Critical Thermal Limits, Temperature Tolerance and Water Balance of a Sub-Antarctic Kelp Fly, Paractora Dreuxi

Journal of Insect Physiology 47 (2001) 95–109 www.elsevier.com/locate/jinsphys

Critical thermal limits, temperature tolerance and water balance of a sub-Antarctic kelp ﬂy, Paractora dreuxi (Diptera: Helcomyzidae) C. Jaco Klok *, Steven L. Chown Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa

Received 11 April 2000

Abstract

Paractora dreuxi displays distinct ontogenetic differences in thermal tolerance and water balance. Larvae are moderately freeze tolerant. Mean larval onset of chill coma was Ϫ5.1°C, and onset of heat stupor was 35.5°C. Larval supercooling point (SCP) was Ϫ3.3°C with 100% recovery, although mortality was high below Ϫ4°C. Starvation caused SCP depression in the larvae. Adults were significantly less tolerant, with critical thermal limits of Ϫ2.7 and 30.2°C, no survival below the SCP (Ϫ9.6°C), and no change in SCP with starvation. Moderate freeze tolerance in the larvae supports the contention that this strategy is common in insects from southern, oceanic islands. Fly larvae survived desiccation in dry air for 30 h, and are thus less desiccation tolerant than most other sub-Antarctic insect larvae. Water loss rates of the adults were significantly lower than those of the larvae. Lipid metabolism did not contribute significantly to water replacement in larvae, which replaced lost body water by drinking fresh water, but not sea water. Kelp fly larvae had excellent haemolymph osmoregulatory abilities. Current climate change has led to increased temperatures and decreased rainfall on Marion Island. These changes are likely to have significant effects on P. dreuxi, and pronounced physiological regulation in larvae suggests that they will be most susceptible to such change.  2000 Elsevier Science Ltd. All rights reserved.

Keywords: Ontogenetic differences; CTMin;CTMax; Freeze tolerance; Osmoregulation; Marion Island; Climate change

1. Introduction contributors to ecosystem functioning (Tre´hen et al., 1985; Crafford and Scholtz, 1987; Ha¨nel and Chown, The majority of studies concerning temperature and 1998). In consequence, there is little information avail- water relations of arthropods in the sub- and maritime able on the way in which these ecologically important Antarctic regions has concerned strictly terrestrial spec- arthropods cope with their often variable environments, ies. With the exception of a few studies of semi-aquatic and how their physiological responses differ to those of ﬂies, beetles and copepods (Chown and Van Drimmelen, arthropods from terrestrial environments in the region. 1992; Chown, 1993; Convey and Block, 1996; Daven- Such comparisons are of considerable interest from port and MacAlister, 1996; Davenport et al., 1997), little three perspectives. First, the ways in which terrestrial emphasis has been given to environmental tolerances of and aquatic species cope with their respective environ- arthropods living in shoreline or semi-aquatic habitats ments are usually quite different (e.g. Lee and Denlinger, (see Block, 1984; Klok and Chown 1997, 1998; Sinclair, 1991; Hadley, 1994), yet many insect species make the 1999). Nonetheless, these habitats, and particularly the transition from one environment to the other as they shore zones, are characterized by high arthropod diver- develop. Although marked ontogenetic differences in sity (Bellido, 1981; Trave´, 1981; Chown, 1990; Marshall physiology have been documented in some insect spec- et al., 1999), of which a variety of species are major ies (Morrissey and Baust, 1976; Tre´hen and Vernon, 1986; Vernon, 1986; Vernon and Vannier 1986, 1996), the subtleties of such differences remain to be thor- * Corresponding author. Tel.: +2712-420-3236; fax: +2712-342- oughly explored (see Spicer and Gaston, 1999 for 3136. discussion). Second, it has been mooted repeatedly that E-mail address: [email protected] (C.J. Klok). the cold hardiness strategy adopted by most mid-latitude

