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1 This article was downloaded by: [University of Helsinki] On: 30 January 2014, At: 21:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Freshwater Ecology Publication details, including instructions for authors and subscription information: Invertebrate Predators and Preservation Time Lags in Zooplankton Samples: Effects on the Estimates of Prey Abundance and Size Anne Liljendahl, Petra Tallberg & Jukka Horppila Published online: 06 Jan To cite this article: Anne Liljendahl, Petra Tallberg & Jukka Horppila (2010) Invertebrate Predators and Preservation Time Lags in Zooplankton Samples: Effects on the Estimates of Prey Abundance and Size, Journal of Freshwater Ecology, 25:4, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

2 Invertebrate Predators and Preservation Time Lags in Zooplankton Samples: Effects on the Estimates of Prey Abundance and Size Anne Liljendahla, Petra Tallbergb, and Jukka Horppilaavc Downloaded by [University of Helsinki] at 21:25 30 January 2014 ABSTRACT The effects of predatory phantom midge (Chaoborusflavicans) larvae on the density and size estimates of crustacean zooplankton with different time lags between sampling and sample preservation were studied. Chaoborid larvae may easily be included in zooplankton samples, especially if samples are taken with net hauls. Five larvae included in a sample had a significant negative effect on the density estimate of daphnids, cyclopoid and calanoid copepods, and cyclopoid nauplii. When chaoborids were included in the samples, a three-hour preservation lag caused a >5% underestimation of population density estimate of cyclopoids and a six hour lag resulted in a 10% error. In field studies, chaoborids usually prefer small cladocerans such as bosminids as prey, but in the restricted space of the sample containers escape ability of cyclopoids was diminished. As a result, cyclopoids were the main prey of chaoborids. Chaoborids had no effect on the mean individual size of any of the taxa, but in the reference samples the mean size of daphnids and calanoids was reduced. This, as well as the reduced density of cyclopid nauplii in the reference samples, was attributed to predation by cyclopoids. The results emphasized that zooplankton samples should be preserved immediately after the sample haul. INTRODUCTION Predatory species may be included in samples taken for the estimation of zooplankton abundance. If the samples are not preserved immediately after sampling, invertebrate predators may continue preying on zooplankton in the sample containers and cause an erroneous impression of the zooplankton assemblage (Swift and Fedorenko 1973, Baier et al. 1997). The problem is known but has very rarely been quantified. We studied the effects of predatory phantom midge larvae (Chaoborusflavicans) on the density and size estimates of crustacean zooplankton with different time lags between sampling and sample preservation. Larvae of C. flavicans form high biomasses in many eutrophic lakes and can, as efficient predators, substantially regulate zooplankton communities (Luecke and Litt 1987, Liljendahl-Nurminen et al. 2003). Chaoborids can be included in zooplankton samples especially if samples are taken by net hauls. Large invertebrate predators are relatively good at avoiding tube samplers but are more effectively captured with plankton net hauls (Hakkari 1978, Horppila 2005). The aim of this study was to find out whether a relatively small number of chaoborids included in zooplankton samples cause a systematic error large enough to be detected among the natural between-sample variation in density and size structure estimates of crustacean zooplankton. METHODS AND MATERIALS The effects of C. flavicans larvae on zooplankton samples were studied with the following four different preservation lagtimes: 1) the samples were preserved adepartment of Environmental Sciences, P.O. Box 65, FI University of Helsinki, Finland. b~epartinent of Food and Environmental Sciences, Environmental Soil Science, P.O. Box 27, FI University of Helsinki, Finland. Corresponding author; jukka.horppila@helsinki.fi 599 Journal of Freshwater Ecology, Volume 25, Number 4 - December 2010

3 immediately after sample hauls (0-h time lag), 2) preservation was delayed until all samples from the station were taken (3-h time lag), 3) the samples were preserved after a 6-h time lag, and 4) the samples were not preserved until in the laboratory (12-h time 1%). Zooplankton samples were taken from the deepest part (depth 33 m) of Lake Hiidenvesi (southern Finland) (60" 24' N, 24" 18' E). Samples were hauled with a tube sampler (h = 1 m, V = 7.5 L) from the 0-5 m depth (water temperature "C) to minimize the inclusion of chaoborids in the samples. In Lake Hiidenvesi, the majority of chaoborids spend the day below the thermocline at > 8 m depth (Horppila et al. 2000, 2004). Each zooplankton sample consisted of five tube sampler hauls, which were filtered through a 50 pm net and