Rapid behavioural and morphological responses of hydropsychid larvae (trichoptera, hydropsychidae) to sublethal cadmium exposure

Environmental Pollution 84 (1994) 291 299

RAPID BEHAVIOURAL A N D MORPHOLOGICAL RESPONSES OF HYDROPSYCHID LARVAE (TRICHOPTERA, HYDROPSYCHIDAE) TO SUBLETHAL C A D M I U M EXPOSURE Kari-Matti Vuori Department of Biology, University of Jyvdskyld, Yliopistonkatu 9, SF-40100 Jyvfiskld, Finland

(Received 8 September 1992; accepted 1 February 1993)

Abstract

& Bartholomew, 1991), replacing or repairing damaged proteins (Bayne et al., 1979) or changing the time of moulting (Bengtsson et al., 1983). It is plausible to presume that, in territorial animals, stress induced changes in self-defence also include behaviourial changes in connection with the defence of a territory. Stress is likely to have a strong affect on the outcome of contests between the owner of a territory and an intruder. Stress may, for example, change the value of the contested resource and/or decrease the resource holding power of the owners (Otto, 1989). In general, the owner of a territory has a better chance of winning contests due to owner-intruder asymmetries, including differences in fighting ability, resource value or ownership status (Leimar & Enquist, 1984). Many aquatic invertebrates survive exposure to extremely high metal concentrations and show behavioural changes at relatively low concentrations. Morphological and physiological changes usually occur at intermediate concentrations (Moore & Ramamoorthy, 1984; Leland & Kuwabara, 1985; Rand, 1985). Despite the obvious importance of behavioural responses, relatively few behaviourial toxicity studies have been conducted with aquatic invertebrates (e.g. Scherer & McNicol, 1986; Muirhead-Thompson, 1987; Heinis et al., 1990) and the ecological impacts of observed changes in behaviour have been poorly evaluated. Behavioural toxicity studies dealing with competitive interactions of freshwater invertebrates are totally lacking. In fish, exposure to zinc and cadmium has been shown to increase aggressiveness of the dominant individuals (Henry & Atchison, 1979). Recently, competition has been suggested to play an important role in determining the community structure and niche relations of lotic invertebrates (see Dudley et al., 1990). Competitive interactions of stream insects commonly involve direct aggressive encounters (e.g. Jansson & Vuoristo, 1979; Hart, 1985; Hemphill, 1988; Dudley et al., 1990). Net-spinning caddis larvae of the family Hydropsychidae constitute an important element of the fauna in many ecosystems throughout the world (Lepneva, 1970; Wiggins, 1977; Davies & Walker, 1986). The density of hydropsychid larvae is

Larvae of Hydropsyche contubernalis and H. siltalai were exposed to sublethal cadmium concentrations of O, 0.012, 0.16 and 10 mg/litre l for 72 h. Linear logit models revealed a significant increase in the frequency and degree of damage of the anal papillae of both species with increasing Cd concentration. Hydropsyche contubernalis showed a stronger and earlier anal papillae response under Cd e.xposure than H. siltalai, whereas in the latter species darkening of the ventral sides of the abdomen was also observed. The lowest Cd level altered the competition behaviour of Hydropsyche contubernalis larvae after only 24 h exposure, yet no visual signs of morphological damage were detected The exposure intruder larvae spent significantly less time trying to enter the nets of resident larvae than did unexposed intruders. In addition, both the exposed intruders and exposed residents pursued different behavioural tactics during the encounters compared to their unexposed counterparts. Keywords." hydropsychidae, cadmium, sublethal exposure, morphology, competition, behaviour

INTRODUCTION Exposure to sublethal concentrations of heavy metals reduces growth rate, fecundity and survival of aquatic organisms. This may be caused by damage to metabolic processes and/or energy investment made by organisms in the detoxification mechanisms (Bayne et al., 1979; Moore & Ramamoorthy, 1984; Leyland & Kuwabara, 1985). In general, reduction in growth induced by environmental stress may be alleviated by spending less energy on metabolic defence and more on growth. Analogously, if stress increases mortality rate, animals should spend more energy on defence. This may be done, for example, by allocating more energy to detoxification and less to growth (see Sibly & Calow, 1989). Animals may defend themselves against metal induced stress by producing metal-binding proteins (e.g. Klerks Environ. Pollut. 0269-7491/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain

