Cold tolerance in Sonoran Desert Drosophilaspecies
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COLD TOLERANCE IN SONORAN DESERT DROSOPHILA SPECIES
by
Lawrence Scott Cleaves
Copyright © Lawrence Scott Cleaves 2002
A Thesis Submitted to the Faculty of the
DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOPHYSICS
In Partial Fulflllnient of the Requirements For the Degree of
MASTER OF SCIENCE WITH A MAJOR IN GENERAL BIOLOGY
In the Graduate College
THE UNIVERSITY OF ARLZONA
2002 UMI Number; 1409486
Copyright 2002 by Cleaves, Lawrence Scott
All rights reserved.
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TABLE OF CONTENTS
ABSTRACT 6
INTRODUCTION AND HISTORICAL PERSPECTIVE 7
SPECIES UTILIZED IN THIS STUDY 13
MATERIALS AND METHODS 20
RESULTS 26
DISCUSSION 49
ACKNOWLEDGMENT 60
REFERENCES 61 4
LIST OF TABLES AND ILLUSTRATIONS
TABLE 1, Drosophila Species Used in This Study 18
TABLE 2, Taxonomy of Drosophila Species Used in This Study 21
TABLE 3, Cold Shock Response in Drosophila Species 27
TABLE 4, Rapid Cold Hardening Response in Drosophila Species 35
TABLE 5, Prolonged Exposure at 0°C Response in Drosophila Species 39
FIGURE 1, Representative Phylogeny of Drosophila 19
FIGURE 2, Cold Baths Used in Experiments 22
FIG. 2a: Ice Bath with Circulator
FIG. 2b: Sub-zero Water/Anti-freeze Bath
FIGURES 3-9, Cold Shock graphs
FIG 3: D. pseudoobscura 28
FIG. 4: D. melanogaster 29
FIG. 5: D. hydeii 30
FIG. 6: D. mettleri 31
FIG. 7; D. mojavensis 32
FIG. 8: D. niff'ospiracula 33
FIG. 9: D. paulistorum 34
FIGURES 10-11, Rapid Cold Hardening graphs
FIG. 10; Males for five species 37
FIG. 11: Females for five species 38 5
LIST OF ILLUSTRATIONS - continued
FIGURES 12-17, Prolonged Exposure at 0°C graphs
FIG. 12: D. pseudoobscura 40
FIG. 13: D. melanogaster 41
FIG. 14: D. hydei 42
FIG. 15: D. metlleri 43
FIG. 16: D. mojavensis 44
FIG. 17: D. paulistorum 45
FIGURES 18-19, Prolonged Exposure at 0°C Comparison graphs
FIG. 18: Males for six species 46
FIG. 19: Females for six species 47 6
ABSTRACT
I examined resistance to cold temperature in seven Drosophila species from
different habitats to determine the lower limits of cold tolerance. Three separate tests
were administered to measure the; 1) response to a cold-shock exposure; 2) extent to
which a short-term survival strategy, rapid cold hardening, was utilized by each species; and 3) degree to which each species would respond to a prolonged exposure at 0°C. As expected, the temperate-montane species, D. pseudoobscura, was the most cold-tolerant, whereas the least cold-tolerant was the tropical species, D. paulistorum. The two cosmopolitan species, D. hydei and D. melanogaster, and the three Sonoran Desert endemic species, D. mojavensis, D. nigrospiracula, and D. mettleri, demonstrated intermediate levels of cold-tolerance. Of the five species tested for rapid cold hardening, all exhibited the response, including the tropical representative. The results for the 0°C test paralleled the results of the cold shock test. The desert species tested proved surprisingly cold-tolerant, especially D. mojavensis. 7
INTRODUCTION AND HISTORICAL PERSPECTIVE
Introduction
Temperature is the most important abiotic factor affecting the survival and
distribution of insects world-wide (Andrewartha and Birch 1954; Cohet et. al 1980;
Morin et al. 1997), and a great deal of work has been focused on the topic, particularly in
the last 50 years. Some of the fundamental questions in thermal biology include: How do
insects adapt to and survive the stresses of temperature extremes? What role has
temperature played in the ecology and evolution of species? What are the biochemical
and physiological mechanisms insects use to tolerate high and low temperatures? What
impact does temperature have on insect development, reproductive capacity, and
behavior? This paper addresses the first two questions above in relation to cold tolerance
in various Drosophila species.
Although significandy more research has focused on the effects of high temperature on insects (Levins 1969; Denlinger et al. 1991; Heinrich 1993; Stratman and Markow
1998; Feder and Hoffman 1999), the last few decades have produced numerous investigations concerning the effects of cold temperature (Parsons 1977; Lee 1989; Lee and Denlinger 1991; Chen and Walker 1994; Gibert et al. 2001). Recent studies on
Drosophila have attempted to identity cold tolerance parameters for a number of species firom a variety of habitats (Cohet et al. 1980; Stanley et al. 1980; Yamamoto and Ohba
1984a, 1984b; Kimura 1988; Hoffman and Watson 1993; Gibert and Huey 2001; Gibert 8
et al. 2001). One group which has not received much investigative attention for cold tolerance, however, is the desert species, which may experience severe frosts during winter months as well as rapid changes in diurnal temperature (Lowe et al. 1967; Heed
1982). The primary purpose of this study is to compare levels of cold tolerance in three species of desert Drosophila with four other species from the same genus, including one temperate, two cosmopolitan, and one tropical representative.
Historical Perspective
Serious investigations in cryobiology began in the mid-19"* century when research focused on the economic importance of producing frost-hardy plant crops (Payne 1926).
Between the latter part of the 19''' and the middle of the 20"* century, insects became popular as subjects of cryobiology research not only because of their obvious impact on plants, but also because of a growing interest in determining the effect of low temperature on insect ecology, geographic distribution, and behavior (Salt 1936, 1950,1956; Novitski and Rush 1949; Knipling and Sullivan 1957). Since the 1950's, investigations in cryobiology have included a wide variety of other poikilothermic organisms (Block 1982;
Zachariassen 1982; Storey and Storey 1988; Loomis 1991) as well as a substantial broadening of research topics. On insects alone, the last 50 years have produced studies which have investigated the genetic and cellular basis for cold tolerance (Marinkovic et al. 1969; Parsons 1973; Tucic 1979; Ring 1980; Lee et al. 1996); specific physiological and biochemical mechanisms that promote cold tolerance (Salt 1961; Somme 1982;
Zachariassen 1985; Lee 1991; Storey and Storey 1991); implications for cryopreservation 9
(Morris 1987; Leopold 1991; Steponkus et al. 1991); potential for using insect cold
tolerance data as a tool for pest management practices (e.g. long-term population
forecasting and the application of natural pesticides which lower insect resistance to
colder temperatures) (Bale 1991, Lee et al. 1993); and ecological and evolutionary basis
for cold adaptation (Baust and Rojas 1985; Bale 1987; Danks 1978, 1991; Somme and
Block 1991).