0022-1910/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S0022-1910(00)00087-1 96 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 southern hemisphere insect species is one of freeze toler- group, and lies 2100 km south east of Cape Town, and ance (e.g. Klok and Chown, 1997; Van der Merwe et to the north of the Antarctic Polar Front. It has a highly al., 1997). This is thought to be a consequence of the oceanic climate with mean monthly temperatures rang- oceanic nature of the region, which leads to generally ing from a winter low of 2°C to a summer high of 7°C moist habitats and, as a result, a high risk of freezing with little annual variation (Schulze, 1971; Blake, 1996). due to external inoculation by ice crystals. Nonetheless, Precipitation is in excess of 2000 mm per yr and the data with which to test this idea are limited, and it has island experiences a high degree of cloudiness (climate not been well-explored in species where adult and larval data from Schulze, 1971; Smith and Steenkamp, 1990). habitats might differ (see Addo-Bediako et al., 2000; Sea surface temperatures vary between 2°C in winter to Convey and Block, 1996 for exceptions). Third, differ- 8°C in summer with an annual mean of 5°C (De Villi- ences in the physiological tolerances of terrestrial and ers, 1976). aquatic species are not only likely to influence the range On boulder beaches, P. dreuxi larvae and adults occur of habitats they can occupy, and hence, ultimately differ- from just above the high water mark to the lower limit ences in their geographic ranges (Chown and Gaston, of closed vascular vegetation, feeding predominantly on 1999), but they may also influence the extent to which decaying kelp deposits, but also, facultatively, on seal changes in the abiotic environment will differentially and penguin carcasses and other carrion (Crafford, 1984; influence these species. The latter is of particular con- Tre´hen et al., 1985). Larval duration is approximately cern in Antarctic and sub-Antarctic environments where two months, of which instar III lasts for 40–50 days and climates are changing rapidly (Smith et al., 1996; Berg- is responsible for most kelp consumption. Mature instar strom and Chown, 1999). III larvae pupariate below the beach surface to a depth In this study we examine thermal tolerances and water of 50 cm. The pupal stage lasts for 30–60 days and the balance in adults and larvae of Paractora dreuxi Se´guy adults can live for 14–21 days (Crafford, 1984), feeding (Diptera: Helcomyzidae), a brachypterous kelp fly that on the slime that covers decaying kelp fronds (personal frequents kelp deposits in the littoral habitats on the observation). The largest biomass of third instar larvae coastlines of the Prince Edward and Crozet islands in is found in the supralittoral zone, 7–9 m from the shore- the sub-Antarctic (Crafford, 1984; Tre´hen et al., 1985; line. Both adults and larvae avoid the intertidal zone but Crafford and Scholtz, 1987). The associations of P. do occur in low densities in the splash zone (the upper dreuxi with kelp deposits differ markedly between the part of the eulittoral zone) situated 3–7 m from the larvae and the adults. The larvae are less mobile than shoreline (Crafford and Scholtz, 1987). Nonetheless, this adults and are mostly confined to kelp, or the substrate species may come into contact with seawater, either as below the kelp, where they either burrow into the fronds, saltspray blown ashore by high winds, or during storms feed between the fronds, or, in the later instars, feed on when large swells, which override the small (71 cm) the underlying detritus (Crafford, 1984; Crafford and tidal range, inundate the boulder beaches well into the Scholtz, 1987). Here they are subject to substantial fluc- supralittoral zone (De Villiers, 1976; Smith, 1977; Craf- tuations in temperature and water availability. In con- ford and Scholtz, 1987). trast, the shorter-lived adults, though brachypterous, are Large wrack beds are known to have stable internal highly mobile as a consequence of their search for ovi- temperatures compared to ambient temperatures position and feeding sites (Crafford, 1984; Tre´hen et al., (Crafford and Scholtz, 1987; Chown, 1996). However, 1985). However, they often take refuge, at least on sunshine, varying wind conditions, rainfall and sporadic Marion Island (Prince Edward group), between the boul- snowfalls do cause wide fluctuations in the microclimate ders below the kelp-substrate interface either when pred- of smaller kelp deposits, especially the smaller strings ators are present, or when weather conditions are that are also utilized by P. dreuxi (Crafford, 1984). Rela- unfavourable to them. Using flies collected at Marion tive to the stable ambient temperatures, temperatures in Island, we test the hypothesis that stage-related differ- kelp deposits on Marion Island can fluctuate widely, ences in association of P. dreuxi with stranded kelp will from below zero (mean of Ϫ2.3±0.5°C, range Ϫ1.3 to result in differences in their environmental tolerances. Ϫ3.0, n=10 observation days with single temperatures We also examine the idea that these differences will recorded during the early morning) in winter, to 29– affect the likely impact of climate change (especially 37°C during sunny days in early spring (Crafford, 1984, declining rainfall) on the stages. C.J. Klok, unpublished data). Furthermore, kelp deposits often desiccate completely despite the high rainfall at Marion Island (Crafford and Scholtz, 1987), although 2. Material and methods they rehydrate after rain, the main source of fresh water 2.1. Study site and animals on boulder beaches. This study was undertaken from May to October Sub-Antarctic Marion Island (46°54ЈS37°45ЈE) is the 1998, a period when low temperatures are common. larger of two islands forming the Prince Edward Islands Blake (1996) has demonstrated that in lowland areas of C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 97

