Primary Research Paper

Freeze tolerance in larvae of the winter-active Diamesa mendotae Muttkowski

(Diptera: Chironomidae): a contrast to adult strategy for survival at low

temperatures

R.W. Bouchard Jr., M.A. Carrillo, S.A. Kells & L.C. Ferrington Jr.*Department of Entomology, University of Minnesota, St. Paul, Minnesota 55108, USA(*Author for correspondence: E-mail: ferri016@umn.edu)

Received 7 December 2005; in revised form 6 March 2006; accepted 25 March 2006; published online 17 June 2006

Key words: supercooling point, lower lethal temperature, cold hardiness, lotic, winter activity

Abstract

The winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae) is freeze intolerant in the adultstage with a low mean supercooling point (SCP) of �)20 �C. However, cold-hardiness strategies forimmatures of this species are unknown. In this study, we measured SCP values for D. mendotae larvae,pupae and adults using surface-contact thermometry. In addition, the lower lethal temperature (LLT) wasdetermined for the larval stage. The mean SCPs for larvae ()7.4 �C) and pupae ()9.1 �C) were relativelyhigh compared to adults ()19.7 �C). Our results indicate that the larvae of D. mendotae are freeze tolerantwith a LLT99 ()25.4 �C), �)10 �C lower than their minimum SCP ()15.6 �C). Freeze tolerance in theselarvae may be a strategy to provide protection from short-term exposures to ice crystals or to permitdiapause within frozen substrates. The change in cold-hardiness strategy from freeze tolerant to freezeintolerant between the larval and adult stages of this species is likely a result of the different habitatsoccupied by these two life stages.

Introduction

In cold-temperate regions such as Minnesota,overwintering insects must have the ability tosurvive periods of low temperatures. Some speciesavoid subzero temperatures (<0 �C) by moving toprotected habitats or by migrating long distances(Lee, 1989; Oswood et al., 1991; Irons et al., 1993).However, many insect species exposed to sub-freezing temperatures must have physiologicalmechanisms to survive these conditions. There aretwo commonly recognized cold-hardiness strate-gies used by insects for surviving subzero temper-atures: freeze avoidance (or intolerance) and freezetolerance (Salt, 1961; Baust & Rojas, 1985; Za-chariassen, 1985; Bale, 1989). Freeze-avoidantspecies cannot survive freezing of body tissues andfluids and generally get protection from freezing

by supercooling and/or by seeking protection insheltered microhabitats (Bale, 1989). Supercoolingis the ability to maintain fluids in the liquid statebelow their normal freezing point and the super-cooling point (SCP) is the temperature at whichspontaneous freezing occurs (Sømme, 1982; Za-chariassen & Kristiansen, 2000). Freeze-avoidantspecies usually lower their SCP by synthesizingcryoprotectants (e.g., polyols, sugars and anti-freeze proteins) or by removing ice nucleatingagents (INAs) through mechanisms such as gutclearing (Zachariassen, 1985; Bale, 1989; Lee,1989). In contrast, freeze-tolerant species can sur-vive extracellular ice formation by using INAs thatincrease the SCP to promote a controlled freezingprocess at high subzero temperatures (Zacharias-sen, 1985; Lee, 1989; Block, 1991). Slow andcontrolled extracellular ice formation prevents

Hydrobiologia (2006) 568:403–416 � Springer 2006DOI 10.1007/s10750-006-0200-6

internal cell damage and provides enough time tomake metabolic adjustments allowing these speciesto withstand freezing (Block, 1991). Similar tofreeze-avoidant species, freeze-tolerant species alsouse cryoprotectants such as polyols and sugars;however, the function of these compounds is notto depress the SCP, but to prevent intracellularfreezing, decrease intracellular fluid loss and toprotect macromolecules (Storey & Storey, 1997).

Most cold-hardiness studies involve terrestrialinsects or the terrestrial stages of aquatic insects(e.g., Carrillo et al., 2004a; Bouchard et al., 2006).Limited studies on aquatic stages are likely a resultof the assumption that insects in an aqueous envi-ronment face less extreme conditions because ofthermal buffering from groundwater inputs, thehigh specific heat of water and insulation fromsurface ice (Frisbie & Lee, 1997; Lencioni, 2004). Inaddition, most studies on the freeze tolerance ofaquatic insects have focused on lentic rather thanlotic habitats (Irons et al., 1993). This is probablybecause insects in lentic habitats are more likely toexperience predictable freezing. However, manylotic habitats also freeze at the surface or alongmargins, although the extent may be less predict-able. Aquatic insects may be subjected to super-cooled flowing waters resulting in exposure toice-crystal formation (i.e., frazil ice) within thesupercooled fluid (Ashton, 1979; Oswood et al.,1991). Invertebrates in intermittent habitats mayalso be exposed to subzero temperatures andexternal ice (Frisbie & Lee, 1997).

Most cold-hardy aquatic insects are inactive atsubzero temperatures, but some species are able tomaintain activity (e.g., crawling, flying, mating andovipositing) under these conditions. There areseveral examples of activity at subzero tempera-tures in the genus Diamesa Meigen (Diptera: Chi-ronomidae). Diamesa arctica (Boheman) has beenobserved swarming at temperatures of )0.4 �C(Young, 1969). Kohshima (1984) reported an un-known Diamesa species in the Nepal Himalayasthat was active at )16 �C. This species was subse-quently determined to be two species, Diamesakohshimai Sæther &Willassen andDiamesa yalaviaSæther & Willassen. In Minnesota, Diamesa men-dotae Muttkowski is a common winter-active chi-ronomid that can be observed crawling across thesnow along stream banks at subzero temperatures(Carrillo et al., 2004a). Previous studies have esti-

mated thatD. mendotae adults have a mean SCP of�)20 �C, a value lower than that reported forother winter-active species (Sømme & Østbye,1969; Baust & Edwards, 1979). Mortality of D.mendotae adults occurs approximately at its SCP(Bouchard et al., 2006), indicating that this speciesis freeze intolerant and low SCPs likely conferenough protection to maintain winter activity.

