Ontogenetic differences in isotopic signatures and ... - Oxford Journals

JOURNAL OF PLANKTON RESEARCH

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Ontogenetic differences in isotopic signatures and crop contents of Chaoborus ANURANI D. PERSAUD* AND PETER J. DILLON DEPARTMENT OF CHEMISTRY, TRENT UNIVERSITY, PETERBOROUGH, ONTARIO, CANADA K9J

7B8

*CORRESPONDING AUTHOR: [email protected] or [email protected] Received May 8, 2009; accepted in principle September 27, 2009; accepted for publication October 1, 2009 Corresponding editor: Mark J. Gibbons

Intra-specific changes in trophic interactions due to ontogenetic transformations in macro-invertebrates can add to complexity of freshwater food webs. The objectives of this study are therefore to examine isotopic signatures and crop contents of different Chaoborus species and life stages, and to determine whether ontogenetic changes in diet are reflected in their isotopic signatures. Different Chaoborus species and life stages were collected from 15 Precambrian Shield lakes for stable isotope and crop contents analyses. In general, early instar Chaoborus d13C and d13CLC (lipid corrected d13C) signatures were enriched in 13C, while their d15NDC (Daphnia corrected d15N) signatures were lower compared to those of late instars and pupae. Larval Chaoborus size was significantly related to their d13C, d13CLC&DC (lipid and Daphnia corrected d13C), and d15NDC isotopic signatures. Chaoborus crop contents varied among species and larval instars. Generally, rotifers were numerically predominant in crops, with declining abundance in late instars. Larger quantities of copepods, nauplii and cladocerans were found in crops of late instars compared with early instars. Overall, early instars consumed more rotifers and phytoplankton and had lower d15NDC, whereas late instars consumed comparatively larger quantities of copepods, nauplii, cladocerans and early instars, and had higher d15NDC. Together, our results show that there are differences among larval Chaoborus life stages. Hence, different instars of these important aquatic predators cannot be grouped together, but should be separated by species and life stage when examining trophic interactions in freshwater food webs.

I N T RO D U C T I O N Trophic relationships play an integral role in structuring aquatic ecosystems and lead to complexity in their food webs. While inter-specific interactions are often the basis for the complexity of aquatic food webs, intra-specific changes in trophic interactions linked to ontogenetic changes can add to this complexity. As animals increase in body size during ontogenetic development, the size and trophic position (TP) of their prey items increase (Moore et al., 1994; Abrusa´n, 2003). Ontogenetic changes in diet are often reflected in the isotopic signatures of aquatic organisms. Variations in d13C signatures linked to ontogenetic changes have been reported for fish (Grey, 2001; Witting et al., 2004),

polychaetes (Hentschel, 1998) and several crustacean species (Dittel et al., 2005; Matthews and Mazumder, 2006; Ventura and Catalan, 2008). Ontogenetic shifts and size-related changes in d15N signatures have been found in fish (Fry et al., 1999; Grey, 2001), several crustacean species (Branstrator et al., 2000; Gorokhova and Lehtiniemi, 2007; Ventura and Catalan, 2008) and Chaoborus trivitattus (Matthews and Mazumder, 2007). On the contrary, no consistent patterns were found between isotopic signatures and animal size and dietary changes for some fish species (Vander Zanden et al., 2000) and bivalves (Rossi et al., 2004). Stable isotope analysis (SIA) is a widely used biochemical technique which allows for rapid evaluation