Marion Island, freezing of the soil surface takes place onset of uncontrolled muscular spasms in the last animal. regularly (weekly, and often more regularly) at this time The experiments were repeated to increase the sample of year, and that temperatures decline to approximately sizes to n=20. Because larvae in the field are often Ϫ4°C on a regular basis. Adults and third instar larvae covered by a thin layer of slime produced by the decay- were collected with an aspirator from decomposing kelp ing kelp, the effects of this slime on larval CTMin was deposited on Trypot Beach. This site was selected also determined by examining a second set of larvae that because kelp deposition is regular (albeit random), and had not been wiped dry. because of its proximity to the laboratory. Live specimens were taken to the laboratory within two hours of 2.3. Supercooling points collection. In the laboratory, the animals were kept in a temperature-controlled cabinet (10±0.5°C, with an Supercooling points (SCPs) of individual larvae (wet 11L:13D photoperiod) in plastic containers and supplied and dry) and adults were determined by placing the ani- with decomposing kelp. Animals were not held for mals tightly in pipette tips and inserting 40 gauge cop- longer than 24 h in the cabinets prior to experiments. In per-constantan thermocouples between the animal and instances where animals were kept for longer periods as the pipette tip wall. The pipette tips containing the ani- part of an experimental protocol the temperature and mals were transferred individually to air filled vials and light regimes were as above. submerged in the water bath at 0°C. After a 15 min equilibration period temperature was lowered at 0.1°C minϪ1 2.2. Critical thermal limits (see Klok and Chown 1997, 1998; Kelty and Lee, 1999). The thermocouples were connected to a Campbell Scien- The methods of Roberts et al. (1991) and Klok and tific CR10 datalogger that logged temperatures every Chown (1997, 1998) were followed to determine critical second and calculated a mean every 15 s. The tempera- thermal minima, identified as the onset of and recovery ture logged just prior to the freezing exotherm was taken from chill coma (CTMin/o for onset and CTMin/r for as the supercooling point. recovery), and critical thermal maxima, identified as the The SCPs of freshly collected larvae and adults were onset of heat stupor (CTMax), of the larvae and adults. determined. To investigate whether feeding state might Eleven larvae (carefully wiped dry using tissue paper, influence SCPs, larvae and adults were kept at 10°C see below), or adults, were placed individually into vials, (11L:13D) for four and three days respectively, with no with one larva, or adult, serving as an operative ther- access to decaying kelp, but access to fresh water. mometer. A 40-gauge copper-constantan thermocouple was inserted into the body core of the larva or the thorax 2.4. Lower and upper thermal tolerances of the adult to measure body temperature (Tb) during the course of the experiment. The Tb of the operative To determine lower lethal temperatures, the methods thermometer was assumed to be representative of the Tb of Worland et al. (1992) and Klok and Chown (1997, of the ten experimental animals. The vials were sub- 1998) were followed. Batches of ten larvae and ten merged in a Grant LTD 6 water bath (0.1°C accuracy) adults were placed in glass vials submerged in the pro- connected to a PZ1 programmable temperature control- grammable water bath. After a 15 min equilibration per- ° ° Ϫ1 ler. For CTMin/o the bath’s temperature was allowed to iod at 0 C, the specimens were cooled at 0.1 C min stabilize for 15 min at 2°C after which it was lowered until the first temperature, Ϫ1°C, was reached. After one at 0.5°C minϪ1 until the onset of chill coma was hour at this temperature, ten specimens were removed recorded in all animals. To identify the onset of chill from the bath and were given 24 h to recover at 10°C. coma in larvae, their anterior parts were pinched gently Subsequently, the temperature was lowered by 1°C inter- using a fine wooden wand. The tip of the latter was vals at 0.1°C minϪ1 until Ϫ6°C was reached in the case retained in the vials to minimize heat exchange. Chill of the larvae, and Ϫ10°C in the case of the adults, corre- coma was indicated by the larvae’s inability to retract sponding to their respective mean field fresh SCPs. The from this pinching and by the adults’ inability to right removal procedure was repeated at each temperature themselves after being turned on their backs. The bath interval. After 24 h, the larvae and adults were assessed temperature was allowed to drop 0.5°C below the last for survival. Those specimens with full motor function ° CTMin/o value measured. The animals were held there for were considered survivors and were kept at 10 C for five minutes to allow all animals to equilibrate, and the seven more days and then reassessed. Lower lethal tem- temperature was then increased at the same rate. The perature was considered that temperature after which temperature at which an individual regained complete survival was consistently less than 100%. motor function was noted as the CTMin/r for that individ- To determine upper lethal temperatures, larvae and ° Ϫ1 ° ual. For CTMax, a similar procedure was adopted. Ani- adults were warmed at 0.1 C min to 30 and 25 C, mals were held for 15 min at 20°C and the temperature respectively. After one hour, ten specimens were was subsequently increased at 0.5°C minϪ1 until the removed and the temperature raised by 1°C. This pro- 98 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 cedure was repeated until temperatures were reached that three rotations (24 h each) of a 2:1 methanol–chloroform corresponded to the CTMax values of the larvae and solution. Individuals were dried to constant mass again, adults respectively. Survival assessment was the same and lipid content was assumed to be equivalent to the as above. difference between dry mass and lipid-free dry mass (Naidu and Hattingh, 1988). 2.5. Desiccation and starvation resistance The remaining individuals of the two groups were numbered, weighed and then transferred to desiccation For desiccation resistance, Chown’s (1993) protocol chambers containing silica gel, which in turn were was followed. Twenty field-collected larvae were given placed in the controlled-temperature cabinets. At 4, 8 access only to fresh water. The animals were kept for and 12 h all individuals were weighed, and at each inter- 24 h in the controlled-temperature cabinet to allow them val ten individuals, of each group, were removed and to clear their digestive tracts. The individuals were num- their haemolymph osmolality, dry mass, and lipid-free bered, weighed (to the nearest 0.1 mg using a Mettler dry mass were determined. After the 12 h interval, the AE163 electronic microbalance) and placed in des- remaining individuals were given access to fresh or sea- iccation chambers containing silica gel which reduced water, according to their original treatments. At 1, 2 and the relative humidity to 5% (measured using a Novasina 4 h intervals after the start of the rehydration period, Thermohygrometer). These were kept in the controlled all individuals were weighed. At each post-rehydration temperature cabinets. Larvae were weighed at 6-h inter- interval, ten individuals of both groups were removed vals until 100% mortality. Maximum tolerable water and their haemolymph osmolality, dry mass and lipid- loss, expressed as the percentage fresh mass lost (% FM) free dry mass determined. In addition, fresh mass, dry and as water loss (mg), time to maximum water loss (h), mass, water content, lipid content and haemolymph and rate of water loss expressed as %FM hϪ1 and mg osmolalities were determined for 10 field-fresh larvae. hϪ1 were determined. These measures were used because The osmolalities of freshly collected seawater, fresh cuticular and respiratory transpiration could not be sep- water and decaying kelp slime were also determined. arated, and they were calculated from the mass recorded Mass loss (mg), lipid content (mg), and water content in the time interval directly prior to death in each indi- (mg) were also corrected for initial mass, using an analy- vidual. Weight loss consisted of incidental and respir- sis of covariance (Packard and Boardman, 1988) before atory water loss (Wharton, 1985) and of minute faecal the means for each desiccation interval were calculated. droplets produced only as a reaction to the disturbance The same was done for the larvae which were allowed during weighing. The effects of body size on the water to rehydrate, but this analysis had to be undertaken sep- loss parameters was determined using least-squares lin- arately because mass gain, rather than mass loss, was ear regression analyses (Sokal and Rohlf, 1995). the variable of interest (see Klok and Chown, 1997). Because of differences in the size of the larvae used in To determine the efficacy of lipid metabolism as a the experiment and the unreliability of ratios, mass loss means of supplementing body water during desiccation, (mg), and rates of mass loss (mg hϪ1) were corrected we followed the method outlined by Naidu and Hattingh for initial mass, using an analysis of covariance (Packard (1988). Both mass loss and lipid content were corrected and Boardman, 1988). This experiment was repeated for larval mass using an analysis of covariance. The cor- with the adults, but because of their tendency to attempt rected values were then used in a least squares linear to burrow through the mesh and into the silica gel, was regression of lipid content on mass loss. terminated after 5.5 h. Larval and adult water loss rate The efficacy of haemolymph osmoregulation was comparisons were made over this period only. determined by comparing haemolymph osmolality values predicted by mean whole body water contents at 2.6. Larval water balance and osmoregulation each time interval, and actual haemolymph osmolality and body water contents. Predicted values were calcu- = Seventy field-collected larvae were given access only lated from pe/pi Vi/Ve (V is volume, p is haemolymph to fresh water and another seventy only to seawater. The osmolality, i and e are initial and experimental values larvae were kept for 24 h in the controlled-temperature respectively; Hadley, 1994), and linear regression lines cabinet to allow them to clear their digestive tracts. At were fitted to both data sets. For reasons outlined above the start of the experiment, ten individuals were chosen we used body water content corrected for initial mass as at random from both groups. They were weighed, a 10 the abscissa. Data for larvae subjected to desiccation, but µl haemolymph sample was extracted with a capillary not given access to water, and those that had access to tube from a small, dorsal cuticular incision, and the lar- water, were treated separately. vae were then dried to constant mass at 60°C. Haemo- 2.7. Larval tolerance of submersion lymph osmolality was determined using a Wescor 5120 B vapour pressure osmometer. Larvae were weighed dry, Tolerance of total submersion of larvae in fresh water broken up, and lipids were subsequently extracted using and in seawater was determined by placing batches of C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 99 ten larvae in sealed vials filled with water. Care was allowed the larvae to free themselves from these sheaths. taken not to include any air bubbles in the vials. These The newly exposed slime layers subsequently froze vials were placed in the temperature-controlled cabinet again, consequently small frozen tunnels marked the and a batch of larvae from both fresh water and seawater movements of the larvae. In some cases, larvae froze at were removed after 6, 12, 18, 24, 48, 72 and 96 h. After the ends of these tunnels, after the onset of chill coma. removal the larvae were placed on decomposing kelp Among the wet larvae, 14 out of 20 specimens froze and assessed for survival after 24 h, and reassessed after and none of these recovered when the temperature was seven days. increased to determine CTMin/r. The dry larvae were not Changes in water content and haemolymph osmolality exposed to this possible external inoculation. Only nine of submersed larvae were also determined. Field-fresh dry larvae froze during the CTMin/o decline in tempera- larvae were weighed and submersed individually, as ture. In contrast with the wet larvae, four of the nine described above, in both seawater and fresh water. Five frozen dry larvae recovered along with the unfrozen indi- larvae from both sea- and fresh water were removed and viduals. However, two of these four specimens appeared weighed again after 2, 5, 10, 20 and 48 h and their hae- much weaker than those that did not freeze. They were molymph osmolalities and dry mass were determined. not considered survivors. None of the adults froze during After the 48 h interval the remaining seawater submersed the course of the CT experiments, but one specimen larvae were weighed and then switched to fresh water. Min failed to recover from chill coma. Both the CTMin/o and The fresh water submersed larvae were switched to sea- CT values of the larvae were significantly lower than water. After the switch, five larvae from both treatments Min/r those of the adults (Table 1). However, when the adults were removed at 0.5, 1, 2, 5, and 24 h, re-weighed, and were removed from the analysis, the slime-covered lar- their haemolymph osmolalities and dry masses determ- vae had significantly lower CT values than the dry ined. We adopted this dual procedure to assess whether Min/o larvae (F =14.28, PϽ0.0005), but there was no dif- larvae showed different responses to the order of treat- (1,38) ference in CTMin/r values between the wet and dry larvae ments (hyper-osmotic to hypo-osmotic or vice versa, see = = Parkinson and Ring, 1983; Chown and Van Drim- that recovered (F(1,17) 0.5, P 0.47). CTMin/o values of melen, 1992). larvae that froze, wet or dry, did not differ significantly from those larvae, in either group, that did not freeze = = = = (F(1, 18)wet 1.61, P 0.22; F(1,18)dry 0.006, P 0.94). 3. Results Paractora larvae had significantly higher CTMax = values (see Table 1) than did adults (F(1,38) 277.73, Ͻ 3.1. Critical thermal limits P 0.0001). The critical temperature ranges of the larvae and the adults were calculated using the CTMin/r values to make the results comparable with those of other similar The results of the CTMin/o,CTMin/r, and CTMax experiments are summarized in Table 1. At temperatures close studies. Larvae have a critical temperature range 8.5°C to the CTMin/o values of the larvae, the outer layer of wider than that of the adults. slime covering the wet larvae froze, forming a sheath around their bodies. The unfrozen inner layer of slime

