Determining habitat partitioning and metamorphosis processes of Odonata through isotope analysis

Melanie Jackson, Alex Lee, Alexandria Novo, Jack Sunderland Abstract Homogeneous habitats may partition into microhabitats for smaller organisms through geographical barriers !e hypothesi"ed Odonata nymphs collected at sites #ith drop$off "ones, #hich are barriers for macroinvertebrates, #ould have a difference in %&'N and % &() isotope values *n addition, #e are also studying if hemimetabolous insects +e g Odonata,undergo isotopic fractionation like holometabolous insects +e g Lepidoptera, -re$ and post$metamorphosis, #e predicted that the isotope values of the Odonata larva #ould e.uate to the combined exuvia and adult, obeying the La# of )onservation of Mass /ive adults and exuviae pairs and three nymphs from the Epitheca genus from South /ishtail 0ay and five P. obscurus nymphs per site from four sites in 1ouglas Lake #ere collected and analy"ed 2ur results indicate three distinct Odonata nymph populations across sampling sites 3he % &() values of the North /ishtail 0ay nymph population #ere most depleted, #hile the %&'N values of the South /ishtail 0ay nymph population #ere most enriched in the four sites 2ur results also indicate significant % &() fractionation during metamporphosis )omparison of means for %&'N of larvae and for combined %&'N of adults and exuviae sho#ed no significant difference Ho#ever, means for % &() of larvae and for combined % &() of adults and exuviae #ere significantly different Lipids #ere extracted from the E. spinigera samples to test if lipids caused the % &() fractionation Statistics sho#ed that lipids #ere not a significant source of fractionation 1ue to the differences in % &() and %&'N, #e conclude habitat partitioning is occurring in 1ouglas Lake No significant %&'N fractionation occurred bet#een the larval and adult stage, but #e found that adults and exuviae #ere significantly more &() $depleted compared to larvae !ith this information, #e can further explore 2donata food #ebs and metamorphosis processes Introduction *n environments that seem homogeneous on a large scale, habitat partitioning may occur for some species on a smaller scale due to dispersal limitation or habitat speciali"ation 3opographical differences can create microhabitats that may affect an organism4s behavior, including its foraging habits +5akareko et al 677', !ant et al 677', 8ey et al 67&6, /or example, among individuals of the same species of )hironomid larvae +C. plumosus, #ithin the same lake, %&'N and % &() values differed for individuals living at different depths +&7 and &' meters, 3he high level of intraspecific variability

#as attributed to prolonged anaerobic conditions at deeper depths +9rey et al 677:, *n another study, t#o invasive species of fish, the monkey goby and racer goby, #ere found to have different diets bet#een the t#o species and #ithin species based on habitat #ithin the ;istula 8iver, -oland +5akareko et al 677', Stable isotopes #ere also used to determine the diet of giant petrels in t#o different populations in Antarctica and southern -atagonia, #here population$specific differences in foraging distribution #as observed #ithin the Antarctic populations +8ey et al 67&6, 0ased on the organism4s dispersal abilities and habitat usage, geographic barriers can cause limitations in an organism4s reali"ed niche even if an environment may seem homogeneous at larger scales Stable isotope ratios have proven a useful tool for ecologists studying a #ide variety of organisms !hen comparing isotope values in organisms the carbon +), and nitrogen +N, levels are calculated relative to the accepted *sotope Notation Standard +/ry 677<, 3he differences in ) and N isotopic signatures of individuals or populations are due to varied environment and diet +8ey et al 67&6, /urthermore, ) and N isotopic signatures of the organism are generally enriched relative to the food=prey the consumer ingests +/ry 677<, Symes et al 67&7, *n addition, isotopic enrichment has been sho#n to increase #hile climbing higher in trophic levels in general communities +3ibbets et al 677>, *f habitat differences result in diet differences among different populations, then stable isotope ratios can be used to detect this variability 0y comparing both %&'N and % &() signatures of individuals from different populations and diet differences, habitat partitioning across a seemingly homogenous environment can be inferred Stable isotopes can also be used in detecting important physiological changes that occur during development /or example, holometabolous insects, such as butterflies, have been sho#n to favor heavy nitrogen isotopes during metamorphosis resulting in relatively &'N enriched adults compared to larvae +3ibbets et al 677>, 3his fractionation during metamorphosis is important to recogni"e #hen constructing food #ebs or conducting studies on the different life stages of insects +3ibbets et al 677>, 3he changes that occur in holometabolous insects during metamorphosis do not completely follo# the same life cycle as hemimetabolous insects, thus similar fractionations during metamorphosis cannot be assumed Hemimetabolous species4 life cycles transition from a.uatic to terrestrial species, unlike holometabolous #hich are constantly terrestrial organisms Hemimetabolous organisms also undergo an egg, larval and adult stage, omitting the pupal stage, #hich holometabolous species don4t 1ue to the lack of a pupal stage in hemimetabolous insects, they are said to undergo an ?incomplete metamorphosis@ +Mead 677A, 3he use of stable isotopic signatures to characteri"e #hether or not isotopic fractionation occurs at metamorphosis for hemimetabolous species as it does in holometabolous species has yet to be thoroughly investigated