stored in a 250 ml sample bottle. For each of the four preservation schemes, 10 such zooplankton samples were assembled. To half of the samples, five instar IV Chaoborusf2avicans larvae were added (CHA samples), while the other half remained as untreated reference samples (REF samples). Samples were preserved with 4% formaldehyde according to the schedule. Thus, each preservation scheme had five replicates with Chaoborusffavicans larvae and five replicates without larvae. The chaoborid larvae were collected with a plankton net (diameter 0.5 m, mesh size 180 pm) from m depth concomitantly with the zooplankton samples. Since the added larvae were lifted from the same location as the zooplankton samples, they were cyclopoida ~~clopoid nauplii , , Calanoida 1 Calanoid nauplii Preservation lag (h) Preservation lag (h) + CHA samples REF samples Figure 1. The effect of preservation lag on the density estimates of different zooplankton taxa with (CHA) and without (REF) chaoborid larvae included in the samples. The linear regression lines are also shown. 600

4 identical to larvae unintentionally included in the samples with respect to their diets and nutrition level. This is important, since the state of hunger affects the prey selection of invertebrates (Pastorok 1980a, Williamson 1980). In the laboratory, zooplankton was counted using inverted microscopy. Cladocerans were indentified to species level and copepods to family level. Cyclopoid and calanoid nauplii were counted separately, but the different copepodite stages and adults were counted as one group. Thirty individuals from each taxon were measured. Daphnids were measured from the center of the eye to the base of the tailspine, and other species from the anterior edge of the carapace to the posterior edge of the carapace. The diets of the added larvae, with the exception of the 0-h treatment, were checked with the method by Swift and Fedorenko (1973). The larvae in the 0-h treatment were not examined, since their diets were likely to include prey captured from the lake. It must also be noted that the diet data are only indicative and do not necessarily reveal the true diet composition, because chaoborids often eject the exoskeleta of prey animals via their mouth and many prey animals may therefore leave little trace (Croteau et al. 2003). The gut passage time in chaoborids is also shorter than our sampling interval (Croteau et al. 2003). The effects of preservation time lag on the density and individual body length of different zooplankton taxa with and without chaoborids were statistically studied with linear regression analysis. Before the analyses, the datasets were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test). Since the assumption of equal variances was not fulfilled in most cases, the datasets were log(x+l) transformed before the analyses. In three zooplankton samples, chaoborid larvae were captured by the tube sampler; these samples were thus excluded from the datasets. RESULTS Daphnia cristata (Sars) and Bosmina coregoni were the main cladoceran species in the samples, and only a few Chydorus sp. and Limnosida frontosa were observed. In the 0-h samples, the density of D. cristata varied from 18 to 35 ind. L-' (Fig. 1). After 12 h, the density was 21 ind. L-' in the REF samples and 13 ind. L-' in the CHA samples. For the CHA samples the slope of the regression line was significantly negative. The density of B. coregoni in the samples varied between 2 and 9 ind. L-'. No systematic change in the density of B. coregoni was detected in CHA samples or REF samples, the regression being insignificant for both sample series. In both copepod families, cyclopoids (mainly Mesocyclops leuckarti) and calanoids (mainly Eudiaptomus gracilis), the preservation time lag had a hlghly significant negative effect on density, while in the reference samples no density reduction was found (Fig. 1). The greatest density reduction in the CHA sam les occurred in cyclopoids, the average density of which decreased from 26 to 7 ind. L- P during the experiment, and the effect of preservation time lag was highly significant. For the density of cyclopoid nauplii, the preservation time lag had a significant negative effect both in the CHA samples and in the REF samples. When different cladoceran species were combined, preservation time lag had a highly significant negative effect on density, while in the REF samples no effect on cladocerans was found. Similar results were obtained when cyclopoids and calanoids were combined to a single group of copepods, as well as when cladocerans and copepods were analyzed together as crustaceans. The prey remains identified from the guts of chaoborids consisted exclusively of cyclopoids. Preservation time lag had no significant effects on the mean individual body length, in any of the taxa in the CHA samples (Fig. 2). In the REF samples, however, the mean individual body length of D. cristata and calanoid copepods increased significantly with increasing preservation lag.