doi:10.1093/plankt/fbp099, available online at www.plankt.oxfordjournals.org. Advance Access publication 2 November, 2009 # The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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and estimation of diet and trophic level in ecosystem studies. As a method for studying trophic interactions, SIA offers the advantage of providing information regarding the diet assimilated over a length of time. On the other hand, the dietary approach (i.e. analysis of stomach /crop contents) is a labour-intensive method that gives a snapshot of an organism’s diet. By applying these two complementary methods, we are able to better interpret and elucidate trophic relationships and strengthen our conclusions. Stable isotope analyses are often made on macro-invertebrates (Branstrator et al., 2000; Gorokhova and Lehtiniemi, 2007; Matthews and Mazumder, 2007; etc), but linking isotopic signatures to actual crop contents is rarely done (Creach et al., 1997; Johannsson et al., 2001; Gorokhova and Lehtiniemi, 2007) and has not been attempted for larval Chaoborus species. It is important that we determine whether their trophic dynamics can be easily predicted using less labour-intensive biochemical methods such as SIA. For larval Chaoborus, developmental changes in diet are largely associated with changes in their gape diameter which occurs during metamorphosis of the four larval stages (Moore, 1988). Consequently, early instars (I and II) feed mainly on phytoplankton and small rotifers, whereas late instars (III and IV) tend to consume larger rotifers, meso-zooplankton and even their own young (Moore, 1988; Moore et al., 1994). In addition to intra-specific ontogenetic changes, inter-specific differences also occur due to differences in size among species (Fedorenko, 1972a; Hanazato, 1990; Moore et al., 1994). Larval Chaoborus play a central role in temperate foodwebs functioning as both predator and prey. When abundant, Chaoborus can control the spatial distribution (Masson and Pinel-Alloul, 1998; Lagergren et al., 2008), relative abundance (Yan et al., 1991; Wissel and Benndorf, 1998; Wissel et al., 2003), life history characteristics (Brett, 1992; Sell, 2000) and presence of their prey (Luecke and Litt, 1987). At the same time, they are preyed upon by both planktivorous fish and other invertebrate predators (von Ende, 1979; Ramcharan et al., 2001; Kehl and Dettner, 2003). For larval Chaoborus, predation pressure can regulate their species composition (Garcia and Mittelbach, 2008), abundance (Wissel et al., 2003) and spatial distribution (Dawidowicz et al., 1990; McQueen et al., 1999). Considering that Chaoborus larvae are pivotal in energy transfer dynamics of temperate lakes (Moore et al., 1994), the objectives of this study are to examine isotopic signatures and crop contents of different Chaoborus species and life stages, and determine whether ontogenetic changes are reflected in their isotopic signatures. Here we primarily focus on Chaoborus punctipennis, the most widespread and smallest Chaoborus species in temperate North American lakes (Borkent, 1981), but

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also present information for Chaoborus flavicans and Chaoborus trivitattus.

METHOD Chaoborus collection and stable isotope sample preparation Chaoborus were collected from 15 Precambrian Shield lakes in the Dorset area of Ontario in July and August 2004. Location, and selected morphometric and chemical characteristics are presented for the study lakes in Table I. The lakes were oligotrophic to mesotrophic with total phosphorus concentrations ranging from 3.4 to 28.1 mg L21. Lake DOC concentrations ranged from 2.7 to 14.1 mg L21. Different Chaoborus species and life stages were sampled using a 150 mm mesh conical net (Table I). In each lake, the net was towed vertically through the water column beginning at 2.5 m from the bottom to the surface at the deepest station after nightfall. All animals were picked by hand from fresh composite samples obtained from several net hauls. First and second instar Chaoborus larvae were grouped as early instars, and third and fourth as late instars. Chaoborus were identified and aged by measuring head capsule length (Borkent, 1981). Chaoborus pupae were also collected when abundant. For each Chaoborus species and life stage, two to four replicate samples consisting of 10– 50 animals were prepared for each lake. The animals were rinsed with Milli-Q water, dried at 508C for 48 – 72 h and then ground to a fine powder for SIA (Grey and Jones, 1999).

Chaoborus crop content analyses For crop content analyses, samples were collected from the deepest station of each lake. Initially, samples were preserved with Lugol’s solution to minimize crop eversion, and then transferred to 4% formalin (within a week) for long-term preservation (Moore, 1988). Subsequently, Chaoborus were identified and aged by measuring head capsule length (Borkent, 1981). Following identification and aging, the crop contents were removed and examined. Crop contents of 10– 37 individuals (mean¼29) of each Chaoborus species and life stage collected from each lake were examined individually under a light microscope at 50 and 100 magnification. Crop contents were placed in five broad categories: rotifers, cladocerans, copepods, nauplii and others (including phytoplankton and early instar Chaoborus species) because mastication and partial digestions did not allow identification to species for most remains. Cladocerans were identified by the remains of