In contrast to adults, the few cold hardinessstudies involving larvae of other chironomid spe-cies suggest that this life stage uses freeze toleranceas a strategy for survival at subzero temperatures(e.g., Scholander et al., 1953; Danks, 1971; An-drews & Rigler, 1985; Lencioni, 2004). The abilityto tolerate freezing is often based on the premisethat these larvae have been extracted from frozensediments, although the state of their body fluidswas not directly determined (Frisbie & Lee, 1997).Determinations of SCPs in chironomid larvaeindicate that most freeze at temperatures between�)11 and )5 �C (Danks, 1971; Baust & Edwards,1979). In some cases, the sediments where larvaeare extracted can be buffered and may not reachtemperatures lower than the SCP of the larvae.However, some studies have identified viable chi-ronomid larvae recovered from lake sedimentswhere temperatures as low as )18 to )20 �C wererecorded (Scholander et al., 1953; Andrews & Ri-gler, 1985). In addition, viable larvae were ob-tained from sediments frozen to )18 �C in thelaboratory (Danks, 1971). Therefore, it is reason-able to assume that some of these larvae werefreezing, but research is needed to provide directevidence for the survival mechanisms of chirono-mid larvae in response to freezing conditions.

In the present study, we hypothesized thatD. mendotae larvae are freeze tolerant and haverelatively high SCPs. Because the pupal stage isnon-feeding and transitional, we also hypothesizedthat pupal SCPs are lower than larval SCPs andcloser to values reported for adults (Carrillo et al.,2004a; Bouchard et al., 2006). To test thesehypotheses, we estimated the SCPs of larvae,pupae and adults of D. mendotae. Adult SCPs weredetermined to confirm previously published esti-mates and to directly compare them with valuesfor larvae and pupae collected from the samelocation at the same time. In addition to SCPs, wedetermined the lower lethal temperatures (LLT)for larvae. However, LLT experiments for pupae

404

were not conducted as a result of the low numberof individuals collected. To our knowledge, this isfirst time SCPs are reported for aquatic pupae andthe first time SCPs and LLTs are reported forlarvae in the subfamily Diamesinae. Our resultswere compared to those obtained from previousexperiments with D. mendotae adults (Carrilloet al., 2004a; Bouchard et al., 2006). Finally, wediscuss the ecological implications of cold-hardi-ness strategies used by this species to surviveexposure to low temperatures.

Materials and methods

Collection and treatment of test specimens

Larvae, pupae and adults of D. mendotae werecollected on 17, 23 and 26 February 2005 fromTrout Brook northeast of Cannon Falls, Minne-sota, USA (44.5453� N, 92.8057� W). Fourth-in-star larvae and pupae were collected from rifflesusing a D-frame dip net with 500-lm mesh. Detri-tus and macroinvertebrates were placed in freezerbags and stored in a cooler containing snow untilreturned to the laboratory. Once in the laboratory,material was poured out into a white plastic panand D. mendotae larvae and pupae were pickedfrom the detritus using soft forceps. Attempts weremade to collect only large, active larvae to insurethat healthy, late instars were tested. Specimenswere picked into 118-ml snap-top jars with damppaper towels and held in a programmable growthchamber (Percival Scientific, Inc., Perry, IA) at6 �C until needed. Adults were located on the snowalongside the stream and scooped into individual3.7-ml snap-top vials with a small amount of snowto prevent dehydration. The vials were stored in acooler containing snow until they arrived at thelaboratory and were held in a growth chamber at6 �C until needed. All experiments were run within72 h of specimen collection, but most were tes-ted<24 h post collection.

Determination of SCPs for larvae, pupae,and adults

For larvae and pupae, specimens were removedfrom the 118-ml snap-top jars using soft forceps,transferred into a white plastic pan with a small

amount of water and then placed briefly on a pa-per towel to remove excess external moisture. Foradults, specimens were removed from each 3.7-mlsnap-top vial by upturning the vial on a piece ofpaper towel. Although vials contained smallamounts of water from thawed snow, there wasless concern about removing external moisturefrom the adults due to their hydrophobic cuticle.Methods for determining SCPs followed Carrilloet al. (2004b). Specimens were attached to 24-gauge copper-constantan thermocouples using athin layer of high-vacuum grease (Dow Corning�,Dow Corning Corporation, Midland, MI, USA).Two insect-thermocouple arrangements wereplaced at the center of a polystyrene container(0.19� 0.19� 0.19 m) with a starting temperatureof � 0 �C and closed with a rubber stopper. Thiswas repeated for a total of five containers (i.e., 10total insects per trial) and placed simultaneouslyinto a )80 �C freezer (Revco Scientific Inc.,Asheville, NC, USA) to cool insects at a rate of� 1 �C min)1. Temperatures were recorded at 1 sintervals with a multichannel datalogger (PersonalDaq/56 data acquisition system IOtech Inc.,Cleveland OH). Specimens were cooled to at least)30 �C before the containers were removed fromthe freezer. Specimens were removed from thethermocouple and preserved in 70% ETOH inindividual 3.7-ml snap-top vials and labeled suchthat each individual specimen could be later mat-ched with their corresponding SCP. The SCPswere estimated as the lowest temperature reachedbefore the release of the latent heat of fusionresulting from the freezing of the insect’s bodyfluids (Lee, 1991). The SCPs of larvae and pupaewere measured together during the same trials;however adults were tested in separate trials. TheSCPs were obtained for a total of 83 larvae, 24pupae and 46 adults (153 total) by repeating thisexperiment 18 times. Supercooling points were notobtained for all 180 specimens tested as thermo-couples did not maintain good contact with � 15%of the insects.

Measurement of larval instar and phase

Instar and developmental phase were determinedfor larvae preserved from the SCP experiments. Todetermine instar, an ocular micrometer (Nikon) ina dissecting microscope (Olympus SZX12) was

405

used to measure larval head capsule width andlength to the nearest 0.01 mm. Head length wasmeasured from the post occiput to the frontocly-peal margin. Head width was measured at thewidest part of the head. Larval development wasestimated with a dissecting microscope for fourthinstar larvae using phases simplified from Wulker& Gotz (1968). Each individual larva was assignedto one of three categories: (1) small imaginal discswithout well-defined mesothoracic leg sheaths (legsheath differentiation may be beginning or com-pletely absent) (L1), (2) well-defined mesothoracicleg sheaths widely separated ventrally (L2) and (3)thorax greatly swollen with large, obvious meso-thoracic leg sheaths meeting or nearly meetingventrally (L3) (Fig. 1).