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often enormous, especially in lake outlets where up to six species may co-exist at the same localities (Bagge, 1989; Muotka, 1990). Chronic exposure to relatively low levels of toxicants may alter behaviour (Spehar et al., 1978), damage anal papillae and tracheal gills (Petersen, 1986; Camargo, 1991) and cause anomalies in the capture nets of the larvae (Petersen & Petersen, 1983). Larvae fight aggressively over the ownership of capture nets and the outcome of such fights is heavily affected by asymmetries between intruders and resident larvae (Jansson & Vuoristo, 1979; Otto, 1989; Englund & Olsson, 1990). Hydropsychids have been recommended as bioindicators of metal contamination in streams (Petersen, 1986; Cain et al., 1992). However, only a few studies give species-specific information on the responses of larval hydropsychids to metal exposure. Hence, the aim of the present study was to evaluate the potential effects of a short-term, sublethal cadmium exposure on the morphology and intraspecific competition behaviour of two hydropsychid species, HydropLLvche contubernalis and H. siltalai. M A T E R I A L S AND M E T H O D S In order to examine the morphological responses of hyropsychid larvae to Cd, ten fifth instar larvae of Hydropsyche contubernalis and H. siltalai were exposed to concentrations of 0 (control), 0-012, 0-016 and 10 mg litre ~ Cd for 72 h. The lower Cd concentrations (0.012, 0.16 mg litre 1) represent the values reported in many freshwaters contaminated by industrial/municipal wastes, whereas the highest value is representative for industrial effluents (Wachs, 1982; Ravera, 1984). The larvae were collected on 24th June 1991 from Siikakoski Rapids, Central Finland (62 ° 40' N, 26 ° 30' E), transported to the laboratory within 30 min and acclimated to test conditions in eight well-aerated 1-1itre polyethylene vials for 24 h, ten larvae in each vial. Only actively moving individuals were chosen for the tests. A net-spinning surface was provided by pieces of plastic grass matting. Test water was taken from Siikakoski and the temperature, pH and oxygen conditions were held constant in each vial (Table 1). After acclimation, Cd was added as 3CdSO4.8H20.Cd concentrations at the beginning of the tests were determined by atomic absorption spectrophotometry (Perkin Elmer 5100 PC) using a H G A 500 graphite furnace. Structural changes in the anal papillae, abdomen and tracheal gills of the larvae were observed under a stereomicroscope immediately following exposure. To evaluate the effects of the 72-h exposure on the survival of the larvae, twenty individuals of both species were transferred to pure test water after exposure, and maintained for 2 weeks. The larvae were fed with aquarium fish food and their mortality was monitored daily. In the second experiment, the effect of a 24-h sublethal Cd exposure (0.012 mg litre ~) on the competitive behaviour of H. contubernalis larvae was observed. Eighty fifth-instar individuals of approximately the

Table 1. Water quality parameters of the test water (Central Finland District Office of Water and Environment). Temperature and pH values are averages (and standard deviations) of the daily measurements in the eight experimental vials. 02 value is the average and SD at the end of the experiment

02(%) pH temperature °C Colour (mg Pt litre 1) Conductivity (25°C) COD (mg 02 litre i) Tot. N (/xg litre i) Tot. P (/zg litre 1)

85 (1.5) 7-3 (0.6) 21-2 (0-9) 30 4.4 7.1 7.7 380~,60 5.8

same size were removed from Siikakoski to two 10-1itre glass aquaria divided into two similar parts by a glass wall. A batch of 20 individuals was allowed to colonize each compartment of the aquaria. A current of approximately 30 cm s i was provided by a magnetic stirrer in the middle of each compartment and oxygenation by an air compressor. Pieces of plastic grass mat on the sides of the aquaria provided the net-building surfaces for the larvae. The larvae were acclimated for 24 h. During the first 6 h, all the individuals had spun their retreats and nets. Cd was added to one part of both aquariums, while the other part was left as a control. After 24 h, 18 exposed larvae and 18 control larvae were removed from their retreats to the other aquarium. Nine larvae within both groups were removed to the exposed part and nine larvae to the control part (Fig. 1). The larvae and the part from which they were removed were selected randomly. Larval behaviour was followed by direct observation through the glass wall and the time expended in fights between the residents and intruders was measured. Larvae were removed one by one. After the first encounter between a new intruder and resident larvae was observed, the intruder was displaced and a new one introduced. The significance of effects of different Cd levels and species on the physiological damage observed was analysed by constructing linear logit models (Cox, 1970). In these models the logarithms of the expected cell frequencies in a multiway contingency table are expressed as linear equations of the model parameters. Furthermore, additional logit models were constructed to reveal the significance of the effects of species and Cd concentration on the degree of physiological Control

Cd Intruders

Residents Control

Cd

Fig. 1. Arrangement of the Hydropsyche contubernalis larvae behavioral experiment.