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CHAOBORUS ISOTOPIC SIGNATURES AND CROP CONTENTS

Table I: Lake location and attributes, and the Chaoborus species (C. punctipennis, C. flavicans and C. trivitattus), life stage (larva or pupa) and larval instar (early, first and second instars; late, third and fourth instars) sampled in the 15 Ontario study lakes Lake

Latitude Longitude 0

Zmax (m)

DOC (mg L21)

TP (mg L21)

Blue Chalk

45812 78856

0

23.0

2.7

3.9

Brandy

458060 798310

7.5

14.1

28.1

Chub

458130 788590

23.0

7.4

3.4

Crosson Crown Devine

458050 798020 458260 788400 458110 798130

25.0 30.0 9.0

6.9 5.2 7.3

6.9 3.1 10.7

Dickie

458090 798050

12.0

6.7

9.8

Fawn

458100 798150

7.9

11.6

13.9

Healey

458050 798110

5.8

8.0

6.6

Heney Mckay

458080 798060 458030 798100

7.0 19.5

4.4 5.9

6.4 3.5

Moot

458090 798100

7.9

8.4

13.1

Plastic

458110 788500

16.3

2.9

4.2

38.0 13.1

3.8 9.4

3.9 12.8

Red Chalk Saw

0

0

45811 78856 458030 798020

Species

Life stage

Larval instar

C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

Larva Pupa Larva Larva Pupa Larva Pupa Larva Pupa Larva Larva Larva Larva Larva Larva Larva Larva Larva Larva Pupa Pupa Larva Larva Larva Larva Pupa Larva Pupa Larva Larva Larva Pupa Larva Pupa

Late

punctipennis punctipennis flavicans punctipennis punctipennis punctipennis punctipennis trivittatus trivittatus trivittatus punctipennis punctipennis trivittatus punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis punctipennis flavicans punctipennis punctipennis trivittatus trivittatus

Early Late Late Late Late Late Late Late Early Late Early Late Early Late

Early Late Early Late Late Late Early Late Late

All sampling was done in July and August 2004.

where Rsample and Rreference were the sample and reference isotope ratios (13C/12C and 15N/14N). International and internal standards were used for calibration of the isotopic data. The calibration standards for nitrogen were USGS 41 (d15N ¼ 47.57‰, National Institute of Standards and Technology), IAEA-N1 (d15N ¼ 0.4‰, International Atomic Energy Agency) and L-glutamic acid (d15N ¼ 2 4.56‰, Aldrich). The calibration standards for carbon were USGS 41 (d13C¼37.76‰), L-glutamic acid (d13C ¼ 2 29.05‰) and caffeine (d13C, 240.25‰, Acros). In addition to calibration standards, D-glutamic acid (Fisher) was used as a quality control standard with d13C and d15N values of 214.12‰ and 22.71‰. Analytical reproducibility was +0.1‰ for C and +0.2‰ for N. Variation in invertebrate lipid content (reflected in their atomic carbon to nitrogen ratio, C:N) can have significant effects on their d13C (Symntek et al., 2007).

their mandibles, carapaces and other appendages. Copepods were identified by the remains of their exoskeletons and appendages. Rotifers were primarily loricate (Keratella spp. and Kellicottia spp.) and therefore easily identified by their distinctive lorica. Using the number of these prey items found per crop, we calculated percentage frequency of occurrence of each prey category in all Chaoborus individuals for a given instar within each lake.

Stable isotope analyses Isotope ratios and percent of carbon and nitrogen of the samples were determined using a Euro Elemental Analyzer and a Micromass IsoPrime Continuous Flow Isotope Ratio Mass Spectrometer in the Worsfold Water Quality Centre at Trent University. Stable isotope values were calculated as follows:

d‰ ¼ ½

Rsample 11000 Rreference

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Lipids are depleted in 13C compared with proteins and carbohydrates, consequently zooplankton with high lipid content will tend to have lower d13C signatures (Matthews and Mazumder, 2005). To account for the confounding effect that zooplankton lipid content can potentially have on their isotopic variability, lipid correction was performed on all Chaoborus d13C isotopic signatures. Chaoborus lipid corrected d13C signatures (Chaoborus d13CLC) were calculated using the following equation:

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consistently occupied the lowest TP (Persaud et al., 2009). Following Daphnia d15N baseline corrections TP was calculated as follows: Trophic position ¼

Mean d15 NDC þ2 3:4

where 3.4‰ is the enrichment factor per trophic level and 2 is the TP of Daphnia which are used for the baseline corrections (Minagawa and Wada, 1984; Peterson and Fry, 1987; Post, 2002).