1ouglas Lake is a ' ( mi6 inland lake in northern$lo#er Michigan that sho#s a great deal of depth variation 3he maBority of the lake does not exceed depths of A & m Ho#ever, there are several deep "ones, reaching depths of 6: : m 3hese "ones may in turn act as geographical barriers to small organisms such as dragonfly +Odonata, nymphs #hich tend to live in shallo# #aters, and result in observable habitat partitioning Nymphs feed on various invertebrates including mos.uito larva, seed shrimp, midges, #ater fleas, other nymphs +/ebria et al 677', and #ill hunt vertebrates such as small fish and tadpoles +Mc9uffin et al 67&6, 1epending on the species, nymphs can spend several months to years in their a.uatic form +Stoks et al 67&&, 2nce ready to emerge, nymphs leave a.uatic habitats to climb onto a vertical surface #here a larval case +i e exuviae, is shed as a teneral dragonfly emerges +Mead 677A, Nymphs also tend to live in areas #ith substrate suitable for their hunting techni.ues, and they may even re.uire micro habitats #ith particular sediment si"e #hich may limit their habitat range +Hager et al 67&6, 1ragonfly nymph dispersal limitation and hemimetabolous life cycle, provides a useful system in #hich stable isotope ratios can be used to +&, characteri"e habitat partitioning across a homogeneous environment +i e 1ouglas Lake, and +6, determine the magnitude of isotopic enrichment or depletion after adult emergence 3o determine #hether or not habitat partitioning occurs in 1ouglas Lake on a small scale, #e compared ) and N stable isotope ratios for one species of dragonfly nymph +P. obscurus, at four sites sho#ing topographic differences across 1ouglas Lake Specifically, #e ask #hether or not there is observable diet variation across dragonfly nymph populations that reside in the same a.uatic ecosystem, but that may experience variation in food sources and=or limited dispersal bet#een populations !e hypothesi"e that %&'N and % &() values for nymphs #ill be different across all sites and #e predict that these differences #ill be most drastic for the nymphs living in South /ishtail 0ay as it is the most geographically isolated *n order to test #hether or not isotope fractionation occurs in Odonata during metamorphosis from nymph to adult, #e compared levels of %&'N and % &() in ne#ly emerged dragonfly adults #ith those of late stage nymphs of the same genus +Epitheca, Specifically #e investigated #hether or not there is a significant enrichment or depletion in %&'N and % &() signatures bet#een nymphs and the combined isotope values of exuviae and teneral adults !e hypothesi"e that fractionation #ill not occur during metamorphosis due to the La# of )onservation of Mass 3herefore the %&'N and %&() values for larvae and for combined exuviae and adults should be the same Methods and Materials

Study system 1ouglas Lake is a kettle lake #hich formed #hen the glaciers receded 3here are seven significant depressions throughout the lake, the deepest at 6: meters +/igure &, 1ouglas Lake is located in )heboygan )ounty, Michigan, covering &,(C( hectares Most of the shores of 1ouglas Lake are not developed and the Dniversity of Michigan 0iological Station o#ns a large portion of shoreline, #hich is dedicated to research 3he lack of development results in high macroinvertebrate diversity in the lake +DM0S 67&6, Samples #ere collected using 1$/rame 1ip Nets at four different locations #ithin 1ouglas LakeE North /ishtail 0ay +N :' '><:6 ! 7>: <'<<C,, South /ishtail 0ay +N :' '<776 ! 7>: <C('6,, Maple 0ay +N :' 'C6:> ! 7>: C6<<',, and -ell4s *sland +N :' 'C(66 ! 7>: C&&6(, +/igure &,