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Table 2. The frequencies of normal (A,B) and damaged (C,D,E) anal papillae of Hydropsyche contubernalis and H. siltalai larvae under different levels of cadmium exposure. A, papillae clear and transparent; B, white but unclear; C, slightly darkened; D, almost totally darkened; E, totally darkened and reduced

H. contubernalis mg Cd/litre ~ 0-00 0.012 0.16 10.00

H. siltalai

N

A

B

C

D

E

A

B

C

D

E

10 l0 10

7 6 5

3 1 0

0 2 2

0 1 1

0 0 2

10 9 8

0 1 1

0 0 1

0 0 0

0 0 0

10

1

0

3

3

3

5

0

1

2

2

damage in the larvae. Contrasts were applied to the statistically adequate models to show the effects of different Cd levels on the physiological damage to the two species. The significance of differences in fighting times between groups of intruder/resident pairs was tested with the Mann-Whitney U-test. RESULTS Morphological responses

Upon exposure to Cd five categories of anal papillae were detected. These were: (A) transparent, (B) white but unclear, (C) slightly darkened, (D) almost totally darkened, (E) totally darkened. Of unexposed larvae, 70% of Hydropsyche contubernalis and 100% of H. siltalai larvae had transparent anal papillae after 72 h (Fig. 2(a). Of the unexposed Hydropsyche contubernalis larvae 30% had white but unclear papillae (Fig. 2(b)). Such papillae also occurred in some individuals of Hydropsyche siltalai exposed to 0.012 and 0-16 mg Cd litre ~. Since specimens collected from unpolluted rivers may display the same range of variation in the transparency of their papillae (Vuori, unpublished), it is presumably of natural origin and does not indicate damage to the anal papillae. Hence, only categories C - E where darkening of the papillae was observed, are referred to as damage (see also Petersen, 1986; Camargo, 1991). Damaged anal papillae were detected only in the larvae exposed to cadmium. These damaged

papillae were more or less darkened and in the most serious cases were almost black and heavily reduced (Figs 2(c)-2(e)). At the highest Cd concentration, 66.7% of the anal papillae of Hydropsyche contubernalis and 80% of those of H. siltalai were totally darkened and also often reduced. The proportion of severely damaged papillae increased with increasing Cd concentration in both species (Table 2). The logit model in which Cd level and species explained the anal papillae damage, was statistically adequate and fitted the data (Table 3, Model 2). The interaction-term Damage. Cd level Species was not statistically significant (SD-- 0.801, df= 3, p > 0.100) and was excluded. The hypothesis 'the higher the Cd level, the greater anal papillae damage in larvae' was tested by contrasting the factor Cd level. This model fitted the data and was statistically adequate (Table 3, Model 3). Hence, an increase in Cd concentrations significantly increases the frequency of anal papillae damage which is, however, species dependent. Hydropsyche contubernalis showed a stronger and earlier anal papillae response to Cd exposure than H. siltalai (Fig. 3), whereas the proportion with severe damage was higher in the latter species (Fig. 4). The degree of anal papillae damage was statistically adequately explained by Cd level alone (Table 3, Model 10). The interaction term damage.sp was not statistically significant (SD = 0.001, df= 1, p > 0.100). By contrasting the variable Cd level it can be seen that higher

Table 3. The parameters, sealed deviances (SD), degrees of freedom (dJ) and P values of the logit models. The models chosen to describe the frequency and degree of anal papillae damage in hydropsychid larvae are indicated by an asterisk, (Sp, species; Cd, cadmium; c, contrasts)