Table 1 ° A,B Summary statistics of CTMin/o,CTMin/r and CTMax ( C) of wet and dry field-fresh P. dreuxi larvae and field-fresh adults. denote significantly Ͻ different means (Tukey HSD, P 0.001). (CTMin/o—onset of chill coma; CTMin/r—recovery from chill coma; CTMax—onset of heat stupor)

± CTMin/o Mean SE Minimum Maximum n

Wet larvae Ϫ5.1±0.09A Ϫ5.5 Ϫ4.0 20 Dry larvae Ϫ4.5±0.13A Ϫ5.9 Ϫ3.3 20 Adults Ϫ2.7±0.19B Ϫ3.7 Ϫ0.9 20 ANOVA: F =30.98, df=2, 57, PϽ0.0001

CTMin/r Wet larvae Ϫ1.3±0.32A Ϫ2.4 Ϫ0.3 6 Dry larvae Ϫ0.3±0.90A Ϫ3.9 +6.5 13 Adults 1.9±0.40B Ϫ2.4 +4.3 19 ANOVA: F=6.25, df=2, 35, P=0.0048

CTMax Critical range Field-fresh larvae 35.5±0.11 34.7 36.6 20 36.8 (40.6)a Adults 30.2±0.27 27.9 31.9 20 28.3 (32.9)a

a Critical temperature ranges are calculated from CTMaxϪCTMin/r to be comparable with previous studies but for the values in parentheses the

CTMin/o of wet larvae and adults were used. 100 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109