Effect of short-term exposure to subzerotemperatures on larval mortality

A method modified from Koch et al. (2004) wasused to determine LLTs. Foam plugs were placedmidway down 16� 150-mm glass test tubes and afilter paper (Whatman grade 1 55-mm circle) wasinserted into each test tube so that they restedagainst the foam insert. Larvae were removed fromthe 6 �C chamber and placed in a white plastic panwith a small amount of water. Larvae were pickedusing soft forceps and placed briefly on a dry papertowel to remove as much external water as possible.Prior testing indicated no mortality when larvae(n = 10) were placed on dry filter paper, main-tained at 6 �C for 1 h and then rewetted. Temper-atures selected for treatment were )5, )10, )15,)20, )25 and )30 �C. For each temperature treat-ment, 10 larvae were placed into each test tube anda 24-gauge copper-constantan thermocouple waspositioned in close proximity to the larvae. Anadditional 10 larvae were added to another test tubeand maintained at 6 �C as a control during theperiod the other treatments were tested. Any mor-tality in the control treatments was used as a cor-rection factor for the subzero temperaturetreatments (Abbott, 1925). The test tubes were thenplaced into a 0.35� 0.35� 0.35-m polystyrenecontainer with an initial temperature of � 6 �C.This initial temperature was attained by placing thecontainer with a starting temperature of � 25 �Cinto a )80 �C freezer until the center of the con-tainer reached� 6 �C. The container with test tubesinside was plugged and then returned to the )80 �Cfreezer. The size of the container maintained a rateof cooling of � 0.3–0.4 �C min)1. Individual testtubes were cooled to designated temperatures of)5, )10, )15, )20, )25 and )30 �C and removedfrom the container 1 min after the target tempera-ture had been achieved. This experiment wasrepeated five times although not all six temperaturetreatments were tested in each experiment, but eachtemperature was tested at least three times. Afterthe larvae were removed from the container, thefilter paper was transferred to a 55-mm Petri dishcontaining � 2 ml of stream water and placedinto the 6 �C chamber. At this time, the controlspecimens were treated similarly. The condition oflarvae was noted immediately after removalfrom the container and then 24 h after removal.

Figure 1. Diamesa mendotae larval developmental phases. msth

lsh = developing mesothoracic leg sheaths; L1: mesothoracic

leg sheaths not obvious or only beginning to differentiate, L2:

large, obvious mesothoracic leg sheaths, and L3: mesothoracic

leg sheaths touching or nearly touching ventrally with thorax

greatly swollen.

406

Survivorship (i.e., lack of moribundity) was as-sessed as the number of larvae active or movingwhen prodded with forceps 24 h post treatment.

To understand the effect of ice formation onlarval mortality, the cumulative proportion ofindividuals freezing (CPIF) was directly comparedto the mean proportion of mortality that resultedafter short-term exposure to subzero temperatures.For a valid comparison between parameters,specimens from the same collection were used inboth experiments and larval SCP experiments(n = 83; see ‘‘Determination of SCPs for larvae,pupae and adults’’) were performed shortly beforeor after mortality experiments. From larval SCPs,the CPIF was determined by calculating thenumber of individuals freezing at or above eachone-degree temperature step and dividing this sumby the total number of individuals tested (Kochet al., 2004; Carrillo et al., 2005a; Bouchard et al.,2006). The CPIF was then plotted against themean proportion of mortality of the six treatmentsfrom the short-term exposure experiments.

Long-term mortality after short-term exposureto subzero temperatures

To determine if freeze or prefreeze injury occurredand caused long-term mortality, larvae that sur-vived short-term exposure to subzero temperaturesand the control treatments were maintained inPetri dishes in a growth chamber at 6 �C. Survi-vorship and life stage were monitored daily.Emerging adults were removed and placed inindividual 3.7-ml vials with a small amount ofstream water and monitored daily. Specimens wereassessed as alive as previously explained. Anylarvae, pupae, or adults that died overnight wereremoved and preserved in 70% ETOH. Theexperiment was terminated after 147 days.

Analysis of data

SCPs. Box plots were created to visualize the dis-tribution of SCPs at different developmental stages(i.e., L1, L2, L3, pupa and adult) (SigmaPlot,2001). Differences among the SCPs for the threelarval phases and the pupae were tested using aone-way analysis of variance (ANOVA) (PROCGLM; SAS Institute, 1998) after applying an x0.25

transformation as recommended by the Box–Cox

procedure (MacAnova, 2002). When significantdifferences were observed at p £ 0.05, Tukey’sStudentized Range Test was used to separate themeans (SAS Institute, 1998). The same proceduresand transformation were used for a second time toidentify any differences in SCPs among the threedifferent larval phases, pupae and adults despitethe fact that SCPs for adults were measured indifferent trials. Because adult SCPs were measuredin different trials, a statistical comparison may notbe considered to be valid by some readers; how-ever, previous experiments showed similar SCPvalues for this developmental stage (Carrillo et al.,2004a; Bouchard et al., 2006). Therefore, it is un-likely that the adult SCPs were an artifact of theprocedure or time of measurement.

Mortality. An ANOVA (PROC GLM; SASInstitute, 1998) on the arcsine-square-root trans-formed proportion of mortality (Southwood &Henderson, 2000) was performed to test for sig-nificant differences in mortality among tempera-ture treatments for the experiments on short-termexposure to subzero temperatures. When signifi-cant differences were identified (p £ 0.05),Tukey’s Studentized Range Test was used to sep-arate the means. Probit analysis (PROC PROBIT;SAS Institute, 1998) was used to estimate thetemperature (95% fiducial limits, FL) at which 50and 99% of mortality occurred (i.e., LLT50 andLLT99). For specimens surviving short-termexposure to subzero temperatures, the arcsine-square-root transformed proportion of larvaepupating and emerging were tested for significancebetween treatments using an ANOVA (PROCGLM; SAS Institute, 1998).