Model Frequency of damage (1) Damage+ Damage.SP+Damage.CD+ Damage.SP.CD (2) Damage+Damage.SP+Damage.CD (3) Damage+Damage.SP+Damage.CD (c)* (4) Damage+Damage.SP (5) Damage+Damage.CD (6) Damage Degree of damage (7) Damage +Damage.Sp+Damage.Cd+Damage.Sp.Cd (8) Damage+ Damage.Sp+Damage.Cd (9) Damage+Damage.Sp (10) Damage+Damage.Cd (11) Damage+Damage.Cd (c)* (12) Damage

SD

df

P

0.000 0.801 1-421 27.912 10.05 34.521

0 3 5 6 4 7

1.000 0.849 0.922 0.000 0-040 0.000

0.000 0.893 2-523 0.895 0.942 2-530

0 2 4 3 4 5

1.000 0.640 0.641 0.827 0.918 0.772

K.-M. Vuori

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(a)

(b)

J

(c)

(d)

Fig. 2. The anal papillae (arrows) of unexposed (a), (b) and exposed (c~(e) larvae of Hydropsyche contubernalis and exposed larvae of H. siltalai (f). (a) and (b) represent normal anal papillae and (c)-(e) damaged papillae (for explanations, see text and Table 2). (f) shows ventral darkening of the 6th abdominal segment and (g) reduced tracheal gills of exposed Hydropsyche siltalai larvae.

Responses of hydropsychid larvae to cadmium exposure

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(r)

(e)

8,

(g) Fig. 2.--contd.

K.-M. Vuori

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I

number of damaged larvae

[--l Hcon Hsil

I

1

I

,

,

2.

3.

4.

20

10815

64.

I.--

10

2 0

• 0

0.012

0.16

0

mg Cd/1

Fig. 3. Number of Hydropsyche contubernalis and H. siltalai larvae with damaged anal papillae under different levels of Cd exposure. The dots are expected frequencies of the logit model fitting the data.

5

0

Cd levels caused significantly more severe damage to anal papillae than the lower concentrations in both species. This model (11) fitted the data, was statistically adequate and so was chosen to describe the data. Hydropsyche siltalai did not have severe damage except in the highest Cd concentration, whereas most of the H. contubernalis larvae were badly damaged at a concentration of 0.16 mg Cd litre 1 (Fig. 4). In all exposed Hydropsyche siltalai larvae, some areas of the ventral sides of the abdomen were darkened and at the highest concentration reduced tracheal gills were detected (Figs. 2(f)-2(g)). However, there was no general trend in the frequency of the darkened areas of the abdomen in relation to the Cd concentration. Usually there were only a couple of dark areas, but in some individuals all of the abdominal segments were ventrally darkened. This phenomenon was not observed in Hydropsyche contubernalis. All larvae exposed to concentrations of 0 and 0.012 mg Cd litre j survived and pupated during the period of 2 weeks after removal to uncontaminated water. Survival was 70% and 50% for Hydropsyche contubernalis and 80% and 60% for H. siltalai larvae exposed to concentrations of 0.16 and 10 mg litre ', respectively. In addition, only 50% of the surviving proportion of bad damages % 80-

I

I Hc°n Hsil

6040:i:i:i:i:i:i:!:i:i:i:l

20- ~

•

+:.-:.:+:<.:.z :::::::::::::::::::::1

iiiiiiiiiiiiii~iiiii] x+~,:+.+:.:.:~

0

0.012

0.16

0

mgCd/1

Fig. 4. Proportion of badly damaged anal papillae (category E, see text) in Hydropsyche contubernalis and H. siltalai larvae under different levels of Cd exposure. The dots are as in Fig. 3.

1.

EXPOSURE

Fig. 5. The average duration (+ SE) of fights between exposed/unexposed intruder and resident larvae of Hydropsyche contubernalis. (1, exposed resident/exposed intruder (n = 9); 2, unexposed resident/exposed intruder (n = 9); 3, exposed resident/unexposed intruder (n = 9); 4, unexposed resident/unexposed intruder (n = 9)). larvae of both species pupated after exposure to 0-16 and 10 mg Cd litre 1. The structure and colour of the anal papillae did not change during the 2 weeks. Behaviourial responses Intruding H. contubernMis larvae exposed to Cd concentrations of 0.012 mg/litre 1 for 24 h expended significantly less time trying to take over the nets of the resident larvae than did the control larvae (Mann-Whitney U-test, U = 22.00, df-- 1, p = 0.001). The average time spent fighting was 1.7 andl4.9 min for exposed and unexposed intruders, respectively. The duration of fights between pairs where the resident larva was exposed to Cd was shorter than for fights between pairs where the resident was unexposed (Fig. 5). However, these differences were not significant ( M a n n - W h i t n e y U-test, p > 0.05). All of the intruders tried almost immediately after introduction to overcome a net of a resident. None of the residents lost their nets. However, the behaviour of exposed intruders and exposed residents clearly differed from that of the control larvae. The exposed intruders usually made one or two attacks, trying to bite the residents and on most occasions spent a few minutes with mandibles locked with the resident in the opening of the net. After this the exposed intruders gave up and started to wander along the aquarium searching for a suitable net-spinning site. The unexposed intruders, on the other hand, exhibited fiercer attacks and then usually adopted a 'sit and wait' -strategy which involved long periods of locking of mandibles with the resident larvae at the opening of the net. During this period both the resident and the intruder bit each other

