Aquat Ecol (2008) 42:141–150 DOI 10.1007/s10452-007-9089-z
Effects of growth factors and water source on laboratory cultures of a northern Asellus aquaticus (Isopoda) population Tiina Hasu Æ Jukka Jokela Æ E. Tellervo Valtonen
Received: 4 July 2006 / Accepted: 22 February 2007 / Published online: 24 March 2007 Springer Science+Business Media, B.V. 2007
Abstract We tested whether two vitamin-based supplements, bright light or water from three different sources affected the survival and reproduction of Asellus aquaticus isopods, and the growth and survival of their laboratory-born juveniles. Isopods were sampled from the littoral zone of a lake in November (L:D = 6:18, temp. = 4C) and brought to laboratory (L:D = 16:8, temp. = 21C), where they started to breed after 5 weeks. None of the tested nutrient or light treatments significantly affected the time females started to carry the eggs in their marsupia, the duration of marsupial stage, the number of egg bearing females, the proportion of females producing live young, or the offspring growth measured as an increase in length. Generation specific survival effects of nutrient and light treatments were found in the two parent generations. However, the reproductive output of adult isopods measured as the mean number of T. Hasu (&) E. T. Valtonen Department of Biological and Environmental Science, University of Jyva¨skyla¨, P.O. Box 35 (YA), Jyvaskyla, 40014, Finland e-mail:
[email protected] J. Jokela EAWAG (Swiss Federal Institute of Aquatic Sciences and Technology), Department of Aquatic Ecology, ETH-Zu¨rich, Institute of Integrative Biology (IBZ), ¨ berlandstrasse 133, Du¨bendorf, CH-8600, U Switzerland
surviving offspring per reproducing female was drastically reduced in tap water treatment, possibly due to the lethal effects of copper in the water for the newborn offspring. Survival of parent isopods did not differ among the water treatments. The lack of response to the tested growth factor treatments suggests that gut and/or endosymbiotic microbes may have an important role in A. aquaticus nutrition.
Keywords Crustacean Offspring Reproduction Survival
Introduction Asellus aquaticus (L.) is a ubiquitous freshwater isopod having several important roles in lake and stream ecosystems. It is a detrivorous shredder (Andersson 1985) that feeds on the bacterial and fungal material associated with decaying organic matter (Rossi and Fano 1979; Grac¸a et al. 1993a, b; Costantini and Rossi 1998), but can also utilise green plant tissue and algae (Moore 1975; Marcus et al. 1978). Moreover, A. aquaticus is an important and preferred prey item for many fish (e.g. Rask and Hiisivuori 1985; Hart and Gill 1992; Jamet 1995) and predatory invertebrates (e.g. Cockrell 1984; Henrikson 1993; MacNeil et al. 2002). A. aquaticus is also a common
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intermediate host for acanthocephalan parasites, such as Acanthocephalus lucii and Acanthocephalus anguillae (Schmidt 1985). Its multifaceted roles in benthic communities make A. aquaticus an appropriate model species in aquatic ecology. Experimental studies with A. aquaticus report that isopods can survive in the laboratory for up to several months (e.g. Ridley and Thompson 1979; Zimmer et al. 2002; Hasu et al. 2006). The wide variety of culture conditions and methods used for laboratory cultures with respect to water and offered food implies that A. aquaticus is a species that can be cultured under a variety of laboratory conditions, as its commonness in the field also suggests. Although, the nutritional preferences and utilisation of different food sources of A. aquaticus are widely studied, our knowledge on detailed nutritional demands of the species are scarce. Yet, the information on possible essential growth factors, required vitamins and amino acids is of great concern, especially for long-term laboratory cultures to be used in ecological investigations. In the present study we specifically tested whether growth factors (vitamins, amino acids) or bright light could influence juvenile growth and survival, or adult reproduction in laboratory cultures of a cold-climate A. aquaticus population (Finland). A major role for L-carnitine (also known as vitamin BT) as an essential growth factor has been reported for some invertebrates (Fraenkel and Friedman 1957; Dias-Wanigasekera et al. 2000), while Franken et al. (2005) found a significant positive effect of light intensity on the growth of A. aquaticus probably due to its effect on some components of biofilms (e.g. algae) that are utilised as food. We also tested, whether different types of water commonly used in laboratory cultures (tap water, borehole water and spring water) could affect the survival, growth or reproduction of isopods. Because the study population came from a shallow littoral zone with rich of alder Alnus glutinosa (L.) trees, providing thick masses of decaying leaves as potential food, conditioned alder leaves were used as food for isopods in these experiments. Isopods were collected in November after the animals had been naturally exposed to winter conditions (L:D = 16:8; temp = 4C). We
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expected that adult isopods having been at least two months in reproductive diapause would start to reproduce within a month in laboratory conditions (L:D = 16:8; temp = 21C) (see Tadini-Vitagliano et al. 1982).
