© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society 301

Physiological Entomology (2007) 32, 301–304 DOI: 10.1111/j.1365-3032.2007.00547.x

Introduction

Ecdysis is a necessary component of the somatic develop-ment of arthropods (Wigglesworth, 1965). Recent studies of sub-Antarctic Collembola have shown that moulting confers a significant increase in their cold hardiness ( Worland, 2005 ; Worland et al ., 2006). Although not considered to be an adap-tive strategy per se , the increases in cold hardiness are linked to the temporary cessation of feeding and the voiding of gut nucleating material ( Worland, 2005 ); the convergent cryoprotection afforded by the moulting state may have im-portant consequences for the low temperature survival of ar-thropods.

At high latitudes, such incidental cryoprotection may be particularly useful, either augmenting overwintering acclima-tization or facilitating low temperature survival in arthropods exposed to challenging diurnal changes of temperature. Arthropod growth in polar environments is constrained by short growing seasons and limited heat budgets ( Burn, 1981 ). The discovery of rapid cold hardening in Antarctic arthro-pods has identified a means by which low summer tempera-tures can be tolerated without sacrificing the active feeding state ( Worland & Convey, 2001; Sinclair et al. , 2003 ). Moulting may represent an additional, if accidental, form of summer cryoprotection for polar arthropods, increasing cold hardiness without the interruption of their life cycles. Furthermore, in that the temporal constraints of growth and

development in Antarctic summers have led to overlapping generations entering the overwintering period ( Block & Convey, 1995 ), animals overwintering in the moult state (which, at high latitudes, may be maintained throughout the winter hibernation period), are not only able to resume activ-ity earlier in the next summer ( Convey, 1994 ), but also may benefit from the cryoprotection afforded by the moult state.

To date, evidence for the effect of moulting on cold hardi-ness has been obtained only for Collembola ( Worland, 2005 ; Worland et al., 2006 ). The present study tests the hypothesis that a similar effect can be observed in mites, specifically the oribatid, Alaskozetes antarcticus (Michael), one of the most common and widely distributed arthropods of the maritime Antarctic ( Block & Convey, 1995 ).

Materials and methods

The study was carried out from December 2004 to March 2005 at the British Antarctic Survey research station at Rothera Point, Adelaide Island, on the west coast of the Antarctic Peninsula (maritime Antarctic) (67°34 ' S, 66°08 ' W). Animals were collected from aggregations under stones from Lagoon and Anchorage Islands. Alaskozetes antarcticus is one of the most abundant microarthropods in the maritime Antarctic, including the study location ( Block & Convey, 1995; Convey & Smith, 1997 ). It is a herbivorous/detrivorous oribatid with six postembryonic life stages: an inactive prelarval stage that develops within the egg, a six-legged larval stage, three eight-legged preadult nymphal stages (proto-, deuto- and tri-tonymphs), and the adult ( Block & Convey, 1995 ).

Moulting reduces freeze susceptibility in the Antarctic mite Alaskozetes antarcticus (Michael)

T . C . H A W E S 1 , J . S . B A L E 1 , M . R . W O R L A N D 2 and P . C O N V E Y 2 1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham and 2 British Antarctic Survey, National

Environment Research Council, High Cross, Cambridge, U.K.

Abstract . The effect of moulting on the cold hardiness of the oribatid mite Alaskozetes antarcticus (Michael) is investigated. Non moulting animals show clear seasonal patterns of cold hardiness with high supercooling points (SCPs) at the peak of summer and an increasing proportion of low SCPs with declining environmental temperatures. By contrast, both field-fresh and laboratory acclimated (5 °C) mites in the moult state are consistently found to have low SCPs regardless of environmental temperature.

Key words . Cold hardiness , cryoprotection , ecdysis , maritime Antarctic , oribatid , overwintering , supercooling point .

Correspondence: T. C. Hawes, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B16 2TT, U.K. e-mail: tinstone12@hotmail.com

302 T. C. Hawes et al.

© 2007 The Authors Journal compilation © 2007 The Royal Entomological Society, Physiological Entomology, 32, 301–304

Supercooling point (SCP) determination was carried out us-ing differential scanning calorimetry ( Worland & Convey, 2001 ). SCPs of field-fresh animals were determined immedi-ately after collection. Laboratory acclimated samples were maintained at 5 °C for 2 weeks after collection, maintained on an ad libitum food supply of the algae Prasiola crispa , before sorting and SCP assays. Mites freezing in ‘high’ and ‘low’ groups were defined, respectively, as animals with SCPs above and below – 15 °C ( Worland & Convey, 2001 ). Because SCPs were distributed bimodally, the Kolmogorov – Smirnov two-sample test was used to compare medians.