During the past three decades in particular, the ecological and evolutionary basis for
cold adaptation in insects has received increasingly significant attention. Many
investigations have attempted to correlate laboratory results with conditions in nature to
establish ecological relevance (Parsons 1978; Hori and Kimura 1998; David et al. 1998;
Kelty and Lee 1999, 2001; Gibert and Huey 2001; Gibert et al. 2001). For example,
researchers have assessed cold tolerance in a wide range of species in a variety of
developmental stages to better understand overwintering strategies and distribution
patterns (Izquierdo 1991; Kimura and Beppu 1993; Kimura et al. 1994; Collett and
Jarman 2001). Furthermore, recent studies have more closely examined both short and
long-term acclimation responses (Lee et al. 1987; Czajka and Lee 1990; Chen and
Walker 1994; David et al. 1998), cooling and warming rates (Czajka and Lee 1990; Kelty and Lee 1999, 2001), and the effects of exposure to cold on fertility and fecundity
(McKenzie 1975; Parsons 1978; Chen and Walker 1993; Huey et al. 1995; Watson and
Hoffman 1996; Collett and Jarman 2001). Many of these investigations used Drosophila 10
species as study organisms, and as expected, much of the work focused on D.
melanogaster.
Around 1910, D. melanogaster was chosen by T.H. Morgan and associates at the
Morgan Fly Lab, Columbia University, as the model eukaryotic organism for study.
Drosophila melanogaster was chosen because it was small and easy to rear, had a
relatively short life cycle, had naturally occurring variants, and could be experimented on
easily (Kohler 1994). From there, intense work on the genetics, population biology,
ecology, developmental biology, and behavior of Drosophila began (Ashbumer et al.
1984; Ashbumer 1989). Work on Drosophila continued to grow and its popularity as a
research organism expanded. By the early 1950's, more than 800 species had been
identified (Patterson 1952), and by the year 2000, the list had grown to include over
1,300 species (Russo et al. 1995).
Early work on the influence of cold temperature was not only conducted on D.
melanogaster (Chiang et al. 1962), but included other species including D. robusta
(Levitan 1951), D. Junebris (Timofeeff-Ressovsky, 1940; Dubinin and Tiniakov 1945,
1946), D. pseudoobscura (Dobzhansky, 1948; Crumpacker and Marinkovic 1967;
Marinkovic et al. 1969), and D. persimilis (Dobzhansky 1948; Mohn and Speiss 1962).
Over time, the number of species used in cold tolerance experiments has increased, and numerous comparative studies have also been undertaken (Parsons 1973; Stanley et al.
1980; Yamamoto and Ohba 1982, 1984; Hori and Kimura 1998; Goto and Kimura 1998;
Gibert, et al. 2001). In many of these experiments, researchers attempted to relate their II
results to natural conditions found in the environment However, as pointed out by
Kimura et al. (1994), laboratory success is somewhat limited because Drosophila, as well as other insects, have ecological and behavioral (and perhaps unknown) means to respond to cold temperatures that are difficult to duplicate in the laboratory. Therefore, making accurate measurements and assessments of cold tolerance in the laboratory which reflect conditions in the natural environment of insects, is a challenging endeavor. Nevertheless, these investigations have provided ecologically relevant information on cold tolerance for a variety of species.
Historical Methods of Measuring Cold Tolerance
A number of methods have been used in the laboratory to assess insect cold tolerance.
Early experiments measured survival from cold shock (Kerkis 1941; Chiang et al. 1962;
Coyne et al. 1983) and long-term cold hardening (Marinkovic et al. 1969; Parsons 1977,
Tucic 1979) responses in a variety of species. In cold shock studies, insects were exposed to a sudden stress at sub-zero temperatures. Long-term cold hardening studies, on the other hand, were designed to test whether insects could improve survival at sub-zero temperatures if they were first acclimatized by a non-injurious, above 0°C pre-treatment generally lasting for days or even weeks.
Another method of assessing cold tolerance in insects is the "rapid cold hardening"
(RCH) response, which measures short-term acclimation to cold. In a landmark experiment (Chen et al. 1987), it was hypothesized that rapid cold hardening was used by 12
insects for survival during diurnal and seasonal temperature fluctuations. Like long-term
cold hardening studies, RCH studies test whether insects could improve survival at sub
zero temperatures if provided a non-injurious above 0°C pre-treatment. However, with
RCH, the pre-treatment lasts only for minutes to a few hours instead of days or weeks.
Present Study
In this study, I measure and compare cold tolerance in seven species of Drosophila,
using three approaches including: an immediate cold-shock response; a short-term
acclimation response (rapid cold hardening); and a response to a prolonged exposure at
0°C. Of particular interest to me was the Sonoran Desert species' response to cold, since
little work of this nature had previously been performed. I also wished to determine
whether most, if not all, species of Drosophila, including the tropical representative,
would exhibit the RCH response. Finally, I wanted to ascertain whether the 0°C test
would prove useful as a tool for measuring cold tolerance, and whether the data obtained
would support the data acquired from the cold shock and RCH tests. 13
SPECIES UTILIZED IN THIS STUDY (Phylogeny, Natural History, and Use as Study Organisms)
Subgenus Sophophora
Drosophila melanogaster: Drosophila melanogaster is a member of the melanogaster species group which contains over 170 species world-wide (Schawaroch
2000; Drosophila Species Workshop manual. Center for Insect Science, University of
Arizona 2001). The origin of the species group is Asia, but D. melanogaster is of tropical
African descent (Cohet et al. 1980). Drosophila melanogaster is currently recognized as one of the "true" cosmopolitan species, and there is considerable speculation regarding its adaptive shift to cosmopolitanism. One hypothesis holds that D. melanogaster owes its global distribution primarily to humans, as providers of "artificial transports" (Cohet et al. 1980). Another hypothesis poses that the tropical/heat-tolerant D. melanogaster, as well as its closely related cosmopolitan sibling, D. simulans, became pre-adapted to colder conditions by ascending into the Afncan mountains (Cohet et al. 1980).