3.2. Supercooling points and temperature tolerances 3.3. Water balance and osmoregulation

All of the field-fresh larvae survived freezing and The large range in the maximum tolerable water loss, recovered completely. The SCPs of the field-fresh wet rate of water loss and time to maximum water loss and dry larvae (Table 2) did not differ significantly values (Table 3) is a consequence of the size range of the = = (F(1,28) 2.624, P 0.116). Of the starved group, nine individual larvae used in this experiment. This is clearly specimens recovered from freezing, but only two of demonstrated by the significant relationships between those survived after 24 h and seven days. None of the initial mass and both mass loss prior to death and rate adults survived freezing. The field-fresh larvae had sig- of water loss (Table 3). Most of the larvae reacted with nificantly higher SCPs than the field-fresh adults jerking movements when handled and some of them = Ͻ (F(2,47) 230.8, P 0.0001). Starvation drastically accompanied these movements by excreting a small depressed the SCPs of the larvae, but had no significant droplet (Ͻ1 mm in diameter) of liquid from the anus. effect on the SCPs of the adults (Table 2). The SCPs of This excretion continued well into the desiccation period kelp slime (Table 2) were significantly lower than those until the weighing intervals preceding death, and thus = of both wet and dry field-fresh larvae (F(2,37) 46.89, the gravimetric water loss provided here may be over- PϽ0.0001). estimates of incidental and respiratory loss. Nonetheless, Percentage survival in the larvae [Fig. 1(A)] showed comparisons of water loss rates for larvae (1.187±0.138 a sudden decline with temperature corresponding to the mg hϪ1) and adults (0.708±0.19 mg hϪ1), corrected by measured CTMin/o and SCP values. All surviving speci- ANCOVA for differences in initial body mass, showed mens remained viable and reacted normally seven days that these rates differed significantly even over short = = after the experiment. In contrast, adult survival declined time periods (ANCOVA F(1,37) 7.136, P 0.011). gradually as temperature declined, although mortality Table 4 provides summary statistics for fresh mass, was high at temperatures higher than their mean field- lipid content, water content and haemolymph osmolality fresh SCPs. At Ϫ5°C the adults suffered further mor- of field-fresh larvae and the osmolalities of kelp slime, talities in the seven days following the experiment. seawater and fresh water. At the start of the osmoregul- Below Ϫ5°C the ‘surviving’ adults did not live for more ation experiments (i.e. after 24 h acclimation in either than 24 h. fresh water or seawater), body mass corrected water con- In the determination of upper lethal temperatures [Fig. tents in the two groups were not significantly different ° = = 1(B)], larvae showed 100% survival at 31 C (24 h and (F(1,138) 1.104, P 0.3). When desiccated for 12 h the 7 days after exposure), whereas the adults had already mass corrected body water contents of the seawater suffered 100% mortality at this temperature. Once again, treated larvae were significantly less than those of the = Ͻ larvae showed a sudden increase in mortality over a fresh water treated larvae (F(1,78) 19.27, P 0.0001, Fig. small temperature range, 32–34°C. Surviving larvae 2). On the other hand haemolymph osmolalities after 12 from all the high temperature intervals were still alive h desiccation were not significantly different = = seven days after exposure. Immediate survival of high (F(1,12) 2.25, P 0.16). During the rehydration period, the temperatures declined more gradually in the adults than mass corrected data showed that the fresh water in the larvae, but further mortalities occurred in the acclimated larvae, when given access to fresh water, adults seven days after the experiment. gained significantly more water than those acclimated in

Table 2 Summary statistics for the supercooling points of P. dreuxi larvae, adults and kelp slime

SCP Mean±SE Minimum Maximum n

Wet ﬁeld-fresh larvaeA Ϫ3.3±0.05 Ϫ3.8 Ϫ2.8 20 Dry ﬁeld-fresh larvaeA Ϫ3.5±0.08 Ϫ3.9 Ϫ3.1 10 Starved larvaeB Ϫ7.4±0.44 Ϫ10.6 Ϫ4.0 20 Adults Ϫ9.6±0.51 Ϫ15.0 Ϫ6.7 20 Starved adults Ϫ9.1±0.39 Ϫ12.0 Ϫ4.7 19 Kelp slime Ϫ5.1±0.28 Ϫ6.6 Ϫ4.1 10

ANOVAs F ratio df P

Larval SCPs 62.98 2,47 Ͻ0.0001a Adult SCPs 0.51 1,37 Ͻ0.48

Wet CTMin/o vs Wet SCP 299.82 1,38 Ͻ0.0001

Dry CTMin/o vs Dry SCP 30.03 1,28 Ͻ0.0001

a Different letters denote signiﬁcant differences. C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 101

Fig. 1. The lower lethal temperatures (A), and upper lethal temperatures (B), of P. dreuxi larvae (slime covered) and adults. Specimens were kept for one hour at each treatment temperature and were assessed for survival at 24 h and 7 days after the experiments. Larval survival for both lower and upper lethal temperatures did not decline after seven days.

= = Ͻ seawater, when given access to seawater (F(1,58) 25.74, water for rehydration [F(1,63) 10.403, P 0.001 and Ͻ = Ͻ P 0.0001). In addition, after rehydration, haemolymph F(1,55) 9.013, P 0.0001, respectively, Fig. 2(B)]. osmolality of the fresh water acclimated larvae was sig- Linear regressions of the observed and predicted nificantly lower than that of the seawater treated larvae values of haemolymph osmolality on mass corrected = that had been given access to seawater (F(1,54) 70.66, water content during the desiccation period, for both the PϽ0.0001). fresh water and seawater treated larvae, were all highly Within the fresh water group, mass corrected water significant. The slopes of observed haemolymph osmol- contents and haemolymph osmolalities, after 12 h des- alities on mass corrected water contents in both the fresh iccation, differed significantly from initial water contents and seawater experiments also differed significantly = Ͻ and osmolalities (F(1,63) 4.646, P 0.0006 and from the slopes derived using predicted haemolymph = Ͻ F(1,59) 9.332, P 0.0001 respectively). After rehydration, osmolalities (Fig. 3). The values of the predicted slopes neither the mass corrected water contents nor the haemo- in both treatments are approximately three times larger lymph osmolalities differed from the initial values [Fig. than what was observed in the experiments. These 2(A)]. The seawater group’s water contents and haemo- regressions indicate that larval haemolymph osmolalities lymph osmolalities also differed significantly from the did increase somewhat with increasing water loss. How- initial values after desiccation for 12 h, but in contrast, ever, these osmolalities were much lower than those pre- remained different when they were given access to sea- dicted from a decline in body water content. In contrast, 102 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109

Table 3 Summary statistics for initial mass, maximum tolerable water loss, rate of water loss and time to maximum water loss, and the least squares linear regressions of maximum tolerable water loss, rate of water loss and time to maximum tolerable water loss on initial mass in P.dreuxi larvae used in the desiccation experiments (n=20)

Variable Mean±SE Minimum Maximum

Initial mass (mg) 41.9±2.6 19.2 61.9 Maximum tolerable 23.4±1.5 7.3 34.2 loss (mg) Maximum tolerable 55.60±1.25 37.24 64.14 loss (%) Corrected 23.4±0.04 20.0 27.1 maximum loss (mg) Rate (mg hϪ1) 0.85±0.07 0.43 1.52 Rate (% hϪ1) 2.07±0.14 1.17 3.58 Corrected rate (mg 0.85±0.05 0.53 1.36 hϪ1) Time (h) 29.30±2.10 16.50 47.50