Materials and methods Experimental animals Decaying leaves and detritus with associated A. aquaticus individuals were collected with a kick net from the littoral area in Lake Niemisja¨rvi (8 km2, mean depth 2.3 m, max. depth 13 m) in central Finland (6216¢ N, 2621¢ E) on 8th November and brought to the laboratory in 60 l buckets along with lake water. Niemisja¨rvi is a typical mesotrophic (total-P 17 lg l–1; chlorophyll-a 10 lg l–1) and mesohumosic (40–50 mg Pt l–1) lake. At the laboratory, body length was determined for each A. aquaticus individual in graduated clear containers (to the nearest 0.5 mm). Animals were kept in aerated, aged tap water and fed with decaying alder (Alnus glutinosa) leaves, which were conditioned for at least two weeks in aerated water enriched with (NH4)SO4 (20 mgl–1) in order to accelerate microbial growth (Costantini and Rossi 1998). After two weeks acclimatisation to laboratory conditions, isopods were divided into two size classes (or parent generations) based on their length from the top of the head to the tip of the telson: size class I, later on ‘‘large’’ isopods (4–9 mm), and size class II, later on ‘‘small’’ isopods (2–3.5 mm). Size class I included the ‘‘older’’ generation, which possibly had already reproduced during the previous summer, while size class II comprised individuals that were reaching or just had reached sexual maturity. Experiment I From size class I 216 isopods were randomly assigned into 18 one-litre containers (12 isopods in each), of which 6 were filled with 0.8 l of tap water, 6 with 0.8 l of borehole water (charcoal filtered water from an artesian well) and 6 with 0.8 l of water from a nearby spring (suitable for potable
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water). All three types of water were first aerated in the laboratory for at least a day before the experiments. Once a week approximately 0.3 l of water from each container was replaced with fresh water of same origin. Isopods were fed ad libitum with conditioned decaying alder (A. glutinosa) leaves. Dead isopods were removed from containers, and their gender and length were checked under a binocular microscope. Gravid (i.e. eggbearing) females were transferred individually into separate one-litre containers, maintained and fed as described above and the time when young were released was recorded. At the end of the experiment, adults and offspring in all containers were counted, sexed and their body length was determined. The experiment started on the 21st of November and continued for 11 weeks at 21C and 16:8 (light: dark) photoperiod. Experiment II Forty-five one-litre containers were filled with 0.8 l of spring water, and 360 large isopods (size class I) were randomly assigned into 30 containers (12 animals per container), and 180 small isopods (size class II) into 15 containers (12 animals per container). Containers were then randomised into five treatments, so that each treatment consisted of six replicates with large isopods and three replicates with small isopods. Changing of water, feeding and other protocols were the same as in experiment I. The five treatments were: 1. 2.
3.
4.
Control; no additional treatments. L-carnitine (vitamin BT, Sigma C-0283) (1 mg ml–1); 2 ml (resulting concentration in a container 2.5 ppm) twice a week per container for the first 4 weeks, and then once a week until the experiment was ended. Vitamin mix (Atvitol, JBL; commercial multivitamin and amino acid product used to maintain the health, increase the appetite and promote natural growth of aquarium fish); 5 ll (resulting concentration in a container 6.25 ll l–1) twice a week per container for the first 4 weeks, and then once a week until the experiment was ended. Treatments 2 and 3 combined: L-carnitine 2 ml (1 mg ml–1) and vitamin mix 5 ll twice a
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week per container for the first 4 weeks, and then once a week until the experiment was ended. Bright light treatment; containers were placed under a Phillips bright light lamp (HF 3301) (7,000–8,000 lux) for 2 h per day during the light period 3 times a week for the first 4 weeks, and then for 2 h once a week until the experiment was ended.