Results

Larval instar and phase

The relationship between larval head capsulewidth and length formed a single cluster suggestingthat all larvae were the same instar (Fig. 2). Onespecimen had a short head length compared to theother larvae, but this specimen was shriveled andhad relatively large imaginal discs and therefore,was not likely to be an earlier instar. We canconclude that all of the larvae tested for SCPs and

407

likely all of the larvae tested for LLTs were in thefourth and final instar. The number of specimensin each larval phase was similar with 28.1, 39.0 and32.9% for the L1, L2 and L3 phases, respectively(Fig. 3).

SCPs

Overall, D. mendotae larvae (n = 83) had a SCP(mean ± SE) of )7.4±0.26 �C with valuesranging from )15.6 to )2.3 �C (Fig. 3). Diamesamendotae pupae (n = 24) had a comparable SCP(mean ± SE) of )9.1±0.58 �C with valuesranging from )15.7 to )4.3 �C (Fig. 3). Adults(n = 46) had a SCP (mean ± SE) of )19.7±0.58 �C with values ranging from )25.8 to)10.5 �C (Fig. 3). Significant differences werefound in the mean SCP among phases of devel-opment when including larvae and pupae(F = 7.49; df = 3, 102; p = 0.0001) and whenincluding all developmental stages (F = 114.54;df = 4, 147; p<0.0001) (Fig. 3). Mean SCPs forimmature stages were relatively high with a steadydecrease in the mean as the insect matures andthen a large decrease in SCP in the adults (Fig. 3).Mean SCPs for phase L1 and L2 larvae were sig-nificantly higher than mean values for L3 larvae

and pupae (Fig. 3). Adult mean SCPs were sig-nificantly lower than those of all other develop-mental stages. There was also a greater amount ofvariability in the SCP of phase L3 larvae, pupaeand adults (Fig. 3).

Effect of short-term exposure to subzerotemperatures on larval mortality

Significant differences in the proportion of larvalmortality among subzero temperatures wereobserved (F = 40.90; df = 5, 19; p<0.0001)(Fig. 4). A significant increase in mortality of� 61% occurred when the temperature droppedfrom )15 to )20 �C. In contrast, larval mortalitywas not significantly different at temperaturesabove )15 �C and remained below � 22%, despitea mean SCP of only �)7 �C (see SCPs results). Byplotting the proportion of mortality with the CPIF,a separation between these curves was apparent(Fig. 4), indicating thatD. mendotae larvae froze athigher temperatures than those causing mortality.The larval LLT50 and LLT99 and their respective95% FL were )17.4 �C ()18.7, )16.2) and)25.4 �C ()30.3, )23.0), respectively. The LLT50

occurred 10 �C below the mean SCP for larvae()7.4 �C). The LLT99 occurred � 10 �C below the

Figure 2. Relationship between head width and length in larvae (n = 81) of Diamesa mendotae. Two larvae from which SCPs were

measured were not included because the head capsule was damaged. Circles represent 61 larvae, with 20 individuals overlapping in

measurement.

408

Figure 3. Effect of developmental stage on the supercooling point (SCP) of Diamesa mendotae. Larval developmental phases as in

Figure 1. The center bars of the box plots represent the median, the upper and lower ends of the boxes represent the 25th and 75th

percentiles, the whiskers represent the 10th and 90th percentiles, circles represent outliers, and the filled squares represent the mean.

Developmental stages with significantly different (p<0.05) mean SCPs are indicated by different letters below each box plot as

determined by Tukey’s Studentized Range Test. Numbers in parentheses represent sample size. One larva from which SCPs were

obtained (n = 83) is not included in the graph because it was too damaged to determine developmental phase.

Figure 4. Corrected mean proportion of mortality (±SE) (closed circles) and cumulative proportion (±SE) of Diamesa mendotae

larvae freezing (open circles, CPIF) after short-term exposure (1 min) to subzero temperatures. Each mean proportion of mortality

represents 31 to 50 individuals in 3 to 5 independent replicates. Mean proportions of mortality for all stages followed by different

lowercase letters are significantly different (p<0.05) as determined by Tukey’s Studentized Range Test.

409

minimum SCP recorded for D. mendotae larvae(i.e., )15.6 �C).

Long-term mortality after short-term exposureto subzero temperatures

Survivorship curves were similar for the 6, )5,)10, )15 and )20 �C treatments with most post-experiment mortality occurring within 30 d afterthe experiment (Fig. 5). However, two larvae sur-vived 143 and 147 days until the experiment wasterminated in the )15 and )5 �C treatments,respectively. Of specimens surviving the short-termexposure to subzero temperature experiments,including the control group (n = 185), 71% pu-pated and 16% emerged as adults. Numericaldifferences in pupation rates were observed; withtreatments 6 and )5 �C ranging from 62 to 64%compared to the )10, )15 and )20 �C treatmentswhich ranged from 75 to 82% pupation. However,no significant differences (F = 1.36; df = 4, 16;p = 0.2905) were observed between the propor-tion of larvae pupating for specimens cooled below(i.e., )10, )15, )20 �C) and above (i.e., 6, )5 �C)the mean SCP for larvae (Fig. 6). In contrast,significant differences were observed for adult

emergence rate across treatment groups (F = 6.02;df = 4, 16; p = 0.0038); however, no particularpattern among treatments was apparent (Fig. 6).

Discussion

SCPs

The mean SCP for adults in this study was similarto values previously reported for D. mendotae(Carrillo et al., 2004a; Bouchard et al., 2006). Amean SCP of )19.7 �C for the Trout Brook pop-ulation was numerically similar to the mean SCPof )21.6 �C recorded from another population ofD. mendotae in Wisconsin. These SCPs were lowerthan values determined for other winter-activeinsects (Sømme & Østbye, 1969). Bouchard et al.(2006) reported freeze intolerance in D. mendotaeadults and hypothesized that low SCPs allowedthese insects to survive and remain active at sub-zero temperatures.