Drosophila paulistorum: Drosophila paulistorum is a "superspecies" member of the willistoni species group which contains six known sibling species. (Ehnnan and Powell
1982). All representatives of this group are neotropical in origin ranging in distribution from the Amazon basin to Central America, and including various islands of the
Caribbean (Ehrman 1960; Perez-Salas et al. 1970). 14
During the 1950's, Dobzhansky studied the life cycle of members of the willistoni group, including D. paulistorum, and compared them with flies of the obscura species group, including D. psetidoobscura (see below) (Dr. W. Heed, personal communication
2001). Since Dobzhansky's work, D. paulistorum has continued to receive significant research attention due to its unique gene flow characteristics, reproductive isolation, and mechanisms of speciation (Ehrman and Powell 1982).
Drosophila pseudoobscura: Drosophila pseudoobscura is a member of the obscura species group and pseudoobscura subgroup. The pseudoobscura subgroup is endemic to the New World with nearctic origins. (Lakovaara and Saura 1982; Barrio et al. 1992;
Barrio and Ayala 1997; Drosophila Species Workshop manual 2001). D. pseudoobscura is generally considered a temperate-montane species, but it occurs in a wide range of habitats beginning at sea level and extending to altitudes of 9,000 feet and above. The species is well known for its tolerance to cold temperature (Crumpacker and Marinkovic
1967; Marinkovic et al. 1969) as well as its long-distance migratory capability, having recently spread to western deserts, rain forests, suburban backyards, and urban habitats
(Coyne etal. 1982).
As a study organism, D. pseudoobscura has been examined extensively beginning in the 1930's with Dobzhansky who studied its morphological and physical characteristics and ecology and evolutionary genetics (Dobzhansky 1935; Dobzhansky and Epling
1944). More recently, D. pseudoobscura and other members of its species group have 15
been used to study gene flow and adaptive geographic variation (Coyne et al. 1983), mechanisms of speciation (Noor 1995), population genetics of polytene chromosome inversions (Popodic and Anderson 1994; Powell 1972, 1992); and phylogeny (Barrio and
Ayala 1997; O'Grady 1999; Drosophila Species Workshop manual 2001).
Subgenus Drosophila, the Repleta Group
The repleta group diverged from the melanogaster group approximately 35 million years ago (Russo et al. 1995). It then divided into five subgroups including the hydei and mulleri subgroups (Wasserman 1992), representatives of which have been used in this study.
The repleta species group, with approximately 100 representatives, has been well studied, but an accurate phylogeny for the group has been difBcult to acquire due to the numerous and complicated chromosomal inversions involved. Only recently have relationships between species been clarifled as newer approaches to phylogenetics, such as DNA sequencing, were applied (Durando et al. 2000).
Drosophila hydei: Drosophila hydei, though now a cosmopolitan species, has origins in the New World, like all members of the repleta species group. Drosophila hydei is a member of the hydei subgroup and hydei complex, an apparently ancient group which. 16
over time, has undergone very little chromosomal change with the exception of the sex
chromosomes (Spicerand Pitnik 1996).
Males of D. hydei have one of the longest sperm of any animal, which has led to
numerous investigations concerning the evolution of reproductive strategies (Spicer and
Pitnik 1996). Research on D. hydei has also focused on deciphering chromosomal
inversions and evolutionary and phylogenetic relationships (Spicer and Pitnik 1996).
Drosophila mettleri, DrosophUa mojavensis, and Drosophita nigrospiracula:
Drosophila mettleri, D. mojavensis, and D. nigrospiracula are all representative species
from the mulleri subgroup of the repleta group (Wasserman 1982, 1992). The mulleri subgroup diverged from the hydei subgroup some 15 million years ago (Russo et al.
1995). All three species are cactophilic and endemic to the Sonoran Desert of the southwestern United States and Mexico. Each species is from a different species complex
(anceps, eremophila, and mulleri respectively), so they are not siblings. Although these species overlap in geographical distribution, they have distinct host plant preferences. D. mettleri and £>. nigrospiracula share the same host plants (cardon and saguaro), but D. mettleri uses the soaked soil beneath the host plant "rot" while D. nigrospiracula utilizes the host plant's necrotic tissue. The primary host plants of D. mojavensis are agria, organ pipe, and the California barrel cactus (Heed 1978, 1982). These three desert fly species, along with another Sonoran Desert endemic, D. pachea, have provided a popular model system in evolutionary ecology and genetics (Barker and Starmer 1982; Powell 1997). 17
For additional information on the taxonomy and phylogeny of these species, see
Table 1 and Figure 1 respectively. 18
Table 1. Taxonomy of 7 Drosophila species used in this study.
Taxon Species Subgenus Sophophora Melanogaster species-group Melanogaster species-subgroup D. melanogaster Willistoni species-group Willistoni species-subgroup D. paulistorum Obscura species-group Pseudoobscura species- D. pseudoobscura subgroup Subgenus Drosophila Repleta species-group Hydei species-subgroup D. hydei Mulleri species-subgroup D. nigrospiracula D. mettleri D. mojavensis Figure 1; Representative Phylogeny ofDrosophila
^ "0
Species used in this study 20
MATERIALS AND METHODS
Insect Rearing
Seven representative species of Drosophila were used, which were classified by their distribution, including: one montane-temperate, three desert, two cosmopolitan, and one tropical species (Collection biformation in Table 2). All flies were reared under normal laboratory conditions at 24°C in half-pint milk bottles. Drosophila melanogaster, D.
pseudoobscura, and D. paulistorum were reared on standard Drosophila commeal medium with added yeast. Drosophila mojavensis was reared on banaoialOpuntia cactus medium with added yeast. Drosophila mettleri, D. hydeii, and D. nigrospiracula were reared on baaaana/Opuntia cactus medium with added yeast and a piece of saguaro cactus.
Upon emergence, virgin adults were separated by sex and transferred to 35ml shell vials all containing fresh commeal medium and yeast. The flies were then maintained for 72h at 24°C, because age could affect cold tolerance (Czajka and Lee, 1990).