Changes in the SCPs of nonmoulting adults from summer to early winter were obtained to give a picture of seasonal variation in cold hardiness. To investigate the effect of moult-ing on the summer cold hardiness of Alaskozetes , compari-sons were made between the SCPs of moulting and nonmoulting adults either from field-fresh aggregations or from the 5 °C acclimation treatment. The latter laboratory acclimation treatment (known to produce animals predomi-nantly in the ‘high’ group) was employed to control for the effects of potential short-term acclimatization (maritime Antarctic arthropods are capable of rapid cold hardening; Worland & Convey, 2001 ) to low summer temperatures in field samples.

Because it is not always easy to discriminate between ani-mals that have just entered or are preparing to enter the pre-ecdysial resting stage from active individuals, comparisons were made between moulting tritonymphs and adults. Moulting represents a transitional stage between the two stages of development. Thus, feeding animals of either stage are suitable controls for comparing cold hardiness. In accord-ance with their lower volumes of body fluids, nymphal stages of A. alaskozetes have slightly lower SCPs ( Pugh, 1994 ), but still have ‘summer’ and ‘winter’ modalities in their SCPs ( Young & Block, 1980 ). In other words, if they are not moult-ing and are sampled in the summer or are acclimated to warm temperatures (e.g. 5 °C) they will have SCPs in the ‘high’ group. Moreover, as adults do not moult, they provide a more reliable reference group for animals in an active, feeding state.

Tritonymphs were distinguished by gross morphological ap-pearance from adults by inspection under stereo microscope. Changes in the cuticle undergone during moulting allowed for ready discrimination of mites from field collections into adults or tritonymphs in the ‘moult’ state, with the former

Fig. 1. (a) Field aggregation of Alaskozetes antarcticus on upturned rock surface.

Fig. 2. Scanning electron micrographs of moulting tritonymph of Alaskozetes antarcticus : (a) whole animal; (b, c) close-ups of tritonymph carapace with ‘double cuticle’ evident. oc, old cuticle; nc, new cuticle. Note the pleated integument of the old cuticle (oc) typi-cal of the nymphals stages of A. antarcticus ( Block & Convey, 1995 ).

Moulting reduces freeze susceptibility in the Antarctic mite 303

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society, Physiological Entomology, 32, 301–304

having shiny black cuticles, and the latter having matt black ( Fig. 1). Moulting tritonymphs were quiescent and, at the end of the pre-ecdysial stage, swollen because increases in haemolymph volume precede ecdysis ( Chapman, 1998 ). In addition, the surface cuticle of moulting animals could be seen, under magnification, to be detaching from the underly-ing epidermis ( Fig. 2), which is the equivalent of the ‘double cuticle’ effect described in Collembola ( De With & Joose, 1971 ).

Results and Discussion

Figure 3 shows snap-shot SCP distributions of nonmoulting Alaskozetes adults from mid-summer to early winter. This is the expected distribution of animals sampled at seasonal scales. Two-sample comparison of medians between each snapshot revealed highly significant differences between the summer-type SCPs of 28 January ( d max = 1.000; P < 0.001) and 7 February ( d max = 0.944; P < 0.001) and the winter-type SCPs of 6 March The effect of moulting on the SCPs of these mites was clear ( Table 1). Whether sam-pled directly from the field (d max = 0.569; P < 0.001) or from temperature-controlled conditions ( d max = 0.533; P < 0.001), the difference between the SCPs of moulting tri-tonymphs and adults was highly significant.

Therefore, moulting significantly effects the cold hardi -ness of A. antarcticus , producing a state of cold hardiness in

animals that is independent of environmental/seasonal tem-peratures. The moulting tritonymphs examined in the present study, whether field-fresh or acclimated at a temperature known to induce ‘high’ group SCPs, had SCPs unambigu-ously skewed towards temperatures below – 15 °C (i.e. the ‘low’ group). In tandem with studies on sub-Antarctic Collembola ( Worland, 2005 ; Worland et al., 2006 ), the present study provides further evidence for the necessity of including moulting among the diverse array of intrinsic fac-tors that need to be taken into account when assessing the cold hardiness of arthropods, particularly at high latitudes.