Chaoborus d13 CLC ¼ Chaoborus d13 C Chaoborus C : N 4:2 þ 6:3 Chaoborus C : N

R E S U LT S

where 6.3 is the average difference between d13C of protein and lipids for zooplankton, Chaoborus d13C and Chaoborus C:N represents the isotopic value and atomic C:N ratio of the Chaoborus with their lipids, and 4.2 is the typical atomic C:N ratio of zooplankton protein (Symntek et al., 2007). The C:N ratios are based on the percentage of sample carbon and nitrogen obtained from SIA. In addition to lipid normalization, we also accounted for differences in d13C at the base of the foodweb among lakes by performing Daphnia d13C correction on lipid normalized Chaoborus d13C (d13CLC&DC) as follows:

Isotopic signatures Among species, Chaoborus d13C and d15N signatures were negatively correlated (Fig. 1). Chaoborus punctipennis late instars and pupae d13C and d15N were significantly different from those of C. trivittatus. d13C of C. punctipennis late instars and pupae were enriched in 13C compared with those of C. trivittatus (one-way ANOVA: F1, 15 68 ¼ 8.7, P ¼ 0.004). In contrast, the d N of C. trivittatus late instars and pupae were enriched in 15N compared with C. punctipennis and the highest among the species and life stages examined in this study (one-way ANOVA: F1, 59 ¼ 16.6, P , 0.0001) (Table II). In general, early instar Chaoborus d13C and d13CLC signatures were enriched compared with those of late instars and pupae (Table II). Specifically for C. punctipennis, d13C, d13CLC and d13CLC&DC signatures varied among life stages but the differences were not significant (one-way ANOVAs, d13C: F2, 67 ¼ 1.2, P ¼ 0.29; d13CLC: F2, 56 ¼ 0.41, P ¼ 0.66; d13CLC&DC: F2, 50 ¼ 0.60, P ¼ 0.55). Early instar C. punctipennis d13C signatures (230.1 + 0.5‰) were slightly enriched compared with those of late instars (230.9 + 0.3‰) and pupae (231.0 + 0.4‰). Like C. punctipennis, C. trivitattus d13C of late instars and pupae were similar with 235.2 + 0.3 and 232.7 + 0.7‰ signatures, respectively (Table II). Chaoborus d15N and d15NDC also varied among lifestage. d15N and d15NDC signatures were significantly different among C. punctipennis life stages (one-way ANOVAs, d15N: F2,58 ¼ 26.2, P , 0.0001; d15NDC: F2,43 ¼ 7.3, P ¼ 0.001). Early instar C. punctipennis d15NDC signatures (2.0 + 0.3‰) were significantly lower compared with those of late instars and pupae ( post hoc Tukey HSD, P , 0.05). However, d15NDC of late instar and pupal C. punctipennis were not significantly different

Chaoborus d13 CLC&DC ¼ Chaoborus d13 CLC Daphnia d13 CLC where d13CLC represents lipid corrected d13C of Chaoborus and Daphnia. To account for differences in d15N among lakes, the 15 d N signatures of Daphnia, collected from each study lake, were used for baseline corrections on all Chaoborus d15N isotopic signatures (Post, 2002). Daphnia d15N baseline corrections were performed on all Chaoborus d15N signatures (d15NDC) as follows: Chaoborus d15 NDC ¼ Chaoborus d15 N Daphnia d15 N Here we chose Daphnia over POM for our baseline correction because Daphnia feeding behaviour appears to be fairly consistent and non-selective in different lakes and seasons, making it an ideal candidate for use as a pelagic baseline (Matthews and Mazumder, 2003). Daphnia are ecologically related to Chaoborus since they are consumed to varying degrees by different species and larval stages (Moore, 1988; Lunte and Luecke, 1990; Moore et al., 1994), and in our study lakes they

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Fig. 1. d13C versus d15N for all macro-zooplankton species (C. punctipennis - C. p., C. flavicans - C. f. and C. trivittatus - C. t.) and life stages (early, late and pupae) found in the 15 study lakes.