Cold Tolerance Experiments
Phase I: Cold-shock Experiment
Flies were lightly anesthetized with CO2, placed in small, plastic microcentrifuge tubes (5 flies per tube), and allowed to recover for 30-60 minutes. The tubes were then racked and partially submerged in a chilled water/antifreeze bath for Ih (Figure 2). Prior 21
Table 2. Drosophila species used in this study. Species are arranged in general order of increasing habitat temperature.
Species Collection Date Collection Location Habitat
D. pseudoobscura July, 1999 Madera Canyon, AZ temperate-montane D. melanogaster January, 2000 Alamos, Sonora, MX cosmopolitan
D. hydei July, 1999 Madera Canyon, AZ cosmopolitan
D. mettleri March, 1997 Lost Dutchman Mine, AZ desert
D. mojavemis May, 2000 San Carlos, Sonora, MX desert
D. nigrospiracula July, 1999 Tucson, AZ desert
D. paulistorum March, 1999 Panama tropical 22
Figure 2: Cold Baths Used in Experiments
Fig. 2a: Ice Bath with Circulator
Fig. 2b: Sub-zero Water/Anti-freeze Bath 23
to submersion, the racks were tapped against the side of the water bath to ensure that flies
were at the bottom of the tubes for initial exposure. After a few seconds in the bath, the
rack was briefly removed to establish that all flies were chill-shocked and at the tube
bottoms. By inserting and sealing a thermistor and monitoring tube temperature, we
ascertained that the temperature at the bottom of the microcentrifuge tubes was equal to
the bath temperature. The bath temperature ranged between -1 and -12°C depending on
the species tested. (Preliminary testing enabled us to determine relevant starting points
for each species.) Immediately after the cold bath exposure, flies were transferred to
clean, empty glass vials and provided a Ih period of recovery at ambient temperature.
This was done to prevent flies from becoming stuck in the food medium. They were then
transferred to 35ml shell vials with commeal and yeast, and survival was monitored and
recorded over the next three days. Survival was assessed as the number of flies that were
able to right themselves and walk after 24h of recovery. I continued monitoring the flies
an additional two days as a precautionary measure. When the preceding steps were
completed, we measured and recorded survival capacity for each species. As a control,
flies from each species were placed in a 0°C ice water bath for Ih and treated with
identical follow-up procedures.
Phase 2: Rapid Cold Hardening Experiment
For the rapid cold hardening assays, five species of Drosophiia {D. nigrospiracula and D. mojavensis not included) were provided with a 1 h sub-lethal pre-treatment at 0,4. or 8°C. After the pre-treatment, the flies were inmiediately transferred to water baths with 24
below freezing temperatures ranging between -1 to -12°C, depending on the species tested. The Ih cold-shock procedure previously described in Phase I was then repeated.
After the sub-zero exposure to cold, the flies were given 24h to recuperate, then survival was measured and recorded.
Phase 3: Prolonged Exposure to (fC Experiment
In order to determine long-term resistance to cold temperatures, I subjected six species of Drosophila (D. nigrospiracula not included) to a O'^C exposure for a time span ranging between I and 192h (eight days). Preliminary testing enabled me to determine relevant exposure times for each species. In this experiment, flies were lightly anesthetized with CO2, placed in small, plastic microcentrifuge tubes (5 flies per tube), and allowed to recover for 30-60 minutes. After recovery, the flies remained in their tubes, were sealed in plastic bags with added weight, and were completely submerged in an ice bath for a specified time. Once removed from the ice bath, the flies were given 24h to recuperate and survival was measured and recorded.
Statistical Analyses
For the cold-shock and 0°C tests, Sigma Plot was used for curve titling and calculating the LT50 values. For the rapid cold hardening tests, JMP 4 (SAS Institute) was used for data analysis. Using JMP 4. I first took the steep part of the fitted curve for each species and performed an ANOVA to determine whether a pre-treatment effect had been produced.
Once it was determined that a pre-treatment effect existed, I performed a post-hoc test to 25
determine which pre-treatment temperatures were significantly dififerent fiom receiving no pre-treatment and the direction of significance. 26
RESULTS
Phase 1: Cold-shock Experiment
In the cold-shock assays, as well as the other assays, "'survival" was defined as a fly's ability to right itself and walk after 24h after exposure. There was no significant change in
LTso values when flies were observed for an additional two days post-exposure. Table 3 provides a comparison of cold-shock survival capacity between sexes as well as between species. Figures 3 through 9 show the cold-shock data for each species tested (including standard errors) from which the LT50 values were derived. To summarize, the LT50 values generated ranged from —10.5°C in temperate-montane D. pseudobscura to —3°C in tropical D. paulistorum. LT50 values for the three desert species tested ranged between ~ -6 and ~ -9''C, while the range for the two cosmopolitan species was between ~ -5 and ~ -TC.
There was no significant sex difference in cold-shock tolerance for any species.
Phase 2: Rapid Cold Hardening Experiment
For the rapid cold hardening assays, flies of five species were provided with Ih pre- treatments prior to transferring them to water baths with sub-zero temperatures. Preliminary testing enabled us to estimate relevant pre-treatment temperatures for each species. Table 4 provides a comparison of LT50 values of flies receiving no pre-treatment prior to cold-shock exposure and flies receiving pre-treatments. Also included in Table 4 are the pre-treatment temperatures, the resultant changes in LTso values, a measure of statistical significance, and standard errors. Males and females of all five species tested demonstrated a positive rapid 27
Table 3. Cold Shock Response in Drosophila species. Species are arranged in general order of increasing habitat temperature. LT5o= time in hours at which 50% of flies survived, with standard errors
Species Male LTso (°C) Female LT50 ("C)
D. pseudoobscura -10.1 (±0.5) -11.2 (±2.3)
D. melanogaster -5.0 (±0.1) -4.9 (±0.3)
D. hydei -7.1 (±0.2) -7.3 (±0.2)
D. mettleri -7.3 (± 0.2) -7.3 (±0.1)
D. mojavensis -9.0 (± 0.2) -9.3 (±0.1)
D. nigrospiracula -5.9 (±0.3) -6.1 (±0.2)
D. paulistorum -3.1 (±0.2) -3.1 (±0.2) Figure 3: Cold Shock
D. pseudoobscura
0.8 -
0.6 -
0.4 -
0.2 -
0.0 14 -12 -10 -8 -6-4-2 0
Temperature (°C)
Males: lTJO = -io.i°c (+o.s)
Females: lTSQ = -i i .2°c (+ 2.3) Figure 4; Cold Shock
D. melanogaster
0.8 -
0.6 -
0.4 -
0.2 -
0.0 -10 8 6 -4 2 0
Temperature (®C)
Males: LTgo = -5.o°c (± o.i) Females: LT50=-4.9°c (+ 0.3) 30
Figure 5; Cold Shock
D. hydei
O) c •> 3 (0 c o "Co Q. o
Temperature ( C)
I— Males: lTJO = -7.i°c (+ 0.2) I - - Females: lj^ = -7.3°c (+ 0.2) 31
Figure 6: Cold Shock
D. mettleri
O) 0.8 -
0.6 -
0.4 -
0.2 -
0.0 12 -10 -8 6 -4 2 0
Temperature (°C)
—O— Males: lTJO=(+0.2)
- - # - - Females: lTJO = -7.3°c (± 0.1) 32
Figure 7; Cold Shock
D. mojavensis
O) 0.8 -
0.6 -
0.4 - Q.
