to be more susceptible than wild-type larvae to predators, including the backswimmer Plea minutissima (Berticat et al., 2004). The avoidance behavior that C. pipiens larvae exhibit in response to N. glauca consists of an overall reduction of move-

Number of living larvae

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ment. However, it can also include a change in habitat use, because the larvae were found to cluster close to refuges in the presence of N. glauca (Sih, 1986). This implies that a mosquito larva might restrict its movements in response to a particular factor to increase its survival. Thus, there are two possible explanations for the relationship between vulnerability and the alarm response that was observed in the present work. First, the larvae that exhibited no alarm response might have been easier prey for N. glauca. Second, the decrease observed in the alarm response might be a sign of the loss of general environmental perception, which larvae need to avoid N. glauca. To elucidate which of these explanations is more relevant, future experiments should investigate the effects of exposure to fenvalerate on other aspects of larval behavior, such as feeding activity. Sublethal levels of fenvalerate contamination were used in the experiments to evaluate the alarm response and larval vulnerability. Mortality only occurred after 48 h of continuous exposure (Fig. 4). No mortality occurred during the alarm response experiment, or after a period of 48 h following 6 h of exposure in the larval survival experiment. In the larval vulnerability experiment, the larvae were also exposed to fenvalerate for 6 h, but the postexposure period was only 6 h. A similar 6-h exposure and a seven times shorter postexposure were applied in the larval vulnerability experiment. This suggests that, in the larval vulnerability experiment, the exposure to fenvalerate did not have any direct effects on larval survival, and the increase in the PT50 was caused exclusively by the increase in vulnerability of larvae to N. glauca. The proportion of larvae that exhibited the alarm response was shown to be very sensitive to exposure to fenvalerate for as short a period as 1 h. The results of the present study show that exposure to low levels of fenvalerate contamination can increase larval mortality due to predation. This might allow the volume of insecticide sprayed over aquatic habitats to be reduced, which would save money and protect natural diversity while still providing effective pest control. However, although in the work reported herein, the effect of exposure to fenvalerate on the predator was not investigated, the mobility of N. glauca was previously found to be extremely sensitive to another pyrethroid, -cyhalothrin, after 48 h of continuous exposure in eld and laboratory experiments (Schroer et al., 2004). Nevertheless, the results of the present work showed clear effects of exposure to fenvalerate on the behavior and vulnerability of the larval prey. The ecological relevance of these effects needs to be investigated further.

Acknowledgements A Postdoctoral scholarship was awarded to S. Reynaldi by the ALECOL program, academic exchange program between the National University of Colombia and the DAAD (Deutscher Akademischer Austauschdienst/German Academic Exchange). References
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