Starvation reduces the supercooling points of many arthro-pods ( Sømme & Block, 1982 ; Cannon, 1986; Leather et al. , 1991 ; but see also Baust & Rojas, 1985 ). The mechanisms responsible for the reduced freeze susceptibility of moulting arthropods (i.e. an absence of gut nucleating material) may be physiologically identical to starvation, but the circum-stances governing this increased cold hardiness are qualita-tively distinct. Starvation can be a stochastic phenomenon, reflecting the last time an animal fed, or it can be a behav-ioural response to low temperatures, particularly as a prelude to the adoption of the overwintering state. On the other hand, the moult state is governed by hormones and occurs in re-sponse to somatic demands ( Wigglesworth, 1965 ). It is a dis-tinct developmental stage in the life cycle and low SCPs are a by-product of A. antarcticus expressing this stage of their developmental program.

Fig. 3. Snap-shot of seasonal changes in the SCPs of adult Alaskozetes antarcticus : open boxes, 28 January; shaded boxes, 7 February; filled boxes, 6 March.

0-0 -4 -7 -10 -13 -16 -19 -22 -25 -28 -31

1

2

3

4

5

6

supercooling point (ºC)

freq

uenc

y

Table 1. Comparison of SCPs of acclimated (5 °C) and fieldfresh moulting tritonymphs and adult Alaskozetes antarcticus . HG, high group; LG, low group.

Treatment Moult status n Median HG : LG Significance (Komogorov-Smirnov two-sample test)

Fieldfresh (13 January to February) Moulting tritonymphs 27 −27.91 (+5.17–0.91) 2 : 25 P < 0.001 Adults 53 −8.37 (+1.68–13.83) 39 : 14

Acclimated at 5 °C Moulting tritonymphs 74 −27.75 (+20.13–1.85) 25 : 49 P < 0.001 Adults 59 −7.38 (+1.39–2.26) 46 : 13

Median error = interquartile range.

304 T. C. Hawes et al.

© 2007 The Authors Journal compilation © 2007 The Royal Entomological Society, Physiological Entomology, 32, 301–304

Although it is unlikely that the cryoprotection afforded to moulting mites and insects is an adaptive trait, the convergent benefits accrued to polar arthropods, in particular, may be impor-tant. Cold hardiness is useful to animals exposed to low summer temperatures and moulting potentially allows A. antarcticus to endure such exposures without interrupting normal development or diverting energetic resources to either rapid or longer-term ac-climatization. Likewise, it may augment seasonal acclimatization processes in mites preparing to overwinter ( Convey, 1994 ).

The relationship between moulting and cold hardiness demonstrated here makes sense of some of the variability that has been seen in natural populations (e.g. when animals pre-dicted to have clearly delineated seasonal cold hardiness, show heterogeneous SCPs; Sinclair et al. , 2003 ). As shown in the present study, moulting can skew such natural variability significantly.

The relative importance of cryoprotection from moulting depends on the coincidence of low temperatures and the moult state. In an overwintering context, there is clearly a correlation between the need for cryoprotection and low environmental temperatures but, although moulting individ-uals form a noticeable proportion of aggregations into early winter (unpublished observations), the actual extent to which populations overwinter in the moult state is unknown. Moult synchronisation before winter would enable nymphs to moult into reproductive adults at the same time at the be-ginning of the next summer, thus maximizing the amount of time available for oviposition and the maturation of off-spring ( Convey, 1994 ). Synchronisation of moulting is com-mon in Collembola ( Leinaas, 1983 ) and has also been noted in the Arctic mite Ameronothrus lineatus ( Sovik & Leinaas, 2003 ). However Sovik et al. (2003) found that A. lineatus synchronised their moulting in mid-summer, with few individuals passing the winter in the pre-ecdysial stage.

Overlapping generations, and the fact that moulting indi-viduals can be found throughout the summer and beginning of winter, suggest that Alaskozetes do not synchronize their moulting at the level of the total regional population (e.g. of Lagoon or Anchorage Island or of Marguerite Bay). Nevertheless, local synchronisation may be important because moulting in these mites is a gregarious phenomenon, with moulting individuals forming dense aggregations under stones throughout the summer ( Fig. 1 ). Although the signifi-cance of cold hardiness accrued from moulting in terms of a population-level effect (i.e. from moult synchronisation) is uncertain, at the individual level, it certainly protects against low temperatures.