5 DISCUSSION The time lag between sampling and sample preservation is usually not reported. Therefore, it is difficult to evaluate how large errors in zooplankton studies are generally caused by preservation time lags. Our results demonstrated that a lag of few hours can substantially bias zooplankton density estimates when large invertebrate predators are included in the samples. According to the regression equations, a three-hour preservation REF Calanoida REF Daphnia spp. Downloaded by [University of Helsinki] at 21:25 30 January 2014 % 100 CHA Calanoida REF Cyclopoida I REF Bosmina spp. 100 CHA Cyclopoida CHA Bosmina spp > Length (mm) Length (mm) Figure 2. The size distribution of zooplankton in the O-h and 12-h samples. 602

6 lag, for instance, caused a >5% underestimation of population density estimate of cyclopoids when chaoborids were included in the samples, and a six-hour lag resulted in a 10% error. Such an effect was caused by five chaoborid larvae per sample, but the inclusion of much higher number of larvae in zooplankton samples can take place when samples are taken with net hauls. In numerous water bodies, the density of chaoborid larvae in the water column is > 1,000 ind. m-2 and densities exceeding 5,000 ind. m'2 are also common (Xie et al. 1998, Rabette and Lair 1999, Liljendahl-Nunninen et al. 2002). If the density of larvae is 2,000 ind. m-', for instance, and zooplankton samples are hauled with a net having a diameter of 30 cm, five chaoborids are included in the sample if only 3.5% of the larvae from the net area are captured. Since chaoborid larvae are also very transparent, their possible inclusion in the samples is very difficult to detect. In lakes, C. flavicans usually select positively for bosminids and negatively for copepods (Elser et al. 1987, Luning-Krizan 1997, Liljendahl-Nurminen et al. 2003). Hence, it could be assumed that chaoborids included in zooplankton samples would bias the density estimates for bosminids most severely and have-only a minorkffect on copepods. However, in the present study copepods were the most severely affected. Additionally, in cladocerans the largest error was observed in daphnids. In lakes, chaoborids usually prefer bosrninids over daphnids (Liining-Krizan 1997, Liljendahl- Nurminen et al. 2003). The results were thus contradictory with many field studies, but on the other hand, they were in concordance with many experimental results. For instance, Sprules (1972), Swuste et al. (1973), and Pastorok (l980b) demonstrated in feeding experiments that chaoborids, including C. flavicans, select copepods over cladocerans when both prey types are abundant. The usually very low percentage of copepods in the natural diets of chaoborids is attributed to their efficient evasive escape reaction (Pastorok 1980b, Riessen et al. 1984, Luning-Krizan 1997). Compared with cladocerans, however, the body shape of copepods makes them easier for chaoborid larvae to handle, and once copepods are caught they are more likely to be swallowed (Swift and Fedorenko 1975, Pastorok 1980b). In the restricted space of the sample containers the advantage gained by the good escape ability of copepods was diminished and, in accordance with feeding experiments, copepods were most frequently successfully preyed upon. To account for the observed density reductions, each chaoborid should have eaten on average 4.8 cyclopoids, 2.0 daphnids, 0.9 calanoids and 4.6 cyclopoid nauplii per hour. Such high feeding rates seem unrealistic when compared with the results of feeding - experiments, where the feeding rates have usually been only a few prey animals per larvae per hour (Kajak and Ranke-Rybicka 1970, Pastorok 1981, Riessen et al. 1988). It must be considered, however, that the prey densities in the feeding experiments have usually been < 100 ind. L' and have very rarely exceeded 150 ind. L-' (Pastorok 1980b with references). In the sample containers, the total density of prey animals was thousands of individuals per liter and the feeding behavior of chaoborids in such circumstances is poorly known. Croteau et al. (2002) showed that when offered prey in excess of their needs, food consumption rate by chaoborids was several times greater than with smaller food rations. Thus, the food consumption rate by chaoborids-in the sample containers was probably much higher than what could be assumed based on most feeding experiments. However, chaoborids alone were not responsible for all the changes in the sample containers. In REF samples, the density of cyclopoid nauplii decreased steeply with increasing preservation. Also the individual mean size of daphnids and calanoid copepods increased significantly with preservation time lag in the REF samples, while chaoborids included in the samples did not affect the mean size of zooplankton. There are two supplementary explanations for this - the size-selective predation and molting effect of the zooplankton.