0.2 -
0.0 12 -10 -8 6 -4 2 0
Temperature (°C)
- Males: lTJO = -9.o°c (+ o.2)
- Females: lTM=-9.3°c (± o.i) 33
Figure 8: Cold Shock
D. nigrospiracula
O) 0.8 -
0.6 -
0.4 -
0.2 -
0.0 -10 8 6 -4 2 0
Temperature (°C)
I— Males: = -5.9°c (± o.3) > - - Females: = -6.i°c (±o.2) 34
Figure 9: Cold Shock
D. paulistorum
1.0
O) 0.8 -
0.6 -
0.4 -
0.2 -
0.0 7 -6 -5 -4 -3 -2 1 0
Temperature (°C)
I— Males: = -3.i°c (± o.2)
I - - Females: lTJO=-s i^c (± 0.2) 35
Table 4. Rapid Coid Hardening Response in Drosophila species.
* = statistically significant
Males, LTso Values Females, LTso Values
Pre -treatment Pre-treatment
No 0°C 4°C s-c No o-c 4°C 8°C
-10.0 -II.5» -10.7* •••• -10.0 -II.O* -10.5* •••• D. pseudoobscura (±0.24) (±0.65) (±0.21) (±0.16) (±0.61) (±0.17)
-5.1 -5.8 -6.0» • * • • -5.1 -5.2 -6.0» D. meianogasler (±0.42) (±0.22) (±0.19) (±0.42) (±0.42) (±0.18) -7.5 -7.S* -7.5 -8.0* -8.2^ D. hydei (±0.24) (±0.12) (±0.11) (±0.17) (±0.16) (±0.17) -7.7 -7.9 -S.2* -7.6 -7.8 -8.1* D. mettleri (±0.17) (±0.15) (±0.21) (±0.20) (±0.15) (±0.26)
-3.2 -2.3 » -3.4» -3.7* -3.1 -1.6* -3.4» -3.1* D. paulislorum (±0.11) (±0.36) (±0.08) (±0.18) (±0.14) (±0.39) (±0.15) (±0.25)
LT50 values for RCH "no pre-treatment" are slightly different than those obtained for the cold shock study because in the RCH assay, the LTso values were calculated using only the steep portion of the survival curve. 36
cold hardening response (LTso values were lowered) for at least one pre-treatment
exposure. Three of the five species demonstrated a positive rapid cold hardening response
for two pre-treatment temperature exposures. In the two species where only one pre-
treatment temperature was significant, D. melanogaster and D. mettleri, males and females
exhibited similar responses: Neither sex had a significant response to a 0°C pre-treatment,
whereas both sexes had a significant positive response to a 4°C pre-treatment. In all of my
RCH tests, only one negative pre-treatment response was shown: the O^C pre-treatment for
D. paulistorum significantly raised the LTso value thereby reducing the fiy's cold-shock
survival capacity. A O^C pre-treatment was apparently too cold for D. paulistorum. Figures
10 and 11 provide a between-species comparison by sex for the rapid cold hardening
response.
Phase 3: Prolonged Exposure to 0°C Experiment
Figures 12 through 17 present the prolonged exposure data for the six species tested
fi*om which the LTso values in Table 5 were derived. Levels of long-term cold endurance
varied greatly between species. The LT50 value for D. paulistorum was reached after only
~3.5h of exposure, whereas the LTso value for D. pseudoobscura was not reached until
~120h (five days) of exposure. Like D. pseudoobscura, D. mojavensis also exhibited long-
term resistance that was more appropriately measured in days than hours. Neither of the cosmopolitan species, D. melanogaster or D. hydei, produced an LTso value above 24h, although the other desert representative tested, D. mettleri, easily survived beyond one day at 0®C. Figures 18 and 19 provide a between-species comparison by sex to long-term 37
Figure 10: Rapid Cold Hardening
D. paulistorum Males for D. hydei D. pseudoobscura D. mettleri D. melanogaster
0
— -2 + -4 £ 3 5 + Q. -6 ^ E 0) h- + (hydai) 8 -8 + +
-10 + / / \ / \
-12
no pretreat OC 4C 8C
Pre-treatment Temperature ( C)
* = pre-treatment exposure temp increased cold tolerance significantly • = pre-treatment exposure temp decreased cold tolerance significantly 38
Figure 11: Rapid Cold Hardening
D. paulistorum Females for: D. hydei D. pseudoobscura D. mettleri • D. melanogaster
-2 -
£ 3 5 • (mattlofi) -10 f -12 1 r no pretreat OC 4C 8C Pre-treatment Temperature (°C) + = pre-treatment exposure temp increased cold tolerance significantly ~ = pre-treatment exposure temp decreased cold tolerance significantly 39 Table 5. Prolonged Exposure to 0°C Response in Drosophila species. LT5o= time in hours at which 50% of flies survived, with standard errors Species Male LTso Female LT50 (hours) (hours) D. pseudoobscura 129.5 (±5.7) 111.5 (±6.7) D. melanogaster 12.0 (± 0.7) 17.3 (±0.8) D. hydei 18.4 (±1.3) 18.8 (±0.7) D. mettleri 31.2 (±4.4) 39.5 (± 5.4) D. mojavensis 72.2 (± 9.7) 82.8 (± 7.2) D. paulistorum 3.6 (±0.1) 3.3 (±0.1) 40 Figure 12; Prolonged Exposure D. pseudoobscura 100 80 - (D I 60 - 3 CO 40 - Q. 20 - 0 40 80 120 160 200 Hours at 0°C # Males: LT^Q = 129.Shr {* 5.7) O Females: = m.shr (+ 6.7) 41 Figure 13: Prolonged Exposure D. melanogaster s (D > 3 CO c (l> 2 10 20 30 Hours at 0°C O Males: lTSQ «i2.ohr {* o.7) # Females: lt^ = i7.3hr (± o.8) 42 Figure 14; Prolonged Exposure D. hydei 100 80 - (0 I 60 - 3 0) c 20 - 0 10 20 30 40 Hours at 0°C O Males: « i8.4hr (+1.3) • Females: lTjo = i8.8hr (+ o.7) 43 Figure 15: Prolonged Exposure D. mettleri 100 CO > 3 0) 0) ok. 20 40 60 80 Hours at 0°C O Males: lTm > 3i .2hr {* 4.4) • Females: LTSO = 39.5hr {* 5.4) 44 Figure 16: Prolonged Exposure D. mojavensis 100 o o g o • 80 O (0 o >€ 60 t 3 CO o c o 20 o 40 80 120 160 200 Hours at 0 C • Males: LT„ « 72.2hr {± S.?) O Females: ltso = 82.8hr {* 7.2) i I 45 Figure 17: Prolonged Exposure D. paulistorum 100 80 - (D I 60 - 3 CO C 20 - 0 2 4 6 8 10 Hours at 0°C O Males: lTsq » 3.6hr (+ o.i) • Females: LTso*3.3hr(+0.1) 46 Figure 18; Prolonged Exposure Comparison —•— - D. paulistorum Males for; —e—- D. melanogaster A - D. hydei 100 A - D. mettleri •— - D.mojavensis —0— - D. pseudoobscura 80 60 - 40 20 - 40 80 120 160 200 Hours at 0 °C 47 Figure 19: Prolonged Exposure Comparison —•—- D. paulistorum Females for: —e—- D. melanogaster A - D. hydei A - D. mettleri • - D. mojavensis ©—- D. pseudoobscura 40 80 120 160 200 Hours at 0 °C 48 exposure to O^'C. This experiment was the first to yield a significant difiference in results between sexes, although it occurred in only one of the six species tested. Females of D. melanogaster produced a significantly higher Ltso value (~ I7.5h) than their male cohorts (~ 12h) for the 0°C exposure. 