Slope±SE Intercept±SE r2 FPdf

Water loss (g) 0.5676±0.0369 Ϫ0.00034±0.0016 0.929 236.32 Ͻ0.0001 18 Rate (g hϪ1) 0.0204±0.0046 0.00001±0.0002 0.517 19.27 Ͻ0.0004 18 Time (h) 81.99±191.99 25.86±8.33 0.01 0.18 0.67 18

Table 4 Summary statistics for fresh mass, water and lipid contents (absolute and percentages) and haemolymph osmolality of ﬁeld-fresh P. dreuxi larvae (n=10 throughout)

Variable Mean±SE Minimum Maximum

Fresh mass (mg) 39.2±3.8 28.0 60.7 Water content (mg) 33.0±3.4 23.1 50.1 Water content (%) 83.84±0.98 79.28 88.27 Lipid content (mg) 2.2±0.5 0.4 5.8 Lipid content (%) (of dry mass) 38.54±3.32 14.29 52.38

Osmolalities (mOsmol kgϪ1)

Field-fresh haemolymph 339.9±8.48 292 390 Kelp slime 691.2±12.01 608 736 Seawater 928.0±0.95 922 932 Fresh water 67.7±0.33 66 0 during the rehydration period of both the fresh and sea- Percentage survival of total submersion in fresh and water treated larvae, neither the observed changes in seawater was high. The few mortalities that did occur haemolymph osmolalities in relation to the increase in did not exhibit any readily comprehensible pattern. Apart mass-corrected water contents, nor the predicted changes from a 20% mortality at 24 h in fresh water, and 10% gave signiﬁcant regressions. mortality at 18 h and 20% at 48 and 72 h in seawater, No changes in the mass-corrected lipid contents all other larvae survived total submersion in fresh and occurred over the 4 h intervals during the 12 h des- seawater up to 96 h without any observable adverse = iccation period in either the fresh water (F(3,36) 0.9, effects, when assessed both 24 h and seven days after = = = P 0.45) or seawater experiments (F(3,35) 1.83, P 0.15) the conclusion of the experiments. (Table 5). The regressions of lipid contents (corrected When larvae were switched from seawater (928 mOs- for mass) on corrected-mass loss in both fresh water and mol kgϪ1) to fresh water (67.7 mOsmol kgϪ1) or vice seawater experiments were likewise not signiﬁcant versa, after submersion for 48 h, this did not lead to (Fresh water; F=0.018, P=0.894: Seawater: F=5.76, isosmotic changes in the haemolymph or pronounced P=0.216). The mass loss observed was thus mostly the changes in water contents of the submersed larvae (Fig. result of water loss. 4). Even within each treatment larval body water con- C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 103

Fig. 2. Mass corrected water contents (᭿) and haemolymph osmolalities (̆)ofP. dreuxi larvae during desiccation and subsequent rehydration (means±SE). (A) Larvae were pre-treated in fresh water and after desiccation were given access to fresh water. The recovery during rehydration of lost water content and lower haemolymph osmolalities to levels not signiﬁcantly different from the initial levels are clearly evident. (B) Larvae were pre-treated in seawater and after desiccation were given access to seawater. Neither lost water content nor haemolymph osmolalities recovered to initial levels.

tents and haemolymph osmolalities did not follow those from the CTMin/o trials, where mean CTMin/o tem- decreasing or increasing trends over time (Fig. 4). peratures were significantly lower than the SCPs determined for both the wet and dry larvae (Tables 1 and 2). These discrepancies in the data are most likely a conse- 4. Discussion quence of differences in experimental protocol. We sus- pect that the primary way in which experimental proto- 4.1. Thermal tolerances col had an influence was through the differences in cooling rate used in the two trials, because it is well The mean supercooling point determined for field- known that higher cooling rates often result in lower fresh larvae (Ϫ3.3°C) was close to larval lower lethal SCPs (Franks, 1985). However, it should also be noted temperatures (20% survival at Ϫ4°C, but no survival that during the SCP experiments larvae were completely Ϫ ° from 5 C onwards). These data contrast strongly with immobilized, whereas during CTMin/o determination they 104 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109

were able to move freely until the onset of chill coma. Thus a difference in movement ability might in some way have affected ice nucleation, because both wet and dry larvae showed the same responses. In addition the decaying kelp slime had a signiﬁcantly lower SCP than either wet or dry ﬁeld fresh larvae, but its SCP was vir-

tually identical to the CTMin/o of slime covered larvae = = (F(1, 28) 0, P 1), and was significantly lower than the = Ͻ CTMin/o of dry larvae (F(1,28) 1,28, P 0.038). Thus for confined larvae, ice inoculation is clearly not initiated externally and does not influence their survival. For free moving larvae, freezing of the external slime layer does have a negative influence on their survival as is evi- denced by the lower survival of frozen slime covered

larvae in the CTMin/o determinations. This suggests that external ice nucleation is not as well tolerated by the larvae as is inoculation via the gut contents. The latter is clearly important for controlled freezing survival because the SCPs of starved larvae are much reduced and they did not survive freezing (see also Strong-Gund- erson et al., 1992; Lee et al., 1993; Klok and Chown, 1998). The initiation of nucleation in the gut, which leads to survival of the larvae is not unique to P. dreuxi, but has also been described in arctiid and tineid caterpillars (Fields and McNeil, 1988; Klok and Chown, 1997), and a tenthridinid wasp (Shimada, 1989). However, the way in which ice nucleation in the gut acts to enhance Fig. 3. Haemolymph osmolalities as a function of body water content, corrected for initial mass, in larvae subjected to desiccation. The freezing survival is not clear (Shimada, 1989), and the hashed lines indicate the regressions fitted to the predicted values (¼) role of the gut contents (or their absence) in cold hardi- in individuals denied access to water and the solid lines indicate the ness remains the subject of some contention (Baust and regression fitted to the observed values (᭺) for individuals denied Rojas, 1985; Parish and Bale, 1990). Thus, while the ᭺ access to water. (A) Larvae treated in fresh water, ( ) possibility exists that movement affects the response of Y=500.57Ϫ5.02X, r2=17.84%, PϽ0.01, n=36 and (¼) Y=951.25Ϫ16.87X, r2=38.65%, PϽ0.0001, n=36. The slopes of the larvae to low temperatures, we have no adequate expla- lines differed significantly (t=6.25, PϽ0.01). (B) Larvae treated in sea- nation for this phenomenon, nor do we have information water, (᭺)Y=654.03Ϫ10.0X, r2=62.77%, PϽ0.0001, n=36 and (¼) at our disposal on whether or not larvae become immo- Y=1378.9Ϫ31.76X, r2=77.94%, PϽ0.0001, n=36. The slopes of the = Ͻ bile at low temperatures in the field. Clearly such behav- lines differed significantly (t 16.46, P 0.001). ioural information is critical for determining larval survival of a low temperature event.