7 The decreasing density of cyclopoid nauplii can be result from high density of predatory cyclopoids in the samples. Prey of cyclopoids include small zooplankters such as rotifers and small cladocerans (Gilbert and Williamson 1978, Gliwicz and Umana 1994, Chang and Hanazato 2003, Brand1 2005) as well as copepoid nauplii (McQueen 1969, Confer 1971, Brand1 and Fernando 1981, Gabriel 1985). However, density of calanoid nauplii, which are usually preferred prey over cyclopoid nauplii by M. leuckarti (Jamieson 1980), was not reduced. This could be connected to the differences in embryonic development in E. gracilis and M. leuckarti. E. gracilis is a coldwater species having a slightly shorter hatching time in OC than M. leuckarti, a warmwater species (Jamieson 1980). Thus, even though the egg development time is around three days (e.g., Jamieson 1980), it is possible that reproduction of calanoids might have concealed the effect of cyclopoid predation in the REF samples. Predation by cyclopoids also helps to explain why the predation lag had a stronger effect on daphnids than on bosminids in the CHA samples. Although often small in size, bosminids are invulnerable to predation by cyclopoids, which is attributed to their hard carapace (Kerfoot 1977, Williamson 1980, Santer 1993) and Mesocyclops eda has been found to attack bosminids only when alternative preys are scarce (Williamson 1980). In REF samples, the mean size of daphnids and calanoids increased, and the length distribution of zooplankton showed some shift to upper end of the distribution in 12 hours, indicating individual growth during the time period. A portion of juveniles may have molted to a larger body size over the 12-hour period, shifting the size distribution and mean individual size even without any influence of size-selective predation, but most probably the selective predation by cyclopoids strengthened the effect. Mesocyclops leuckarti prefers small-sized prey, and a prey size limit of 0.5 mm for efficient predation has been suggested (Jamieson 1980, Chang and Hanazato 2003). Among crustacean zooplankton, small stages of large-bodied cladoceran species and calanoid copepods are especially vulnerable for predation by cyclopoids (~nderson 1970, Jamieson 1980, Chang and Hanazato 2003). Thus the predation pressure in the REF samples was targeted to the lower end of the size spectrum of these two groups. The growth in the mean body size or shift in the size distribution of daphnids and calanoids did not occur in CHA samples. In fact, the size distribution showed some evidence of selective feeding of intermediate sized calanoids and daphnids, which is typical of C. jlavicans (Dodson 1970,1974, Liljendahl-Nurminen 2003). Chaoborusflavicans were able to feed on practically all the zooplankters in the samples, since only at a size exceeding ca 1.4 mm are daphnids protected from chaoborid larvae (Spitze 1985, Elser et al. 1987). Almost all the zooplankters were much smaller than this and thus were vulnerable to predation by the larvae. A few chaoborid larvae unintentionally included in zooplankton samples can substantially bias zooplankton density estimates if the time lag between sampling and preservation is in the scale of hours. Zooplankton samples should thus be preserved immediately after the sample haul. In the sample containers, the prey animals are aggregated into a small volume of water, which diminishes their possibilities to escape predators. Therefore, the diets of invertebrate predators in the sample containers may be very different from those reported in field studies. In the present study, the effects of chaoborid larvae in the samples were strongest on cyclop~id copepods, although in the lake chaoborid predation is mainly targeted on cladocerans. Feeding in the sample containers could thus mislead researchers about the diet of chaoborids. Additionally, cyclopoid copepods in the samples may have effects on the density and size estimates of copepod nauplii, daphnids and calanoids. ACKNOWLEDGMENTS The study was supported by the Academy of Finland (projects and ).