Responses of hydropsychid larvae to cadmium exposure occasionally while struggling violently. Usually the fight ended with the last fierce attack made by the intruder. After this, the unexposed intruder gave up and almost always tried to attack another resident. The longest observed encounter between an unexposed intruder and a resident larvae lasted 46.9 min, whereas the longest time for exposed intruders was 5.4 min. Exposed residents were surprisingly active during the encounters and they seemed to attack intruders more fiercely than did the unexposed residents. Furthermore, while the locking of mandibles occurred in all fights between unexposed residents and unexposed intruders, and in most cases between unexposed residents and exposed intruders, it happened only occasionally in fights between exposed residents and unexposed or exposed intruders. DISCUSSION Morphological responses Cadmium has been suggested to damage ion-regulation mechanisms of aquatic organisms rather than respiratory or nervous functions (Hellawell, 1986). This is supported by the observed damage to the hydropsychid anal papillae. In hydropsychids these papillae function as ion regulatory organs (Wichard, 1976). The anal papillae of freshwater caddisfly larvae possess specialized cells which are able to absorb electrolytes and thus compensate for the loss of ions due to the hypotonic environment (Ntiske & Wichard, 1972). Hence, the rapid darkening of the hydropsychid and papillae under Cd exposure may be caused by active electrolyte absorption and, consequently, precipitation of Cd in the epithelial cells of the papillae. In addition to anal papillae damage, Hydropsyche siltalai larvae showed a strong effect of Cd on the ventral sides of the abdomen. The tracheal gills of Hydropsyche siltalai were also reduced at the highest levels of Cd. The observed differences in the morphological responses of Hydropsyche contubernalis and H. siltalai to Cd are presumably caused by differences in their metabolic adaptations. Hydropsyche contubernalis has a low oxygen consumption and tolerates oxygen deficiency quite well, whereas H. siltalai has a high oxygen demand (Philipson & Moorhouse, 1974; Becker, 1987; Roux et al., 1992). H. siltalai also prefers faster currents than H. contubernal& (Bagge, 1989; Joensuu & Vuori, 1992). As Hydropsyche contubernalis has a higher total amplitude of metabolism than H. siltalai (Roux et al., 1992), it seems to be better adapted to variable environmental conditions. However, the present results suggest that H. contubernalis is less tolerant to short-term Cd exposure than H. siltalai. Petersen (1986) reported darkened anal papillae in Hydropsyche angusipenn& and H. saxonica larvae upon sublethal copper exposure. The latter species was less tolerant of Cu and showed earlier anal papillae damage than H. angustipennis (Petersen, 1986). Camargo (1991) reported darkening of the anal papillae and reduced tracheal gills in Hydropsyche pellucidula larvae upon