Table 5 Summary statistics of changes in lipid contents during the 12 h desiccation periods of P. dreuxi larvae treated with fresh water and seawater

Fresh water Mean±SE Minimum Maximum n

Initial lipid content (mg) 2.1±0.5 0.4 5.9 10 Corrected initial lipid content (mg) 3.1±0.4 2.4 6.9 10 Dehydrated lipid content (mg) 3.6±0.3 2.7 4.9 10 Corrected dehydrated lipid content (mg) 3.2±0.2 2.2 4.6 10

Seawater

Initial lipid content (mg) 2.2±0.5 0.4 5.8 10 Corrected initial lipid content (mg) 3.2±0.3 1.8 5.3 10 Dehydrated lipid content (mg) 2.9±0.3 1.5 3.8 9 Corrected dehydrated lipid content (mg) 2.5±0.2 1.8 3.5 9 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 105

Fig. 4. Haemolymph osmolalities (᭿) and water contents (̆) (means±SE), of P. dreuxi larvae initially submersed in water of a given osmolality (freshwater or seawater) and then switched to water with a different osmolality (seawater or fresh water). Both the haemolymph osmolalities and the water contents of the larvae in either the fresh water to seawater (A), or the seawater to fresh water (B) submersion experiments, did not show any isosmotic changes corresponding to the osmolalities of the submersion media.

If these contradictory results are assumed to be a theless sufficiently well-developed for their particular consequence of differences in experimental design, and thermal conditions in kelp depositions. This level of if the SCP and lower lethal temperature experiments are freeze tolerance in P. dreuxi larvae is similar to the situ- accepted as a reasonable indication of the cold hardiness ation in the South Georgian beetle, Hydromedion spar- strategy adopted by P. dreuxi larvae, then they must be sutum (Block et al., 1998), and some New Zealand wetas considered moderately freeze tolerant insects (see Sin- (Sinclair et al., 1999), and may reflect a general strategy clair’s 1999 cold hardiness classification scheme which amongst sub-Antarctic insects, which are exposed to low is essentially an extension of the original one proposed intensity freezing events on a regular basis (see Klok by Bale (1993, 1996)). Even so, field-fresh P. dreuxi lar- and Chown, 1997; Sinclair et al., 1999 for further vae had remarkably high SCPs compared to most other discussion). In the case of P. dreuxi larvae, it is signifi- freeze tolerant insects. Of a total of 53 freeze tolerant cant that habitat temperatures are unlikely to decline insect species discussed by Sinclair (1999), only three below approximately Ϫ3toϪ4°C (see Materials and other species had SCP values higher than P. dreuxi’s Methods and Blake, 1996). Nonetheless, it should be Ϫ3.3°C. Thus the relatively high SCP and proximity of borne in mind that most other studies have used cooling the SCP and lower lethal temperatures in P. dreuxi lar- rates substantially greater than those here, and conse- vae suggest that their moderate freeze tolerance is none- quently report much lower SCPs (see Franks, 1985; Kelty and Lee, 1999) 106 C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109