The estimated mean SCP for larvae ()7.4 �C)was significantly higher (� 12 �C) than valuesdetermined for adults of this species. In aquaticinsects, this pattern of higher SCPs in larvae

Figure 5. Long-term survivorship of Diamesa mendotae larvae after surviving short-term exposure to subzero temperatures. Survi-

vorship curves for )25 and )30 �C treatments were not included due to low post-experiment survival (0–2.5%). Each treatment

consists of 7 to 50 surviving individuals combined from 2 to 5 replicates.

410

compared to the adults has been identified pre-viously (Moore & Lee, 1991). These larval SCPsare comparable to SCPs of Chironomidae larvaeusing different experimental approaches (Danks,1971; Baust & Edwards, 1979; Oswood et al.,1991; Frisbie & Lee, 1997). One exception to thisis the marine midge, Paraclunio alaskensis Co-quillett, where mean SCPs as low as )14.2 �Cwere reported by Ring (1989). For the pupalstage, the relatively high mean SCP ()9.1 �C)contradicted our original hypothesis, being closerto the larval stage than to the mean SCP for theadult stage. This result contrasts with reports ofpupal SCPs for the majority of terrestrial insects,which generally have SCPs below )20 �C(Sømme, 1982; Bale, 1989). The low pupal SCPsare generally attributed to the fact that the pupais a non-feeding stage with fewer INAs comparedto the feeding stages. To our knowledge, this isthe first measurement of SCPs in an aquatic pupaand the lack of information for this stage may bea result of the fact that most aquatic insectsoverwinter as eggs or larvae (Moore & Lee,1991); therefore, the generally short-lived pupalstage does not experience long-term exposure tolow temperatures. However, in the case of D.mendotae, pupae may be exposed to ice crystals

and/or subzero temperatures as a result of thewinter emergence of this species.

The determination of SCPs for differentdevelopmental stages of D. mendotae indicatesthis insect begins to decrease its mean SCP late inthe larval stage into the pupal stage (Fig. 3). Themean SCP drops abruptly from )9.1 to )19.7 �Cbetween the pupal and adult stages; however, it isnot clear at which stage this large decrease in theSCP occurs. The lower mean and minimum SCPvalues through development suggest that therecould be physiological preparation late in thefourth instar and pupa for the transition to in-creased supercooling capabilities in the adultstage.

Evidence for larval freeze tolerance

According to Bale (1991), freeze tolerance orintolerance for an organism can be determinedby exposing insects to temperatures above andbelow the mean SCP for a given species. Bycomparing the cumulative proportion at whichindividuals freeze (i.e., CPIF) to the rate ofmortality at different subzero temperatures, onecan determine if a species is tolerant or intoler-ant of freezing. For D. mendotae, comparison of

Figure 6. Pupation and emergence proportions for Diamesa mendotae larvae surviving short-term exposure to subzero temperatures.

Treatments consist of a total of 7 to 50 individuals from 2 to 5 replicates.

411

the proportion of mortality and CPIF curves(Fig. 4), indicated that mortality occurred wellbelow larval SCP temperatures and that theselarvae were freeze tolerant. However, this rela-tionship between mortality and CPIF alone isnot a determinant of freeze tolerance (Kochet al., 2004; Carrillo et al., 2005a). For example,in Koch et al. (2004) and Carrillo et al. (2005a)a similar gap between these two curveswas observed for the freeze-intolerant Harmoniaaxyridis (Pallas) (Coleoptera: Coccinellidae) andPlodia interpunctella (Hubner) (Lepidoptera:Pyralidae). However, in these studies, differencesbetween the two curves were attributed to shortexposures (i.e., 1 min) to subzero temperaturesand the relatively large mass of the specimensresulting in some specimens not fully freezing.Nevertheless, these insects generally had 100%mortality close to their 100% CPIF despite theirlarge mass. According to Salt (1953) survival offreeze-intolerant species after minimum exposure(e.g., 1 min) to temperatures corresponding totheir SCP may be an artifact of laboratoryexperiments. In addition, it has been demon-strated for freeze-intolerant insects that theproportion of mortality increases with time attheir SCP (Block et al., 1988; Koch et al., 2004;Carrillo et al., 2005a).

For smaller, freeze-intolerant organisms suchas D. mendotae adults (Bouchard et al. 2006)and other species like the adults of the parasitoidHabrobracon hebetor (Say) (Hymenoptera: Bra-conidae) (Carrillo et al., 2005b), the mortalityand CPIF curves were much closer compared toeither H. axyridis or P. interpunctella. For D.mendotae larvae, the fourth instar is presumablysimilar in mass to the adult and we would haveexpected closer mortality and CPIF curves if thestage was freeze intolerant. Even for freeze-intolerant insects with greater body mass, like H.axyridis and P. interpunctella, their LLT99 oc-curred within only 2 to 5 �C below the temper-ature at which the CPIF curve reached 100%(Koch et al., 2004; Carrillo et al., 2005a). Incontrast, the LLT99 for D. mendotae larvae oc-curred at � 10 �C below the temperature atwhich the CPIF curve reached 100% andtherefore we conclude that D. mendotae larvaeare freeze tolerant and not an artifact of themethodology.

Long-term mortality after short-term exposureto subzero temperatures

Although insects may be freeze tolerant, mortalitywill still occur at temperatures below the SCP(Sinclair, 1999). Most freeze-tolerant organismscan convert up to 65% of the body water into icewithout negative effects (Storey & Storey, 1997).An excessive and uncontrolled water loss in thebody causes an increase in solute concentrationand cell shrinkage that may result in cell injury anddeath (Lee, 1989). In general, the probability ofmortality increases at lower temperatures belowthe SCP. Although we observed a similar pattern ofincreasing mortality at lower temperatures, larvaesurviving a short exposure to different tempera-tures above or below their SCP exhibited a similarlong-term survival (Fig. 5). If irreversible freezeinjury occurred, the effect was measurable within24 h post exposure. Therefore, we can concludethat the assessment of mortality 24 h post treat-ment was sufficient to determine mortality ratesresulting from subzero-temperature exposure.