Figure 1E Map of 1ouglas Lake Study sites outlined #ith yello# boxes Study sites include four Locations +from left to right,E Maple 0ay, -ell4s *sland, North /ishtail 0ay, South /ishtail 0ay Nymph Collection !e collected five teneral and exuvia pairs, of the species Epitheca spinigera +spiny baskettail,, and one nymph of the same species !e also collected t#o nymphs of the same genus Epitheca princeps +prince baskettail, 3he ne#ly emerged dragonflies and their exuviae and the nymphs #ere collected along the banks of South /ishtail 0ay and fro"en #ithin the hour /ive P. obscurus samples #ere captured from each of the four sites Specimens #ere placed into a controlled a.uarium habitat

containing only tap #ater #hich #as replaced every t#enty$four hours for t#o days to purge gut contents After the t#o day period, the specimens #ere fro"en over$night at a temperature of $<' F) and placed into a lyophili"er for 6: hours After removing the li.uid content, the samples #ere ground using a mortar and pestle %&(C and %&'N analysis 3o analy"e %&() and %&'N signatures, each sample #as first divided into t#o parts 2ne part #as acid$treated using :77 Gl of 7 6'M hydrochloric acid to remove calcium carbonate build$up on the nymphs4 body to account for any influence of calcium carbonate on %&() values 3he second part #as processed #ithout acid to measure %&'N values ) and N isotopic content #as measured using a /innigan 1elta -lus HL Mass Spectrometer 3o calculate % &() and %&'N isotopic signatures, sample isotope ratios of ) and N #ere compared to those of standardsE -ee 1ee 0elemnite +&() , and air +&'N,, respectively 0y comparing the differences in rates in #hich the isotopes travel, relative to the accepted scientific standard values, isotope fractionation #as measured Mean values are listed as meansIS J M unless other#ise noted 3he alpha level for statistical significance #as set at 7 7' Lipid Extraction Preparation 3o test for fractionation in % &() isotope values, a lipid extraction #as performed #ith the acid$ treated E. spinigera adults and nymph samples 2ne mL of chloroform methane #as added to each acid$treated sample in a vial 3he vials under#ent mixing using a test tube mixer and then placed into a sonic bath for ten minutes After the bath, each vial #as centrifuged for one minute Dsing a pasteur pipette, the li.uid substrate +the lipids, #as extracted 3he #hole process #as repeated t#o additional times in order to ensure maBority of the lipids #ere extracted Statistical Analysis *n order to ensure a normal distribution of samples, K$K -lots #ere generated in S-SS -rogram 3he habitat partitioning section of our data #as analy"ed using an one$#ay AN2;A analysis #ith the 3ukey post hoc test to compare the %&() and %&'N for each location 3he data #as then plotted on a scatter$plot to #ith %&() on the x$axis and %&'N on the y$axis 3he isotopic fractionation section of our data #as analy"ed using *ndependent$Samples 3$tests to compare the significance bet#een mean isotope values amongst the samples of each life stage *n order to compare isotope ratios from Epitheca larvae #ith the combined ratios of E. spinigera adults and exuviae, both mass and elemental percentage had to be accounted for 3his #as

achieved using the follo#ing e.uationsE %&'N adultLexuviaM +NNadult,+Madult,+ %&'Nadult, L +NNexuvia,+Mexuvia,+ %&'N exuvia, +NNadult,+Madult, L +NNexuvia,+Mexuvia, 3his value should theoretically e.ual % Nlarva due to )onservation of Mass +MMdry mass,
&'