Table II: Mean and SE (standard error) of d13C, d13CLC (lipid corrected d13C), d13CLC&DC (lipid and Daphnia corrected d13C), d15N, d15NDC (Daphnia corrected d15N) and TP (trophic position) for different Chaoborus species and life stages d13C

d13CLC

d13CLC&DC

d15N

d15NDC

Species

Instar

Mean

SE

Mean

SE

Mean

SE

Mean

SE

Mean

SE

TP

C. punctipennis

Early Late Pupae Early Late Pupae

230.1 230.9 231.0 231.1 232.5 232.7

0.5 0.3 0.4 0.7 0.3 0.7

229.4 229.9 229.9 230.2 231.0 231.1

0.6 0.3 0.5 0.6 0.4 0.8

0.6 20.5 20.3 20.2 23.1 22.6

0.9 0.7 0.6 0.2 1.4 1.9

4.6 6.4 6.2 4.9 7.4 7.3

0.2 0.1 0.2 0.1 0.3 0.6

2.0 3.6 3.7 1.3 4.2 4.0

0.3 0.2 0.3 0.2 0.2 0.9

2.6 3.1 3.1 2.4 3.2 3.2

C. flavicans C. trivittatus

d15NDC signatures were positively related to size: d15NDC ¼ 0.4 þ 2.8*(Head Capsule Length), r 2 ¼ 0.57, P , 0.001 (Fig. 3).

with 3.6 + 0.2 and 3.7 + 0.3‰, respectively ( post hoc Tukey HSD, P.0.05). Like C. punctipennis, C. trivitattus d15NDC of late instars and pupae were similar with 4.2 + 0.2 and 4.0 + 0.9‰ signatures, respectively (Table II). When all larval Chaoborus are grouped, their d13C, 13 d CLC&DC and d15NDC signatures were significantly related to Chaoborus head capsule length (Figs 2 and 3). Chaoborus d13C and d13CLC&DC signatures were negatively related to head capsule length as follows: d13C ¼ 2 29.6 – 1.5*(Head Capsule Length), r 2 ¼ 0.15, P ¼ 0.05, and d13CLC&DC ¼ 1.9– 2.6*(Head Capsule Length) (Fig. 2), r 2 ¼ 0.26, P ¼ 0.01. In contrast, their

Crop contents Rotifers were numerically predominant in the crops of larval Chaoborus, except for late instar C. trivittatus (Fig. 4). In early instar stages of C. punctipennis and C. flavicans rotifers accounted for 78% and 64% of the crop contents, respectively. Flagellated phytoplankton such as Ceratium spp. (indicated by “Others”) were important for C. flavicans.

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Fig. 2. Chaoborus size (early and late instars of C. punctipennis - C. p., C. flavicans - C. f. and C. trivittatus - C. t.) versus (a) d13C and (b) d13CLC&DC (lipid and Daphnia corrected d13C) signatures. Average head capsule length (mm) is an indication of animal size. Data are presented for early and late instars found in the 15 study lakes.

Generally, fewer rotifers but larger quantities of copepods, nauplii and cladocerans were found in the crops of late instar Chaoborus larvae. This was clearly the case for late instar C. punctipennis. However, for late instar, C. trivitattus evidence of intra-guild predation was also found since early instar Chaoborus (as indicated by “Others”) were most common in their crops.

Linking isotopic signatures and crop contents Among the various prey items found in Chaoborus crops, rotifers (negative trend) and copepods ( positive trend) were significantly correlated with their d15N, but none of the prey items were significantly correlated with their

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Fig. 3. Chaoborus size (early and late instars of C. punctipennis - C. p., C. flavicans - C. f. and C. trivittatus - C. t.) versus their d15NDC signatures. Average head capsule length (mm) is an indication of animal size. Data are presented for early and late instars found in the 15 study lakes.