An unexpected result, although not significant,was the numerically greater pupation rates ofspecimens cooled below the mean SCP comparedto those cooled above this temperature (Fig. 6).However, there was no pattern for adult emer-gence. These results are difficult to explain withoutfurther investigation. In addition, we must notethat these specimens were not maintained undernatural conditions in the laboratory and lackedphotoperiod and thermal cues; which potentiallyaltered their behavior. However, relatively highpupation and emergence rates in a laboratorysetting are interesting as they may prove to be aneffective method for rearing specimens to obtainassociated material for cold-adapted chironomidspecies. A similar methodology is described forrearing Simuliidae from mature larvae (Adleret al., 2004).

Ecological implications of freeze tolerance inD. mendotae larvae

The larvae of most aquatic insects are not gener-ally exposed to low subzero temperatures as a re-sult of the thermal buffering from water or theability to find safe refugia (e.g., hyporheic zone)during periods when the surrounding water may

412

freeze (Moore & Lee, 1991; Irons et al., 1993;Lencioni, 2004). Therefore, the ability of D. men-dotae to survive the freezing of body fluids may bean evolutionarily conserved characteristic wide-spread in many chironomid subfamilies, includingthe Diamesinae, which are considered to havediversified in cold-water habitats (Brundin, 1966).In fact, Baust & Lee (1981) concluded that freezetolerance in Belgica antarctica Jacobs was evolu-tionarily conserved because the larvae do not ap-pear to be exposed to low subzero temperatures.Other species of Diamesa seem to be freeze toler-ant (e.g., D. zernyi Edwards; Lencioni, 2004) andthis trait has not been lost in D. mendotae althoughit may not be as critically important in thermally-buffered habitats such as groundwater-influencedstreams. In surface-water dominated or intermit-tent streams experiencing ice-crystal formation,D. mendotae may migrate into the buffered hyp-orheic zone to avoid ice inoculation. The ability totolerate freezing conditions in D. mendotae larvaemay improve their ability to survive in variable orunpredictable habitats. In D. mendotae larvae,freeze tolerance could be a mechanism to (1)remain active in habitats that can undergo shortperiods of freezing temperatures or (2) enterdiapause during long periods of low temperatures.

Winter activity. The ability to tolerate freezingmay be advantageous for organisms active duringcold periods in an aquatic environment. Evidencesuggests that some winter-active species occurringin streams can undergo development during thewinter. Berg & Hellenthal (1991) reported thatDiamesa nivoriunda (Fitch), a species developingfrom autumn to spring, contributed 33.9% to thetotal chironomid production. Nolte & Hoffman(1992) determined that Diamesa incallida (Walker)can undergo 7 to 8 generations per year despite lowwater temperatures. In Trout Brook, D. mendotaemay also be multivoltine and contribute greatly tosecondary production as water temperatures arelow in the winter, but remain above freezing yearround (Bouchard et al., 2006) due to groundwaterinputs. However, D. mendotae has also been col-lected from streams that are not greatly buffered bygroundwater (Bouchard, unpublished data) andmay experience supercooling conditions increasingthe risk of spontaneous ice inoculation in unpro-tected aquatic insects. Supercooling of water oc-

curs in areas of swift flow or turbulence and canresult in the formation of ice crystals called fraziland anchor ice (Oswood et al., 1991; Lencioni,2004). Frazil and anchor ice can be dangerous tostream invertebrates as it might cause ice inocula-tion even if water temperatures are above the SCPof the organism. In laboratory experiments, chi-ronomid larvae exposed to external water (e.g., incontact with wet filter paper) have much higherSCPs than those free of external water (Danks,1971; Frisbie & Lee, 1997); therefore, the ability towithstand these short-term exposures to frazil oranchor ice may be beneficial. This capability wouldpermit species to survive and remain active duringcold periods without requiring migration into thehyporheic zone or the construction of winter co-coons. Such mechanisms may allow these species tobe flexible and take advantage of these habitatsearlier in the spring or during warm periods in thewinter.

Diapause. Freeze tolerance can permit chirono-mid larvae to diapause during relatively longperiods of low temperatures when conditions arenot suitable for activity. Many chironomid specieshave been determined to use diapause in arcticponds (Scholander et al., 1953; Danks, 1971) andglacial streams (Lencioni, 2004). For example,Lencioni (2004) collected apparently frozen D.zernyi from frozen glacial streams which couldthen be thawed and revived in the laboratory.Despite surface or edge ice, liquid water is usuallypresent in most perennial, surface-water domi-nated streams in temperature zones, but the shal-low margins of these habitats can experiencefreezing (Olsson et al., 1981). Intermittent streamsmay experience more widespread freezing condi-tions depending on the depth of moisture andfreezing in hyporheic during the winter. In Min-nesota, D. mendotae has been collected from sur-face-water dominated streams and other Diamesaspecies have been collected from intermittentstreams. In these habitats, Diamesa larvae couldbe exposed to freezing substrates (Bouchard,unpublished data) and experience long-termfreezing. In these conditions, the ability to over-winter by diapausing as larvae could allow survivalin freezing conditions and increase the range ofhabitats available to D. mendotae. However, fur-ther research is needed to determine if D. mendotae

413

larvae occupy such marginal habitats during thewinter and if diapause is a strategy for wintersurvival.

Larva to adult: changing cold-hardiness strategies

Based on previous and current results, it is clearthat D. mendotae uses two different cold hardinessstrategies through its life cycle. According toSømme (1982), so far all arthropod eggs testedhave been freeze intolerant; therefore, we presumethis to be the case for D. mendotae eggs. From ourcurrent results we classify larvae of D. mendotae asfreeze tolerant, whereas Bouchard et al. (2006)classified the adult stage as freeze intolerant. Asimilar shift in strategies across life stages has alsobeen observed in the Antarctic chironomid,B. antarctica (Baust & Edwards, 1979); howeveradult D. mendotae have a much lower SCP. InD. mendotae, both the larval and adult life stagesappear to be well-adapted to cold temperatures,but shifts from one strategy to another seem to bethe result of the different environments in whichthese life stages inhabit.