% &()adultLexuviaM +N)adult,+Madult,+ % &()adult, L +N)exuvia,+Mexuvia,+ % &()exuvia, +N)adult,+Madult, L +N)exuvia,+Mexuvia, 3his value should theoretically e.ual % &()larva due to )onservation of Mass +MMdry mass, Dsing these corrected values for %&'N adultLexuvia and % &()adultLexuvia *ndependent Samples 3$tests #ere performed using S-SS to compare these values #ith % &()larva After lipid extraction, -aired$Samples 3$tests #ere used to determine #hether extraction has a significant effect on % &() values in adults A -aired$Samples 3$test #as performed on pre$ and post$ lipid extraction samples to detect #hether or not extraction made a significant difference in &()$ depletion of combined adult and exuvia samples 3o determine #hether or not lipid extraction eliminated fractionation, an *ndependent$Samples 3$test #as used to compare the isotope ratios of larvae #ith those of extracted adults mathematically combined #ith exuviae

Results Habitat Partitioning ) and N isotopic signatures sho# clustering of nymphs collected across sites, suggesting three distinct populations +i e North /ish 3ail 0ay population, South /ish 3ail 0ay population, and the Maple 0ay=-ell4s *sland population, and habitat partitioning across 1ouglas Lake +/igure 6, %&() values of the North /ishtail 0ay nymph population #ere more depleted that those of populations from all other sites +p O 7 7',, South /ishtail 0ay, -ell4s *sland, and Maple 0ay nymph populations had statistically similar % &() values +South /ishtail x -ell4s *sland p M AC6, South /ishtail x Maple 0ay p M & 777, and -ell4s *sland x Maple 0ay p M A'>, +3able &, %&'N values of the South /ishtail 0ay nymph population #ere more enriched than those of populations from all other sites +p O 7 7', All other nymph populations had statistically similar %&'N values +-ell4s *sland x North /ishtail 0ay p M A>(, -ell4s *sland x Maple 0ay p M A<>, North /ishtail 0ay x Maple 0ay p M >:', +3able &,

Figur e 2E % &() and %&'N isotope values comparing P. obscurus nymphs from all four locations

Site %&'N +No, %&() +No, North /ishtail 0ay : C +7 (,b $6' A6 +7 :A,a South /ishtail 0ay ' : +7 (,a $66 A'+7 <6,b b -ell4s *sland : < +7 (, $66 CA +7 <&,b Maple 0ay : ' +7 :,b $66 AC +7 'A,b Table 1: Mean %&'N and % &() values for each sample site ;alues in parentheses represent I & standard deviation +S 1 , )omparisons made using 2ne$!ay AN2;A +%&'NE /M< A:<, d f M(, pM7 77(P % &()E /M(( CA(, d f M(, pO 77&, and -ost Hoc 3ukey Analysis Lo#er case letters represent significant differences +pO7 7', ractionation in Nymphs, Adults and Exu!iae in Epitheca )omparison of means for %&'N of larvae +meanM: & I7 >No, and for combined %&'N of adults and exuviae +meanM: C I7 'No, sho#ed no significant difference +tM7 <C(, d f M<, pM '6', Ho#ever, means for % &() of larvae +meanM$6: :7 I7 <<No, and for combined % &() of adults and exuviae +meanM$6C <A I7 6>No, #ere significantly different +tM$: &66, d f M <, pM 77',

3he adult and exuviae combined samples before extraction +meanM$6C <A I7 :>No, #ere significantly more &()$depleted than the samples +meanM$6C :6 I7 :<No, after extraction +tM$' 767, d f M:, pM7 77C, )omparison of means of % &() for larvae +meanM$6: :7 I7 <<No, #ith those of exuviae and extracted adults +meanM$6C :6 I7 :CNo, sho#ed a significant difference +tM$( >'<, d f M<, pM7 77A, Larvae remained &()$enriched compared to adults and exuviae after lipid extraction /igure ( displays % &() means for larvae, adults and exuviae before, and adults and exuviae after #ith error bars of t#o standard errors