49 DISCUSSION A number of reviews on cold tolerance in insects (Baust and Rojas, 1985; Bale 1987) have recommended that laboratory work be combined with field work to clearly establish the ecological relevance of the research. Accurate assessment of an insect's cold tolerance should consider both biotic and abiotic factors. For example, insects can use behavioral options such as migrating out of cold habitats (Monarch butterflies), or finding warmer microclimates to avoid cold extremes. Abiotic factors such as the duration of cold periods, the rapidity and extent of temperature changes, elevation and photoperiod may also affect survival. Although recent studies have attempted to include many of these variables in order to adequately address ecological relevance, the time and resources required to control all variables are excessive. Therefore, in my initial study of cold tolerance in Drosophila, I chose to take a "snapshot" of a variety of species in order to establish a general measure of cold tolerance in the three desert species chosen. Cold Shock Resistance My results were usually similar to those of previous studies. I observed a sharp decline in survival with a relatively small change in temperature (I-2°C) in all species tested. Cold shock experiments on D. melanogaster have produced LTso values ranging from -5°C as in my experiment (ref) to -TC (Chen and Walker 1993). Intermediate results included; Novitski and Rush (1949), in which D. melanogaster had 95% survival at -5°C for Ih but only 25% at -5°C for 2h. Resultant LTso's fiwm Chiang et al. (1962) were lower, with flies 50 surviving a Ih exposure at -6°C. In more recent work, Czajka and Lee (1990) found almost 100% mortality at an exposure of -5°C for 2h and nearly 0% mortality at -4°C for 2h; and in the cold-shock portion of an experiment performed in 1999, Kelty and Lee observed 70% survival for Ih at -6°C. The discrepancy between results in these investigations can best be explained by variable rearing conditions, dififering experimental protocols, and use of different fly strains. I expected D. hydei to exhibit an LTso value near that of D. melanogaster, but it performed considerably better. In the only previous cold shock studies for D. hydei, Yamamoto and Ohba (1984b) produced an LTso value of 10.6h at -4°C. This study is not directly comparable to my study, but the combined results do suggest that D. hydei is fairly cold tolerant. Drosophila f^dei also outperformed D. melanogaster in the other two kinds of tests in this study, suggesting that may be the more cold tolerant of the two species. Cold shock results for D. pseudoobscura have varied significantly among investigations. My study found an LT50 value of about -10.5°C for Ih. Coyne et. al (1983) observed an average of 80% survivorship at -lO^C for 3.5h using seven different strains, and Crumpacker and Marinkovic (1967) observed 50% survival at -10°C for four days. In the Crumpacker study, wild-caught flies from Colorado living at an elevation of almost 6,000 feet were used. These flies may have been more cold tolerant because they had acclimated to colder temperatures. I expected D. pseudoobscura to be more cold-shock resistant than the desert species selected and my results bear out this prediction. 51 Little research has been conducted on cold tolerance in tropical and subtropical species; however, there are a few exceptions (Stanley et al. 1980; Cohet et al. 1980; Kimura et al. 1994). As expected, D. paulistorum, a tropical species from Central and South America, did not perform well in my cold shock study. This species, however, would probably never experience such a dramatic shift in temperature (from 24°C to sub-zero temperatures) in its natural habitat. Drosophila paulistorum did produce an LTso value of for Ih and an LT100 value of-I°C for Ih, implying that even tropical species have some resistance to sub- freezing temperatures. The three desert species used in this experiment, D. nigrospiracula, D. mettleri, and D. mojavemis, have a wider range of temperature with which to contend on an annual basis (< 0°C to > 40°C) than the other four species selected. They also occupy the smallest geographic niche of any species utilized in the study, as all are host-specific cactophiles of the Sonoran Desert. Fhese species are therefore under intense selection pressure for temperature extremes, as well as for desiccation resistance. Other studies have already demonstrated the ability of these species to withstand extreme heat and desiccation stresses (Stratman and Markow 1998; Krebs 1999; Gibbs and Matzkin 2001). When hypothesizing about the cold tolerance of these species, 1 assumed that if there were a metabolic cost associated with the aforementioned stresses, then the desert flies would likely '"spend" more resources on heat and desiccation resistance than on cold stress. I therefore anticipated low to moderate cold tolerance in the desert flies, at a level above the tropical species but below the cosmopolitan species and well below the temperate-montane species. 52 The three desert species were surprisingly resistant to cold shock, with D. nigrospiracula, D. mettleri, and D. mojavensis producing LT50 values of -6, -7.5, and -9°C respectively. The cold shock response of D. mojavensis almost equaled that of D. pseudoobscura. Based upon a cold shock study by Lowe et al. (1967) on D. nigrospiracula, my LTso values may well underestimate these flies' ability to resist to below-fieezing temperatures. In Lowe's experiment, D. nigrospiracula which were wild-caught in February (after months of previous acclimation) and transferred directly to the laboratory produced an LT50 value of -7.75°C Oudged as "conservative" by the author). Survival was measured as flies that could fly (instead of standing upright as in my experiment), and most significantly, fly survivors of -8°C had received previous successive Ih exposures at -6 and -TC. Another difference between this experiment and mine was that in Lowe's experiment, flies were slowly cooled at .08°C/min prior to the sub-zero exposure(s), which more closely resembles what the flies would experience in nature. The LT100 in the Lowe experiment was -9.5°C for 5h of sub-zero exposure whereas the LTioo in my experiment was -8°C for Ih. By slowly cooling the flies before the sub-zero exposure(s), Lowe may have induced a rapid cold hardening response which may have dramatically increased the