Fig. 4. Crop contents for Chaoborus species and life stages (C. punctipennis - C. p., C. flavicans - C. f. and C. trivittatus - C. t.). Others consisted mainly of phytoplankton (for early C. p. and C. f. and late C. p.) and early Chaoborus instars (for C. t.). % Occurrence of crop contents was determined for individual larvae, then means and standard errors were calculated for the “n” number of larvae examined.

d13C signatures (Table III). For only C. punctipennis larvae, a positive trend was also found between copepods and their d15N signatures, but none of the crop contents were significantly correlated with their d13C signatures.

DISCUSSION The results presented here go beyond previous research to illustrate clearly that there are ontogenetic differences in both isotopic signatures and crop contents for larval

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Table III: Correlations between the Chaoborus isotopic signatures and the occurrence of different prey items in their crops All Chaoborus larvae d13C

Cladocerans Copepods Rotifers Nauplii Others

C. punctipennis only d15N

d13C

d15N

r

P-value

r

P-value

r

P-value

r

P-value

20.06 20.15 0.15 0.06 20.13

0.76 0.44 0.42 0.78 0.52

0.32 0.62 2 0.45 0.25 20.12

0.16 0.003 0.04 0.27 0.49

20.06 20.12 0.13 20.02 20.11

0.80 0.60 0.58 0.95 0.65

0.28 0.53 20.41 0.15 20.21

0.26 0.03 0.09 0.55 0.42

Significant correlations are highlighted.

exploiting primary producers in the surface waters and allochthonous food sources (Matthews and Mazumder, 2006). Late instars, on the contrary, perform diel vertical migrations in order to avoid extensive predation (Dawidowicz et al., 1990; McQueen et al., 1999), and can thereby feed on zooplankton in surface waters at night and also take advantage of zooplankton in deeper waters during daytime (Moore, 1988). Such deep water feeding by zooplankton grazers has been linked to lower d13C because of grazing on deep water authochthonous food sources (Matthews and Mazumder, 2006). Ontogenetic-related changes in carbon and nitrogen isotopic signatures have also been reported for other aquatic biota (Branstrator et al., 2000; Gorokhova and Lehtiniemi, 2007; Ventura and Catalan, 2008). Several studies have reported that Daphnia pulicaria d13C and d15N were related to their size (Matthews and Mazumder, 2007, 2008; Ventura and Catalan, 2008). More directly related to our study, Matthews and Mazumder (Matthews and Mazumder, 2007) found that C. trivittatus d15N was related to their head capsule length, but did not examine or relate crop contents to their isotopic signatures. By linking isotopic signatures to crop contents, it is apparent that the size-based increases in Chaoborus d15NDC signatures are indeed indicative of an increase in the trophic level at which they are feeding. In general, early instars consumed more rotifers and phytoplankton and had lower d15NDC, whereas late instars consumed comparatively larger quantities of copepods, nauplii, cladocerans and early instar Chaoborus, and had higher d15NDC. Together the isotopic and crop contents data also demonstrate that there are contrasting feeding strategies such as omnivory and carnivory among the various Chaoborus species and life stages. Chaoborus punctipennis larvae (early and late) are more omnivorous in their feeding, taking prey from multiple trophic levels, phytoplankton, rotifers, nauplii, cladocerans and copepods, with larger prey items such as cladocerans and

Chaoborus. Furthermore, it becomes evident that isotopic differences between early and late instar larvae are linked to differences in their diet, with the older Chaoborus larvae feeding at higher trophic levels compared with younger instars. Ontogenetic differences in d15NDC among larval instars are evident within and between Chaoborus species. Early instar C. punctipennis are at a lower trophic level compared with late instar C. punctipennis. We believe that such larval differences are largely driven by changes associated with growth, specifically increases in gape diameter, since the strong positive relationship between head capsule length and Chaoborus d15NDC accounts for over 50% of the variability in our d15NDC data. An increase in gape diameter enables Chaoborus larvae to take larger prey items, presumably from higher trophic levels (Fedorenko, 1972a; Moore, 1988). Fedorenko (Fedorenko, 1972a) linked C. trivitattus gape diameter to the size of prey items commonly found in their crops, and later Moore (Moore, 1998) clearly showed that prey body width is correlated with gape diameter of C. punctipennis. In addition to our hypothesis of gape diameter being the driving force behind the trend we report between Chaoborus d15NDC and their size, it is possible that other processes such as age (Matthews and Mazumder, 2008) and growth rate (Trueman et al., 2005) are also contributing factors since these parameters are also related to animal size (Borkent, 1981; Peters, 1983). The negative relationships between Chaoborus size and their d13C and d13CLC&DC suggest that Chaoborus are selecting prey with lower d13C as they grow. It is possible that prey availability associated with habitat separation could have contributed to these relationships and the differences in d13C among instars since different Chaoborus species and instars migrate to varying degrees in the water column in order to cope with predation pressure. Early instars spend more time in surface waters (Fedorenko, 1972a; Moore, 1988; Lagergren et al., 2008), and hence feed on grazers that are