Figure 3: % &() and %&'N mean values for larva and pre and post extraction adultsLexuviae Discussion Habitat Partitioning 1ue to the differences in % &() and %&'N #e conclude that there is habitat partitioning in 1ouglas Lake that may be caused by the drop offs or the differences in vegetation surrounding the lake Maple 0ay and -ell4s *sland do not seem to be distinct habitats #hich is unsurprising because Maple 0ay and -ell4s *sland are the closest to each other, and there are no deep drop offs bet#een the t#o sites that could inhibit the movement of both nymph populations and their prey 2nly North and South /ishtail 0ay habitats are distinct from one another and from Maple 0ay and -ell4s *sland 3his is likely

because South /ishtail 0ay is only shallo# for a couple feet, follo#ed by a very deep drop off #hich #ould separate invertebrates North /ishtail 0ay is isolated from the other locations geographically #hich could explain the partitioning for an organism as small as a nymph and its prey 0y comparing % &() and %&'N values for different samples, conclusions can be made about the source of energy +% &(), and the trophic level of the sample +%&'N, +-onsard et al 6777, !e found evidence that the P. obscurus nymph population living in North /ishtail 0ay has a different energy source compared to populations living in other locations in 1ouglas Lake 2ne explanation for the difference in % &() values may be that the primary consumers, #hich the nymph population predates, are consuming different organic material at North /ishtail 0ay compared to the other locations Jvery plant has a different carbon value, and terrestrial plant life and suspended organic matter are &()$depleted compared to algae, #hich may explain our results +9rant 67&6, 3he bank of North /ishtail 0ay had a higher abundance of pine trees relative to the other three sites 3he other sites had an observable composition of deciduous trees #hich may also reflect result in the carbon difference #e noticed !hen trees and leaves fall into the #ater, organisms begin to decompose the organic material and their % &() levels change Normally algae and )( plants have a different % &() value, but if the primary consumers are eating both, their % &() levels #ill reflect the relative proportions corresponding to the diet -rimary consumers eating more algae #ill be more &()$enriched and those eating more )( plants #ill be more &()$depleted +9rant 67&6, 3he diet of these primary consumers #ill then be reflected in the % &() levels of the nymphs #hich are normally secondary predators 3he nymph population collected in South /ishtail 0ay has a higher %&'N value #hich suggests that they are higher on the trophic level compared to the nymph populations in the other locations 1ifferent %&'N values reflect at #hat trophic level a consumer exists at, and the higher the value, the higher the sample is on the trophic level +!ebb et al &AA>, !e kno# that nymphs are at least secondary consumers, and since the nymph population collected at South /ishtail 0ay have such a higher %&'N value, members of these populations may act as tertiary consumers in that part of 1ouglas Lake ractionation bet"een Epithica Li#e Stages A previous study has demonstrated %&'N fractionation bet#een larvae and adults of holometablous insects +3ibbets et. al 677>, !e investigated possible fractionation in both %&'N and %
&(

) for hemimetabolous dragonflies from the genus Epitheca No significant %&'N fractionation

occurred bet#een the larval and adult stage Ho#ever, analysis of % &() fractionation gave us an unexpected result )ontrary to our prediction that combined adults and exuviae #ould have similar %

&(

) values compared to larvae due to )onservation of Mass, #e found that teneral adults and exuviae 3his unexpected result suggests the possibility that the accumulation of lipid reserves during

#ere significantly more &()$depleted compared to larvae metamorphosis to be used by the emerging adult could be the cause of this depletion because lipids tend to be &()$depleted compared to the #hole body +!ebb et. al &AA>, *n a study focused on territorial damselflies +Calopteryx splendens xanthostoma, it #as discovered that young pre$territorial males of this species had larger fat reserves than older males 3hey also sho#ed that the ability to succeed in fights, and therefore gain territory, #as correlated #ith energy reserves +-laisto# Q Siva$ Jothy &AA<, 3hus perhaps as in damselflies, fat reserves are important for the success of young E. spinigera dragonflies and the ne# energetic demands they face in a terrestrial environment !e postulated that perhaps the need to store energy as fat for flight #ould result in the conversion of stored carbohydrates to lipids during emergence *n order to test #hether or not excess lipid in the adults #as the reason for this fractionation #e extracted the lipids from the adult samples and ran them through the mass spectrometer again 3he lipid extraction resulted in a significant change in % &() before and after for combined exuvia and adult samples After extraction, the sample #as significantly less &()$depleted Ho#ever, the lipid extraction did not account for the drastic fractionation initially observed !hen compared #ith the larvae, the post$extraction adult and exuvia combined % &() values #ere still significantly lo#er 3hus, our hypothesis that dragonflies create excess lipid reserves during metamorphosis #hich accounts for their &()$depletion is not supported /urther experimentation is needed to explore the cause of &()$depletion in adult dragonflies Sources o# error 3he four locations chosen for testing habitat partitioning of 1ouglas Lake only included the southern side of the lake *f sampling #as done on the northern side, the experiment #ould account for a higher percentage of the lake area /urthermore, more geographical barriers and distance bet#een sites #ould likely sho# higher variation in the isotopic signatures of nymph populations 2ur results for isotope values in nymphs from -ell4s *sland and Maple 0ay #ere similar due to the close proximity of the t#o sites from each other 2ther locations such as North /ishtail, #hich #as more spatially and geographically isolated from the other tested sites, had more a significant difference from the other three sites 2nly one nymph sample #as captured for the matching teneral E. spinigera and the exuvia 3he other t#o nymph samples #ere of the same genus, Epithica Ho#ever, to ensure accurate data, a single species #ould have been more beneficial *n addition, the mass spectrometer sho#ed a standard