flies' ability to survive cold exposures. Rapid Cold Hardening (RCH) Response The first rapid cold hardening experiment was conducted by Atwal (1960) on pupae of the moth species Anagasta (Ephestia) kuniella. In Atwal's experiment, pupae were pre conditioned at temperatures ranging between 5-25°C for 1, 2, 4, 8 and 16h prior to a sub 53 zero exposure of -IS^C for 4h. One of the more significant aspects of the study was the discovery of an RCH "Svindow", or optimal pre-conditioning temperature or temperature range. For Anagasta (Ephestia) kuniella, the optimal pre-treatment temperature prior to the -15°C exposure was 10°C. A pre-treatment temperature of 5°C was too low for this species, whereas a pre-treatment temperature of 15°C or higher proved to be too high. Rapid cold hardening work expanded in the 1980's beginning with the landmark experiment by Chen et al (1987) on the flesh fly, S. crassipalpis. In Chen's experiment, it was discovered that a pre-treatment exposure of only 10 minutes would significantly increase the number of survivors to a sub-zero exposure. These researchers also determined the optimal RCH pre-conditioning range for this species at 0-6®C. A rapid cold hardening experiment on D. melanogaster (Czajka and Lee 1990) produced results similar to those in our study. In this experiment, flies cold shocked at -S°C for 2h yielded no survivors, whereas flies receiving a pre-treatment at 5°C for Ih yielded 90% survival. In my experiment, flies cold shocked at -5°C for Ih yielded 50% survival (LTso value), and a pre-treatment at 4°C for Ih lowered the LT50 value to -6°C. Similar results for RCH in D. melanogaster were observed by Chen and Walker (1993,1994). With little research having been conducted on the rapid cold hardening response in Drosophila species other than D. melanogaster, I was not sure what to expect. I anticipated observing the RCH response in the temperate-montane species, D. pseudoobscura and in the other cosmopolitan species, D. hydei, but was uncertain regarding the desert and 54 tropical species. Since the latter species live in warmer environments, they may have lost the ability for RCH. I did, however, find the RCH response operating in all species tested, including a tropical representative D. paulistorum and a desert fly D. mettleri. (Unfortunately, I was unable to include desert species D. nigrospiracula and D. mojavensis in this phase of the experiment; however, the fact that all other flies tested exhibited RCH led me to conclude that the RCH response was likely present in D. nigrospiracula and D. mojavensis as well.) The degree of the RCH response was not demonstrated quite as significantly in the tropical (LT50 improvement +0.5°C) and desert species (LT50 improvement -K).5°C) as it was in the temperate (LT50 improval +1.5°C) and cosmopolitan representatives (LT50 improval +0.7 to 1°C). This result was expected as the temperate and cosmopolitan species likely experience greater short-term fluctuations in temperature than the tropical or desert species. Under those conditions, the temperate and cosmopolitan representatives would likely be genetically selected for a better RCH response. As in prior rapid cold hardening experiments on insects, an optimal RCH temperature was present for each species tested in my study. This optimal temperature seemed to shift in a parallel manner to the cold shock temperature. For example, in D. pseudoobscura, the 0°C pre-treatment was more effective than a 4°C pre-treatment, which makes sense considering that this species is capable of survival at -10°C and below. In D. paulistorum, the 8°C pre-treatment produced better survival than either 4 or 0°C. The O^C pre-treatment 55 actually decreased survival in a subsequent below-zero exposure for D. paulistorum. For the desert and cosmopolitan species tested, which all exhibited intermediate levels of cold tolerance, the intermediate RCH temperature of 4°C was the most efifective pretreatment exposure. More extensive work is required to determine the RCH pre-treatment temperature range for each species. Prolonged Exposure at 0°C A few studies have investigated prolonged exposure at or near 0°C in Drosophila, primarily in D. melanogaster, and the results have varied considerably. In my study, D. melanogaster males survived approximately 12h while females survived about 17h. Other studies have found greater survival, up to 72h in some cases (Stanley et al. 1980). In D. pseudoobscura, work by Marinkovic et al. (1969) produced LT50 results of twelve days at -3'^C with pre and post-treatment conditioning. Flies from that study were reared from wild-caught adults from mountains in Colorado. With no pre or post-conditioning, the lab-cultured flies in my study exposed to 0°C survived about 5 days. Obviously, cold hardiness in this species, as with others, is dependent upon rearing conditions, including temperature and strain. My study is the first to examine prolonged exposure to 0°C in tropical or desert species. As in the first two phases of my experiment, the tropical species performed the worst of any species tested, and the desert species exhibited intermediate responses. Of the two 56 desert species tested for prolonged exposure, D. mojavensis greatly outperformed D. mettleri, just as it had in the cold shock assay. By surviving three days at 0°C, D. mojavensis approached the prolonged exposure level of five days exhibited by D. pseudoobscura. In general, the prolonged exposure test produced results that paralleled the cold shock and rapid cold hardening tests. Prolonged exposure studies have produced conflicting results regarding differences between sexes. Like mine, other studies have shown that D. melanogaster males are less cold tolerant to long-term exposure than females (Stanley et al. 1980; Izquierdo 1991; Yamamoto and Ohba 1984; David et al. 1998). On the other hand, a number of studies have shown no significant difference between the sexes (Crumpacker and Marincovic 1%7; Parsons 1977). For the other species used in my study, there was no significant difference between sexes. However, Yamamoto and Ohba (1984) reported that D. hydei females were more tolerant to prolonged exposure than