Compared to the larvae, P. dreuxi adults were more and other insect species living in habitats with high salt sensitive to low temperatures. Additionally they proved concentrations. In general, these species can maintain to be intolerant of freezing. The mean SCP of field-fresh stable haemolymph osmolalities in media with widely adults (Ϫ9.6°C) and the decline in adult survival during varying osmolalities (0 to 1500 mOsmol kgϪ1) (Nemenz, the LLT determinations, at temperatures well above the 1960; Shaw and Stobbard, 1963; Barnby, 1987; Herbst mean SCP, suggest that the adult response is typically and Bradley, 1988; Herbst et al., 1988). However, P. that of a summer active insect with no cold hardiness dreuxi larvae differ substantially from several aquatic capacity (Zachariassen, 1985). This response does not dipterans (mostly Chironimoidae, Culicidae and appear to be influenced by feeding state, presumably Ephydridae) that live in brackish water and survive as either because the gut is not wholly evacuated during osmoconformers when the osmolality of the medium starvation, if nucleation is initiated there (see Parish and rises above that of their haemolymph (Shaw and Stob- Bale, 1990), or because, like many other insects, bard, 1963; Garrett and Bradley 1984, 1987; Herbst and nucleation is initiated in the haemolymph or tissues, and Bradley, 1988). gut contents have no influence over this process Gainey (1984) compared the efficiency of osmoregul- (Block, 1990). ation in various insects using the slope of the regression of haemolymph osmolality on total dissolved solids. The 4.2. Desiccation resistance and osmoregulation slope of this regression for strong osmoregulating Dip- tera species, that are thought to be amongst the insects Survival time and water loss rates calculated for the with the most pronounced osmoregulatory ability larvae are slight under-, and overestimates, respectively. (Gainey, 1984; Barnby, 1987; Herbst et al., 1988; This is because some excretion took place during hand- Schwantes and Schwantes, 1990), ranged from 0.02 to ling. Nonetheless, they are unlikely to be greatly in error 0.64. If the media in which P. dreuxi larvae were kept due to the small amounts of liquid excreted. Bearing this are regarded as a solution of NaCl ions, then the slope in mind, it appears that P. dreuxi larvae are not parti- of this regression for P. dreuxi is 0.27. This places P. cularly desiccation resistant compared to many other dreuxi amongst these strong osmoregulating Diptera. insect species on Marion Island (Chown, 1993; Klok and Such a pronounced osmoregulatory ability of the non- Chown 1997, 1998). Larval survival time (ca 30 h) was aquatic P. dreuxi larvae should not only enable them to even shorter than that found for desiccation intolerant survive brief periods of inundation with seawater, but Pringleophaga marioni caterpillars (Lepidoptera: should also ensure their survival of the high osmolalities Tineidae) (ca 59 h) (Klok and Chown, 1997). In the case associated with sporadic desiccation of kelp, if they are of the adults, mass corrected water loss rates over a short trapped inside the kelp fronds. period (5.5 h) were significantly lower than those of the Although P. dreuxi larvae can maintain haemolymph larvae. Although neither the adults’ maximum tolerable osmolality at benign levels when exposed to seawater water loss nor their survival time was estimated, this they do not utilize seawater to replenish lost body water lower water loss rate suggests that P. dreuxi adults are during non-lethal periods of desiccation, nor do they considerably more desiccation resistant than larvae. appear to supplement their free water by metabolism of Given the adults mobile and exposed lifestyle, this dif- lipids. In the latter case they are like many non-desert ference is not entirely unexpected. Nonetheless, it insects (e.g. Klok and Chown, 1997; Le Lagadec et al., remains clear that neither the adults nor the larvae of 1998), but remarkably different to Anatalanta aptera,a P. dreuxi are particularly desiccation resistant, especially sub-Antarctic sphaerocerid fly that is remarkably des- when their mean mass corrected water loss rates are iccation tolerant, and which is thought to metabolize lip- compared with P. marioni (0.66 mg hϪ1) and E. hal- ids to survive dry conditions (Tre´hen and Vernon, 1986; ticella (0.49 mg hϪ1) larvae over similar short periods Vernon, 1986; Vernon and Vannier, 1986). (see Klok and Chown 1997, 1998). An ANCOVA showed that water loss rates of both P. dreuxi larvae 4.3. Physiological tolerances, ontogenetic variation and adults do not differ significantly from those of the and climate change desiccation prone P. marioni larvae, but that they are significantly higher than those of the desiccation resist- Our study of P. dreuxi has provided limited, additional = Ͻ ant larvae of E. halticella (F(3,95) 6.78, P 0.001). support for the contention that in the moist higher lati- Despite the fact that P. dreuxi is not truly aquatic, it tude environments of the southern hemipshere, freezing does have a remarkable osmoregulatory capability. The tolerance is likely to predominate as a cold hardiness largely terrestrial P. dreuxi larvae were able to osmoreg- strategy in insects (Klok and Chown, 1997; Addo-Bedi- ulate in both hypo- and hyper-osmotic media. In ako et al., 2000). Nonetheless, to date, Diptera have addition, it appears that the larvae are able to osmoregul- appeared to constitute an exception to this trend, with ate, at least to some extent, during desiccation. In this most species being freezing intolerant (e.g. A. aptera) respect they are similar to a variety of aquatic Diptera (Vernon and Vannier, 1996). Although freezing toler- C.J. Klok, S.L. Chown / Journal of Insect Physiology 47 (2001) 95–109 107 ance is absent in P. dreuxi adults, and not particularly adults and larvae, coupled with ontogenetic differences well-developed in the larvae, its presence in this species in their physiological tolerances, may result in differen- provides an exception to the general tendency towards tial susceptibility of these stages to climate change. Elev- freezing intolerance amongst sub-Antarctic and Antarc- ated mean annual temperatures, especially if tic Diptera, and supports the broader latitudinal pattern. accompanied by increased sunshine durations (see Smith It also suggests that the relationship between habitat and Steenkamp, 1990) and decreased rainfall, are likely moisture content and cold hardiness strategy is an to mean high microclimate temperatures (Chown and important one. Antarctic and sub-Antarctic flies living in Crafford, 1992; Blake, 1996), and pronounced kelp des- dry environments tend to be freezing intolerant, whereas iccation. The desiccation intolerant P. dreuxi may well those from moist environments are freezing tolerant be affected substantially by this general drying out of (Convey and Block, 1996; Vernon and Vannier 1986, habitats. The less mobile larvae are likely to be most 1996). affected, particularly because they require fresh water to We also found pronounced ontogenetic differences in replenish that lost due to desiccation. Nonetheless, given physiological tolerances in P. dreuxi. Adult flies are not the nature of our initial physiological work, these predic- cold hardy, and generally have a reduced thermal toler- tions must remain largely speculative. Thus we suggest ance range compared to the larvae. In addition, they are that further, more careful investigations of the relation- somewhat more desiccation resistant, although not to the ships between climate change, environmental suitability, extent that they differ from the larvae when compared and physiological tolerances of indigenous species in the with other insect species on Marion Island. Such onto- sub-Antarctic should be undertaken (see also Bergstrom genetic differences in physiological tolerance are not and Chown, 1999). uncommon in Diptera. For example, they have been found in the partially freezing tolerant Tipula excisa, from high altitudes in Norway (Todd and Block, 1995), Acknowledgements and in the goldenrod gall fly, Eurosta solidaginis from North America. Paractora dreuxi differs from these K. Storey, J. Storey and W. Block are thanked for species in that it has overlapping generations that are their helpful insights on critical minimum temperatures, present throughout the year (Crafford, 1984; Crafford et mobility and sub-zero freezing. M. McGeogh, J. Bar- al., 1986), in a habitat that shows only limited tempera- endse, R. Mercer and A. Addo-Beddiako commented on ture variation compared to high latitudes in the northern an earlier version of the manuscript. C. Jacobs provided hemisphere (see Gaston and Chown, 1999). However, in partial assistance in the laboratory. B. Sinclair and an its case, although the adults and larvae share the same anonymous referee provided useful comments on an habitat, they behave quite differently. The larvae are earlier version of the ms. This research was supported much less mobile than the adults, and this means that by the South African Department of Environmental they are unlikely to be as capable as the adults of pro- Affairs and Tourism (SADEA&T) and the University of nounced behavioural responses to altered environmental Pretoria. Logistic support at Marion Island is provided conditions (Tre´hen et al., 1985; Crafford and Scholtz, by the SADEA&T. 1987). Thus larvae are able to tolerate a broader range of conditions than the adults, at least as far as temperature is concerned. 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