deviation of 7 6 for %&'N and 7 &' for % &(), #hen measuring a control Odonata sample, #hich may cause marginal ske#ing of the data 1ue to the mass spectrometer margin of error, repetition of each individual sample in the mass spectrometer should be employed, using an average of the isotope values for a single sample uture Explorations 2ur experiment studying habitat partitioning in 1ouglas Lake only tested a small fraction of the complex interactions #hich exist in an ecosystem 3here is still uncertainty regarding #hether nymphs or their prey that are more affected by the drop$offs in 1ouglas Lake /uture experiments should consider testing prey isotope values along #ith the vegetation in and around the #ater, looking further at different species4 behavior and the substrate at a location 3he difference in carbon values can be explained by the vegetation in the lake, either from algae, suspended organic matter, or terrestrial plant life *n future studies, vegetation should be tested to determine the cause of the carbon values -revious studies have sho#n that even #ithin a genus, different species have evolved their o#n niches based on substrate +9ibbs 677:, 3he behavior of different nymph species affects their dispersal 3he P. obscurus nymphs are ambush predatorsP they bury themselves in the sand and #ait for prey to come to them #hich means that they travel much Ho#ever, other nymphs, like the Epitheca, tend to cra#l along the lake bed and are more likely to have a homogeneous population across 1ouglas Lake due to a larger range Also, nitrogen isotope values can be further studied to understand #hat trophic levels nymphs are feeding at 2verall, there is much more information that could be studied in order to completely understand the interactions in the different areas of 1ouglas Lake, and specifically the importance of nymphs to this a.uatic ecosystem 2ne aspect of our experiment that #e #ere unable to further explore #as the fractionation in %
&(

) from dragonfly nymphs to teneral adults and exuviae !e #ere able to determine that fractionation

#as not caused by the creation of lipid reserves during metamorphosis 2ne possible future experiment to determine the cause of fractionation #ould be to raise numerous dragonfly nymphs in a controlled habitat to ensure diet is not a confounding variable *n addition, the teneral adults should be sampled after a set amount of time once emerged 3his is to ensure tenerals are the same age and are in the same stage of development A controlled environment could also allo# for the collection of )26 during emergenceP this )26 could be tested for carbon isotope ratios *f there is a significant amount of )26 excreted after or during emergence, and this )26 is relatively &()$enriched, then this could account of the &()$depletion #e have #itnessed in adults 3he importance of this research is critical to understanding food chains and trophic levels

amongst organisms in a community Dnderstanding isotope fractionation is especially important #hen studying insects at different life stages because fractionation could impact these comparisons 0etter comprehension of ho# organisms interact, allo#s for better conservation of natural areas, especially fresh#ater ecosystems $ #hich can be fragile !e have sho#n evidence that supports habitat partitioning in 1ouglas Lake Additional testing #ith assorted food sources may reveal the diet of nymphs 3he more #e kno# about the diet of dragonfly nymphs, #ho are both consumers and prey, the more #e can learn about the other organisms in this ecosystem and the interactions that occur

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&(

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