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Precambrian shield lakes with a wide range in DOC concentration, there are substantial ontogenetic differences in their isotopic signatures which are associated with changes in larval size and crop contents. We also show that stable isotope information can be used effectively to study the trophic interactions of larval Chaoborus. Based on our results, we recommend that different species and instars of Chaoborus larvae be separated when examining trophic dynamics because there are significant differences in trophic status among them. Together, the differences found in this study give insight into the complexity of freshwater food webs in which larval Chaoborus are found.

copepods being more abundant in the crops of later instar stages. Unlike C. punctipennis, late instar C. trivittatus larvae are carnivores and intra-guild predators. This is supported by their higher trophic status and the abundance of early instar Chaoborus in their crops. While other zooplankton prey items may also occur in the diet of C. trivittatus, it is clear that intra-guild predation is primarily contributing to its trophic status since on average they are 0.7 and 0.9 trophic levels higher than early instar C. punctipennis and C. flavicans, respectively. Among Chaoborus species and instars, copepods and rotifers in their crops were closely related to their d15N. Previous studies have also reported that these are important prey items for Chaoborus species, with relative importance being dependent on species and life stage (Fedorenko, 1972a, b; Moore, 1988; Moore et al., 1994). Of direct relevance, (Persaud and Dillon, under review) reported that late instar C. punctipennis and C. trivitattus d13C signatures are positively correlated with cyclopoid d13C, and late instar C. punctipennis d13C is positively correlated with calanoid d13C. Furthermore, Persaud et al. (Persaud et al., 2009) found that cyclopoid copepods collected from these study lakes generally had higher d15N and lower d13C. This could likely explain why older, larger larvae that feed more on copepods (Fig. 4) have higher d15N and lower d13C. We are limited in any inferences regarding the dietary importance of rotifers, since we found primarily loricate rotifers in Chaoborus crops. It is likely that the importance of hard-bodied, loricate rotifers may have been overestimated since crop content analysis tends to be biased towards foods that are harder to digest (Hyslop, 1980; Sutela and Huusko, 2000). Consequently, even though soft-bodied and weakly loricate rotifers may be important components of Chaoborus diets (Moore, 1988) their occurrence was underestimated. The lack of a significant difference between late instars and pupae d15NDC suggests that metamorphosis from the larval to pupal stage appears to be primarily structural with a negligible effect on nitrogen enrichment. Tibbets et al. (Tibbets et al., 2008) reported similar results where there was little or no difference in d15N for larvae and pupae of some Leptidoptera (Galleria mellonella, Manduca sexta and Vanessa cardui), Coleoptera (Sarcophaga species) and another Diptera species (Teneborio molitor). This is expected since feeding ceases when Chaoborus larvae metamorphose into the pupal stage. Later, as adults emerge from their exuviae significant differences in d15N can be found between the emerging adult and the remaining exuviae (Tibbets et al., 2008). Previous research focusing on larval Chaoborus did not examine both isotopic signatures and crop contents. Here we illustrate that in oligotrophic and mesotrophic

AC K N OW L E D G E M E N T S Thanks to staff of the Ontario Ministry of the Environment in Dorset, Ontario and Worsfold Water Quality Center at Trent University for their assistance. Jane Gowland assisted in all data collection in the field.

FUNDING This project was supported by several scholarships and awards including a Natural Science and Engineering Research Council (NSERC) postgraduate scholarship (CGS-D), an IODE War Memorial scholarship, Canadian Water Resources Association award and an Ontario Graduate Scholarship in Science and Technology awarded to A.D.P.

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