297

exposure to chlorinated tap water. Significantly higher frequencies of darkened anal papillae in the larvae of Hydropsyche eontubernalis and H. pellueidula were also detected by Vuori (1992) in rapids contaminated by waste waters from a paper pulp mill compared to uncontaminated rapids. Thus, the anal papillae damage of hydropsychid larvae seems to be a general indication of physiology stress caused by poor water quality. As the damage remained after transfer to uncontaminated water, it could potentially reflect past exposure conditions. Furthermore, as 50% of the larvae exposed to high Cd levels had retarded pupation, severe damage to anal papillae most likely indicates failure in reproduction. Behaviouriai responses There was a radical Cd-induced decrease in the time spent fighting by intruder larvae of Hydropsyche contubernalis (Fig. 5). The exposed residents also tended to spend less time in fighting. It is reasonable to presume that Cd exposure induced a physiological stress which led larvae to change their behaviour so that less time and energy was spent fighting. Grellmann (1991) found that starvation stress reduced time spent in the fights between resident and intruder larvae of Plectrocnem& conpersa. Neither exposed nor unexposed resident larvae lost their nets to intruders. This implies a simple residency asymmetry commonly observed in the territorial encounters (e.g. Maynard Smith & Parker, 1976; Gribbin & Thompson, 1991). The resident hydropsycids evidently have a better chance of winning the encounter than the intruders (Jansson & Vuoristo, 1979). Leimar & Enquist (1984) have shown that, in asymmetrical owner-intruder conflicts with matched opponents, the owners win most of the fights and that the longest fights occur when the intruder is slightly stronger than the resident. At the beginning of the study, exposure was expected to weaken the residents and increase their probability of losing the fights against unexposed intruders, and the longest fights were expected to occur between exposed residents and unexposed intruders. However, this was not the case. Instead, the longest fights occurred between matched opponents, unexposed residents and unexposed intruders and the shortest fights between exposed residents and unexposed intruders. This is probably because Cd exposure has a different effect on the pay-off of the contests for the intruders than for the residents. In general, the costs of pursuing different behaviourial strategies have a strong effect on the conflict behaviour of animals (Maynard Smith, 1982). It must be noted that both intruders and residents had already made an investment on silk synthesis and net building, which is energetically quite costly (Dudgeon, 1987). For the exposed intruder, considering the low probability of winning the contest (e.g. Englund & Olsson, 1990), it would be more advantageous to spin its own capture net, even at a less valuable site, instead of spending time and energy in a risky fight. For the exposed residents the optimal

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response may instead be the aggressive and metabolically less expensive defence of the established residency. In addition, the risk of being wounded or killed is high, especially for the intruders (see Otto, 1989). Hence, the Cd-induced behaviourial change can be viewed as decreasing risks involved in fighting. Hydropsychid species have different tolerances to metal contamination, which partially explains the changes in the guild structure and relative abundances of the species in streams polluted by metals (Petersen, 1986). Petersen also concluded that heavy metal pollution not only eliminate sensitive species but also changed the competitive equilibrium between species. He suggested that the elimination of the keystone species (sensu Paine, 1966), Hydropsyche pellucidula, released the more resistant species from competitive pressure, leading to changes in the guild structure and in the niche relations of the remaining species. Speciesspecific differences in behaviourial responses may be an important mechanism in such changes. The present results suggest that sublethal Cd exposure may reduce intraspecific competition in hydropsychids. This is manifested in the inactivity of exposed intruders in trying to encounter the nets of residents and in the increased aggressiveness of exposed residents defending their residencies. This rapid behavioural change may make it possible for the individuals to alleviate a short-term chemical stress by spending less energy in fighting. The ecological consequences of these kinds of behavioural responses to sublethal metal exposure should be evaluated by experiments covering the whole life cycle of the study organisms. ACKNOWLEDGEMENTS The author thanks the personnel of Konnevesi research station for all their support during the study and especially Mrs E.-L. Pakarinen for constructing the bottoms of the aquaria. He also thanks Dr Lena Petersen and Dr R. C. Petersen for introducing him to the ecotoxicology of hydropsychids, J. Taskinen for statistical advice and participants of The Round Table in Jyv/iskyl/i and two anonymous referees for constructive criticisms on the manuscript. Special thanks to P. Sevola and S. Jokela in Vaasa and Kokkola District Offices of Water and Environment for working facilities. Dr Roy Siddall kindly checked the language. This study was partly funded by the Maj and Tor Nessling Foundation. REFERENCES Bagge, P. (1989). Communities and habitats of filter feeding caddisflies in the lake outlet biocoenoses of Central Finland. In Proceedings of the 6th International Symposium on Trichoptera, Lodz-Zakopane (Poland), 12 16 September 1989, ed. C. Tomaszewski. Adam Mickiewicz University Press, Warsaw, pp. 95-9. Bayne, B. L., Moore, M. N., Widdows, J., Livingstone, D. R. & Salkeld, P. (1979). Measurement of responses of individuals to environmental stress and pollution: studies with bivalve molluscs. Phil. Trans. R. Soc. London, 286B, 563-81.

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