8 Uusimaa Regional Environment Centre helped with field equipment, and Jyrki Lappalainen gave helphl comments on the manuscript. Downloaded by [University of Helsinki] at 21:25 30 January 2014 LITERATURE CITED Anderson, R.S Predator-prey relationships and predation rates for crustacean zooplankters from some lakes in western Canada. Can. J. Zool. 48: Baier, C.T. and J.E. Purcell Effects of sampling and preservation on apparent feeding by chaetognaths. Mar. Ecol. Prog. Ser. 146: Brandl, Z Freshwater copepods and rotifers: predators and their prey. Hydrobiologia: 546: Brandl, Z. and C.H. Fernando The impact of predation by cyclopoid copepods on zooplankton. Verh. Int. Ver. Limnol Chang, K.-H. and T. Hanazato Vulnerability of cladoceran species to predation by the copepod Mesocyclops leuckarti: laboratory observations on the behavioural intercations between predators and prey. Freshwater Biol. 48: Croteau, M.-N., L. Hare, and P. Marcoux Feeding patterns of migratory and nonmigratory fourth instar larvae of two coexisting Chaoborus species in an acidic and metal contaminated lake: Importance of prey ingestion rate in predicting metal bioaccumulation. Arch. Hydrobiol. 158: Croteau, M.-N., L. Hare, and A.Tessier Influence of temperature on Cd accumulation by species of the biomonitor Chaoborus. Limnol. Oceanogr Confer, J.L lntrazooplankton predation by Mesocyclops edax at natural prey densities. Limnol. Oceanogr. 16: Dodson, S.I Complementary feeding niches sustained by size-selective predation. Limnol. Oceanogr. 15: Dodson, S.I Zooplankton competition and predation: an experimental test of the size-efficiency hypothesis. Ecology 55: Elser, M.M., C.N von Ende, P. Sorranno, and S.R.Carpenter Chaoborus populations: response to food web manipulations and potential effects on zooplankton communities. Can. J. Zool. 65: Gabriel, W Overcoming food limitation by cannibalism: A model study on cyclopoids. Arch. Hydrobiol. Beih. Ergebn. Limnol. 2 1 : Gilbert, J. J. and C.E. Williamson Predator-prey behavior and its effect on rotifer survival in associations of Mesocyclops edax, Asplanchna girodi, Polyarthra vulgaris, and Keratella cochlearis. Oecologia 37: Gliwicz, Z.M. and G. Umana Cladoceran body size and vulnerability to copepod predation. Limnol. Oceanogr. 39: Hakkari, L On the productivity and ecology of zooplankton and its role as food of fish in some lakes in Central Finland. Biol. Res. Rep. Univ. Jyvaskyla 4:l-87. Horppila, Project background and lake description. Arch. Hydrobiol. Spec. Issues Advanc. Limnol. 59: Horppila, J., A. Liljendahl-Nurminen, and T. Malinen Effects of clay turbidity and light on the predator-prey interaction between smelts and chaoborids. Can. J. Fish. Aquat. Sci. 61 : Horppila, J., T. Malinen, L. Nurminen, P. Tallberg, and M. Vinni A metalimnetic oxygen minimum indirectly contributing to the low biomass of cladocerans in Lake Hiidenvesi - a diurnal study on the refuge effect. Hydrobiologia Jamieson, C.D The predatory feeding of copepodid stages I11 to adult Mesocyclops leuckarti (Claus). Pages In: W.C. Kerfoot (ed.), Evolution and ecology of zooplankton communities. University Press of New England. Hanover. Kajak, Z. and B. Ranke-Rybicka Feeding and production efficiency of Chaoborus 605

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