At the end of the experiment, adults and offspring in all containers were counted, sexed and their body length was determined. The experiment started on the 21st of November and was continued for 11 weeks at 21C and 16:8 (light:dark) photoperiod. Data analyses Differences in all measured variables between the different treatments were analysed with ANOVA after checking the normality and homogeneity assumptions (Levene’s test). If assumptions were not met, data were ln-transformed, or non-parametric tests were used. Cumulative survival analysis of adult isopods was conducted as a Cox regression analysis with treatment and gender, and in experiment II with the size class, as covariates. The forward stepwise method was used to build the best statistical model. Survival models were compared using likelihood ratio tests. Independent variables were entered in the model if they improved the model significantly (probability limit, P < 0.10) and excluded from the model if they did not explain a significant proportion of variation in survival (P-limit, P > 0.15). In experiment I, offspring length differences between treatments were analysed using untransformed offspring mean length as a dependent variable. In experiment II we corrected the effect of offspring age (time from hatching to the end of experiment) on the offspring length at the end by plotting ln-transformed mean offspring length against offspring age in linear regression and using the resulting residual as a dependent variable in one-way ANOVA with treatment as a factor and Tukey HSD as a Post Hoc test. Residuals had homogenous variances. When
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testing the incubation time as a dependent variable in experiment II the data were ln-transformed to homogenise the variances and one extreme case was excluded as an outlier. Lntransformation was also used when testing the number of living offspring per reproducing female (i.e. gravid female that was seen to release live offspring) in experiment II. Figures represent untransformed data, unless else is notified. All statistical analyses were performed with SPSS for Windows (SPSS Inc. 1989–2002).
P < 0.001). Most reproducing females in tap water had lost all their young, while the proportions of such females in borehole water and spring water treatments were 10% or less (Fig. 1a). Hence offspring produced in borehole or spring water had 3-fold higher survival than those produced in tap water (ANOVA: F2,31 = 3.818, P = 0.033; Fig. 1b). Offspring in the tap water treatment were significantly younger (the time from hatching to the end of the experiment) compared to offspring in borehole and spring water treatments (Kruskal–Wallis: H = 8.293; df = 2; P = 0.016; Table 1), but note that offspring of most females in tap water had died during the experiment leaving only two broods for length comparisons in the tap water treatment. Offspring length at the end did not differ significantly between water treatments (ANOVA: F2,22 = 0.118, P = 0.890; Table 1). No difference was found in the number of brooding females (ANOVA: F2,15 = 0.254, P = 0.779), or in the proportion of reproducing females (ANOVA: F2,12 = 1.025, P = 0.388)
Results Experiment I The proportion of reproducing females (i.e. eggbearing females, that were also seen to release free swimming young) with live offspring at the end of the experiment was significantly lower in the tap water treatment than in the borehole or spring water treatment (ANOVA: F2,11 = 25.903,
a)100 Females that lost all their young (%)
Fig. 1 (a) The proportion of reproducing females (egg-bearing females that were seen to release freeswimming offspring, % in treatment group) that lost all their young during the experiment. (b) Mean number (±SE) of surviving offspring per reproducing female isopod in different water treatments at the end of the experiment
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0 Tap water
Borehole water
Spring water
Tap water
Borehole water
Spring water
Surviving offspring / reproducing female
b) 14
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12 10 8 6 4 2 0
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Table 1 Parameters for reproductive traits of brooding A. aquaticus females in experiment I and II Experiment
I Tap water Borehole water Spring water II Size >4 mm Size 0.088). No statistically significant differences were found in the time it took females to start brooding among treatments (ANOVA: F4,36 = 0.716, P = 0.587) or between the two isopod size classes (T-test: t = –1.291, df = 39, P = 0.204). The duration of marsupial stage (incubation time) did not differ significantly between the treatments (ANOVA: F4,35 = 0.146, P = 0.963) or between the isopod size classes (T-test: t = 0.832, df = 38, P = 0.411). The number of egg-bearing (i.e. gravid) females or the proportion of gravid females having produced offspring did not vary
The first brooding (egg-bearing) females were found on 12th December, after 5 weeks in the laboratory and 3 weeks from the beginning of the experiments. The first free-swimming progeny of a female was recorded on 4th January, three weeks after the first gravid female was observed. A total of 115 females were found bearing eggs during the experiments, but only 75 of them were found to release live offspring. The rest 40 of egg-bearing females discarded their eggs at an early developmental stage. In 63 out of those 75 cases the incubation time (from female gravity to the release of offspring) was 3–4 weeks. This 18 16 14 12 10 8 6 4 2 0
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Survivng offspring / reproducing female
Fig. 2 Mean number (±SE) of surviving offspring per reproducing female in different treatments at the end of experiment II
Sexual activity
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Table 3 Survival analysis of adult A. aquaticus isopods in the nutrient experiment was conducted as a Cox regression analysis with treatment, gender, and size class as covariates Model development step/term entered
Change in v2
df
P
1. Size class 2. Size class * Treatment Terms in the final model Size >4 mm Treat Size 4 mm Gender Gend * Treat Size 4 mm at the start) and (b) size class II (