The larval stage presumably remains in contactwith water and potentially ice crystals and is morelikely to experience freezing as a result of iceinoculation. A freeze-avoidant strategy usinglower SCPs would be less effective in an aqueousenvironment where inoculation from external icecrystals could result in the freezing of body fluidsand consequently death (Moore & Lee, 1991;Frisbie & Lee, 1997). Therefore, a freeze-tolerancestrategy with high SCPs may allow these larvae tosurvive short-term exposures to ice crystals ordiapause for longer periods during these condi-tions. The physiological mechanisms increasingSCPs in this stage is unknown; however, as afeeding stage it is more likely to possess greateramounts of INAs (Salt, 1953; Leather et al., 1993).A possible production of cryoprotectants, asreported for the larvae of B. antarctica (Baust &Edwards, 1979), could aid in protecting the cellsduring the freezing of extracellular fluids.

All of the literature on winter diapause in theChironomidae focuses on the larvae and there isno evidence that Chironomidae diapause as pupaeor adults. In general, the pupal stage lasts less than72 h (Langton, 1995), although pupal stage dura-tion has not been tested for D. mendotae under

natural conditions and may require longer periodsas a result of low water temperatures during thewinter. Nolte & Hoffman (1992) determined thatD. incallida pupae required 72 h at 8 �C for eclo-sion. The short pupal stage does not appear to beutilized as an overwintering stage in Chironomidaeas it is in many terrestrial insects. We hadhypothesized, based on the literature available forterrestrial insects, that the pupa would have SCPscloser to the adults. Whether the pupa stage isfreeze tolerant or intolerant is still unknown, butthe high SCPs and the aquatic habitat shared withthe larvae suggests that freeze tolerance may be abetter strategy due to the threat of freezing bodyfluids through ice nucleation.

For D. mendotae adults, the ability to greatlylower their SCP allows them to be active in sub-zero conditions, a requirement for mating andovipositing during the winter (Carrillo et al.,2004a). The physiological mechanisms underlyingthe ability of D. mendotae adults to depress SCP totemperatures of �)20 �C is also unknown. How-ever, these adults are presumably non-feeding andtherefore could potentially have fewer INAs intheir bodies. According to Zachariassen et al.(2004), the removal of all INAs in an insect’s bodycan reduce their SCP by as much as 10 �C. Theproduction of antifreeze compounds such aspolyols, sugars, thermal hysteresis proteins hasalso been implicated in lowering SCPs in manyfreeze-avoidant species (Leather et al., 1993) andcan not be excluded as a mechanism in D. men-dotae adults.

The present study increases our understandingof the cold hardiness of D. mendotae. The ability ofD.mendotae larvae to tolerate freezingmay increasethe range of habitats this species can occupy (e.g.,groundwater dominated and surface-water domi-nated streams). More research is needed to deter-mine how the distribution and success of this speciesin different habitats is regulated by cold-hardinessmechanisms in all life stages of this insect. Ourresults, together with those of Bouchard et al.(2006), provide strong evidence for a change instrategies between the larval and adult stages.However, it is not clear which physiological mech-anisms are used to acquire and maintain cold har-diness in this species, or when the change instrategies occurs between life stages. Future re-search needs to determine the presence, effect and

414

timing of cryoprotectants, INAs and gut contentsfor larvae, pupae and adults to assess if thesemechanisms play a role in cold hardiness of thisspecies.

Acknowledgements

We would like to thank two anonymous reviewersfor critical comments on an earlier version of thismanuscript. This work was partially funded by aUniversity of Minnesota Doctoral DissertationFellowship (R.W.B. Jr. and M.A.C.).

References

Abbott, W. S., 1925. A method of computing the effectiveness of

an insecticide. Journal of Economic Entomology 18: 265–267.

Adler, P. H., D. C. Currie & D. M. Wood, 2004. The black flies

(Simuliidae) of North America. Cornell University Press,

Ithaca.

Andrews, D. & F. H. Rigler, 1985. The effects of and Arctic

winter on benthic invertebrates in the littoral zone of Char

Lake, Northwest Territories. Canadian Journal of Zoology

63: 2825–2834.

Ashton, G. D., 1979. River ice. American Scientist 67: 38–45.

Bale, J. S., 1989. Cold hardiness and overwintering of insects.

Agricultural Zoology Reviews 3: 157–192.

Bale, J. S., 1991. Implications of cold hardiness for pest man-

agement. In Lee, R. E. Jr. & D. L. Denlinger (eds), Insects at

Low Temperature. Chapman & Hall, New York: 461–498.

Baust, J. G. & J. S. Edwards, 1979. Mechanisms of freezing

tolerance in an Antarctic midge, Belgica antarctica. Physio-

logical Entomology 4: 1–5.

Baust, J. G. & R. E. Lee, 1981. Environmental ‘‘homeothermy’’

in an Antarctic insect. Antarctic Journal of the United States

15: 170–172.

Baust, J. G. & R. R. Rojas, 1985. Review - insect cold hardiness:

facts and fancy. Journal of Insect Physiology 31: 755–759.

Berg, M. B. & R. A. Hellenthal, 1991. Secondary production of

Chironomidae (Diptera) in a north temperate stream.

Freshwater Biology 25: 497–505.

Block, W, 1991. To freeze or not to freeze? invertebrate survival

of sub-zero temperatures. Functional Ecology 5: 284–290.

Block, W., Y. Z. Erzinclioglu & M. R. Worland, 1988. Survival

of freezing in Calliphora larvae. CryoLetters 9: 86–93.

Bouchard, R. W. Jr., M. A. Carrillo & L. C. Ferrington Jr.,

2006. Lower lethal temperature for adult male Diamesa

mendotae Muttkowski (Diptera: Chironomidae), a winter-

emerging Diamesinae. Aquatic Insects 28: 57–66.

Brundin, L., 1966. Transantarctic relationships and their signifi-

cance, as evidenced by chironomid midges with a monograph

of the subfamilies Podonominae and Aphroteniinae and the

australHeptagyiae.KunglicaSvenskaVetenskapsakademiens

Handlingar 11: 1–472 pp + 30 plates.

Carrillo, M. A., C. A. Cannon & L. C. Ferrington Jr., 2004a.

Effect of sex and age on the supercooling point of the winter-

active Diamesa mendotae Muttkowski (Diptera: Chironom-

idae). Aquatic Insects 26: 243–251.