In conclusion, although the cryoprotection afforded by the moult state is probably ‘non-adaptive’, moulting may benefit A. antarcticus exposed to low summer temperatures or enter-ing the Antarctic winter. These findings allow further discrimi-nation of the factors, both intrinsic and extrinsic, that contribute to the cold hardiness of polar arthropods and contribute to a more comprehensive delineation of the factors responsible for the natural variability of SCPs in summer populations.

Acknowledgements

T.C.H. is supported by a BBSRC studentship. The NERC Antarctic Funding Initiative (CGS6/13) funded fieldwork at Rothera Research Station.

References

Baust , J.G. & Rojas , R.R . ( 1985 ) Insect cold hardiness: facts and fancy . Journal of Insect Physiology , 31 , 755 – 759 .

Block , W . & Convey , P . ( 1995 ) The biology, life-cycle and ecophysi-ology of the Antarctic mite, Alaskozetes antarcticus , Journal of Zoology , 236 , 431 – 449 .

Burn , A.J . ( 1981 ) Effects of temperature on the feeding activity of Cryptopygus antarcticus . Comité National Français Des Recher-ches Antarctiques , 51 , 209 – 216 .

Cannon , R.F.C . ( 1986 ) Diet and acclimation effects on the cold tol-erance and survival of an Antarctic springtail . British Antarctic Survey Bulletin , 71 , 19 – 30 .

Convey , P . ( 1994 ) Growth and survival strategy of the Antarctic mite, Alaskozetes antarcticus . Ecography , 17 , 97 – 107 .

Convey , P . & Smith , R.I.L . ( 1997 ) The terrestrial arthropod fauna and its habitats in northern Marguerite Bay and Alexander Island, maritime Antarctic . Antarctic Science , 9 , 12 – 26 .

De With , N.D. & Joose , E.N.G . ( 1971 ) The ecological effects of moulting in Collembola . Revue D’ecologie et de Biologie Due So-logical, T , 8 , 111 – 117 .

Leather , S.R. , Walters , K.F.A. & Bale , J.S . ( 1991 ) The Ecology of Insect Overwintering . Cambridge Univeristy Press, U.K .

Leinaas , H.P . ( 1983 ) Synchronised moulting controlled by communi-cation in group living Collembola . Science , 219 , 193 – 195 .

Pugh , P.J.A . ( 1994 ) Supercooling points and water content in Acari . Acta Oecologica , 15 , 71 – 77 .

Sinclair , B.J. , Klok , C.J. , Scott , M.B . et al . ( 2003 ) Diurnal variation in supercooling points of three species of Collembola from Cape Hallet, Antarctica . Journal of Insect Physiology , 49 , 1049 – 1061 .

Sømme , L . & Block , W . ( 1982 ) Cold hardiness of Collembola at Signy Island, maritime Antarctic . Oikos , 38 , 168 – 176 .

Sovik , G . & Leinaas , H . P . ( 2003 ) Long life cycle and high adult suvival in an arctic population of the mite Ameronothrus lineatus (Acari, Oribatida) from Svalbard . Polar Biology , 26 , 500 – 508 .

Sovik , G . , Leinaas , H.P. , Ims , R.A. & Solhoy , T . ( 2003 ) Population dynamics and life history of the oribatid mite Ameronothrus lin-eatus (Acari, Oribatida) on the high arctc archipelego of Svalbard . Pedobiologia , 47 , 257 – 271 .

Wigglesworth , V.B . ( 1965 ) The Principles of Insect Physiology , pp. 741 . Methuen, London.

Worland , M.R . ( 2005 ) Factors that influence the supercooling point of the sub-Antarctic springtail Tullbergia antarctica . Journal of Insect Physiology , 51 , 881 – 894 .

Worland , M.R. & Convey , P . ( 2001 ) Rapid cold hardening in Antarc-tic microarthropods . Functional Ecology , 15 , 515 – 524 .

Worland , M.R. , Leinaas , H.P . , Chown , S.L . ( 2006 ) Supercooling point frequency distributions are affected by moulting. Functional Ecology , 20 , 323 – 329 .

Young , S.R. & Block , W . ( 1980 ) Experimental studies on cold toler-ance of Alaskozetes antarcticus . Journal of Insect Physiology , 26 , 189 – 200 .

Accepted 22 August 2006First published online 10 September 2007