Carrillo, M. A., N. Kaliyan, C. A. Cannon, R. V. Morey &

W. F. Wilcke, 2004b. A simple method to adjust cooling

rates for supercooling point determination. CryoLetters 25:

155–160.

Carrillo, M. A., C. A. Cannon, W. F. Wilcke, R. V. Morey,

N. Kaliyan & W. D. Hutchison, 2005a. Relationship

between supercooling point and mortality at low tempera-

tures in Indianmeal moth (Lepidoptera: Pyralidae). Journal

of Economic Entomology 98: 618–625.

Carrillo, M. A., G. E. Heimpel, R. D. Moon, C. A. Cannon &

W. D. Hutchison, 2005b. Cold hardiness of Habrobracon

hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of

pyralid moths. Journal of Insect Physiology 51: 759–768.

Danks, H. V., 1971. Overwintering of some north temperate

and arctic Chironomidae. The Canadian Entomologist 103:

1875–1910.

Frisbie, M. P. & R. E. Lee Jr., 1997. Inoculative freezing and

the problem of winter survival for freshwater macroinver-

tebrates. Journal of the North American Benthological

Society 16: 635–650.

Irons, J. G. III, L. K. Miller &M. W. Oswood, 1993. Ecological

adaptations of aquatic macroinvertebrates to overwintering

in interior Alaska (U.S.A.) subarctic streams. Canadian

Journal of Zoology 71: 98–108.

Koch, R. L., M. A. Carrillo, R. C. Venette, C. A. Cannon &W.

D. Hutchison, 2004. Cold hardiness of the multicolored

Asian lady beetle (Coleoptera: Coccinellidae). Environmen-

tal Entomology 33: 815–822.

Kohshima, S., 1984. A novel cold-tolerant insect found in a

Himalayan glacier. Nature 310: 225–227.

Langton, P. H., 1995. Chap 8: The pupa and events leading to

eclosion. In Armitage, P. D., P. S. Cranston & L. C. V.

Pinder (eds), The Chironomidae: Biology and Ecology of

Non-biting Midges. Chapman & Hall, London: 169–193.

Leather, S. R., K. F. A Walters & J. S. Bale, 1993. The Ecology

of Insect Overwintering. Cambridge University Press,

Cambridge.

Lee, R. E. Jr., 1989. Insect cold-hardiness: to freeze or not to

freeze. BioScience 39: 308–313.

Lee, R. E. Jr., 1991. Principles of insect low temperature tol-

erance. In Lee, R. E. Jr. & D. L. Denlinger (eds), Insects at

Low Temperature. Chapman & Hall, New York: 17–46.

Lencioni, V., 2004. Survival strategies of freshwater insects in

cold environments. Journal of Limnology 63: 45–55.

MacAnova, 2002. MacAnova for Windows, version 4.12. Uni-

versity of Minnesota, School of Statistics. Available online

at: http://www.stat.umn.edu/macanova/download.html.

Moore, M. V. & R. E. Lee Jr., 1991. Surviving the big chill:

overwintering strategies of aquatic and terrestrial insects.

American Entomologist 37: 111–118.

Nolte, U. & T. Hoffman, 1992. Fast life in cold water: Diamesa

incallida (Chironomidae). Ecography 15: 25–30.

415

Olsson, T. I., 1981. Overwintering of benthic macroinverte-

brates in ice and frozen sediment in a North Swedish river.

Holarctic Ecology 4: 161–166.

Oswood, M. W., L. K. Miller & J. G. Irons III, 1991. Over-

wintering of freshwater benthic macroinvertebrates. In Lee,

R. E. Jr. & D. L. Denlinger (eds), Insects at Low Temper-

ature. Chapman & Hall, New York: 360–375.

Ring, R. A., 1989. Intertidal Chironomidae of B.C., Canada.

Acta Biologica Debrecen Oecologica Hungarica 3: 275–288.

Salt, R. W., 1953. The influence of food on cold hardiness of

insects. The Canadian Entomologist 85: 261–269.

Salt, R. W., 1961. Principles of insect cold-hardiness. Annual

Review of Entomology 6: 55–74.

SAS Institute Inc., 1998. Statistical Analysis System, version

6.12. SAS Institute, Cary.

Scholander, P. F., W. Flagg, R. J. Hock & L. Irving, 1953.

Studies on the physiology of frozen plants and animals in the

arctic. Journal of Cellular Comparative Physiology 41: 1–56.

SigmaPlot, 2001. SigmaPlot 2001 for Windows, version 7.0.

SPSS Inc, Chicago.

Sinclair, B. J., 1999. Insect cold tolerance: how many kinds of

frozen?. European Journal of Entomology 96: 157–164.

Sømme, L. & E. Østbye, 1969. Cold-hardiness in some winter

active insects. Norsk Entomologisk Tidsskrift 16: 45–48.

Sømme, L., 1982. Supercooling and winter survival in terrestrial

arthropods. Comparative Biochemistry and Physiology A:

Physiology 73: 519–543.

Southwood, T. R. E. & P. A. Henderson, 2000. Ecological

Methods. Blackwell Ltd, Oxford.

Storey, K. B. & J. M. Storey, 1997. To freeze or not to freeze -

the dilemma for life below 0 �C. The Biochemist 19: 8–13.

Wulker, W. & P. Gotz, 1968. Die Verwendung der Imagi-

nalscheiben zur Bestimmung des Entwicklungszustandes von

Chironomus-Larven (Dipt.). Zeitschrift fur Morphologie der

Tiere 62: 363–388.

Young, R. M., 1969. Field observations on a midwinter

breeding flight of Diamesa arctica (Diptera: Chironomidae).

Annals of the Entomological Society of America 6: 1204.

Zachariassen, K. E., 1985. Physiology of cold tolerance in in-

sects. Physiological Reviews 65: 799–832.

Zachariassen, K. E. & E. Kristiansen, 2000. Ice nucleation and

antinucleation in nature. Cryobiology 41: 257–279.

Zachariassen, K. E., E. Kristiansen & S. A. Pedersen, 2004.

Inorganic ions in cold-hardiness. Cryobiology 48: 126–133.

416