males. In that experiment, flies were acclimated at 10°C for two weeks to two months and then cold shocked at -4 and at - 8''C. In another long-term acclimation study, Collett and Jarman (2001) reported that D. pseudoobscura females were more tolerant than males. In that study, mated flies were subjected to months at 57 Conclusion As expected, for all three phases of my experiment, the temperate-montane fly, D. pseudoobscura, was the most cold tolerant while the tropical fly, D. paulistorum, was the least. The two cosmopolitan species, D. hydei and D. melanogaster, proved to be fairly cold tolerant, which was somewhat expected considering their wide distribution and availability of prior research on cold tolerance in D. melanogaster . The three Sonoran Desert species, D. mojavensis, D. nigrospiracula, and D. mettleri, were surprisingly cold tolerant considering that they rarely, if ever, are exposed to such low temperatures in nature and would likely never experience cold shocks with an immediate 20-30°C change in temperature. Particularly notable was the ability of D. mojavensis to withstand a cold shock exposure of ~~9°C for Ih and survive three days at 0°C without prior acclimation. All species used in the rapid cold hardening experiment demonstrated the response, with the temperate-montane and cosmopolitan species utilizing RCH to a greater extent than the desert and tropical species tested. Stanley et al. (1980) reported that Australian desert cactophile, D. buzzatii, an introduced species from arid regions of Argentina (Barker and Mulley 1976), and also a member of the muileri subgroup, was very resistant to temperature extremes and desiccation. The author states "... the flies are far less able to avoid environmental extremes by habitat selection than cosmopolitan species. Necessarily, therefore, there would be intense natural selection pressure for resistance to desiccation and temperature stresses." And in Lowe (1967) on D. nigrospiracula, the author concludes "the adults are S8 fully capable of successful winter survival and activity on nearly all of the days of most winters at Tucson, Arizona." My findings on cold tolerance for the three Sonoran Desert species tested, backed by supporting evidence for heat and desiccation resistance (Stratman and Markow 1998; Gibbs and Matzkin 2001), support the conclusions of both authors. Desert Drosophila can tolerate a very wide range of environmental conditions. Observed differences between the Sonoran Desert species for the three tests are difficult to explain. These species overlap in their normal geographic distributions and all exhibit intermediate levels of cold tolerance. The range between these flies' responses, however, was considerable. The three species could generally be ranked for cold tolerance as follows: D. mojavensis > D. nigrospiracula > D. mettleri. A possible explanation for this observation has to do with the microhabitats and microclimates these species experience. D. nigrospiracula lives in the giant cardon cactus, the mass of which may limit thermal extremes. Therefore, selection in this species is for a narrower thermal range. On the other hand, D. mojavensis feeds and breeds primarily on far less massive cacti, such as organ pipe, where greater temperature extremes may prevail. As a soil specialist, D. mettleri might inhabit the soil during periods of cold as temperatures there change slowly and could be far less extreme than inside a cactus. For future research on cold tolerance in Sonoran Desert Drosophila, I recommend the following. First, since the desert species studied are relatively closely related, measure cold tolerance in non-desert members of the repleta group besides D. hydei as well as 59 species from other deserts for comparative purposes. . Second, perform cold-tolerance studies on D. pachea, the only other Sonoran Desert endemic, and member of the subgroup nannoptera. Third, acquire rapid cold hardening data for D. nigrospiracula and D. mojavensis. It would be interesting to determine the optimal RCH temperature for these species (0 or 4''C), particularly for D. mojavenis since its degree of cold tolerance approaches that of D. pseudoobscura. Fourth, assess changes in fertility/fecundity in each species after exposure to cold, since the most accurate measure of survival in insects is the successful passing on of genetic material. Fifth, repeat the prolonged exposure at experiment, but first provide an acclimation period of days to weeks at a higher temperature (10-15°C) to more closely approach natural winter conditions. Finally, test for cold tolerance in other developmental stages for each species to determine the most cold resistant stage and the stage(s) for which overwintering would likely occur. 60 ACKNOWLEDGMENT I thank Dr. Therese Markow for providing Drosophila species and laboratory equipment. For comments and editorial suggestions I thank Dr. Lisa Elfring, Dr. Allen Gibbs, Dr. Margaret Kidweil, and Dr. Therese Markow. Special thanks to Dr. Allen Gibbs for providing technical assistance and advice on experimental design and data analysis. This work was supported by the National Institute of Health Education Partnership: Award # RR1564-01. 61 REFERENCES Andiewartha, H.G., and Birch, L.C. (1954). The Distribution and Abundance of Animals. (Chicago: University of Chicago Press). Ashbumer, M., Bodmer, M., and Lemeunier, F. (1984). On the evolutionary relationships of Drosophila melanogaster. Developmental Genetics 4:295-312. Ashbumer, M. (1989). Drosophila: A Laboratory Handbook. (New York: Cold Spring Harbor Laboratory Press). Atwal, A.S. (1960). Influence of temperature, duration of conditioning, and age of Anangasta (Ephestia) kuhniella (Zell.) (Lepidoptera: Pyralididae) on acclimation to a sub-zero temperature. Canadian Journal of Zoology 38: 131-141. Bale, J.S. (1987). Insect cold hardiness: freezing and supercooling - an ecophysiological perspective. Journal of Insect Physiology 33: 899-908. Bale, J.S. (1991). Implications of cold hardiness for pest management. In: Insects at Low Temperature, pp. 461-498, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Barker, J.S.F., and Mulley, J.C. (1976). Isozyme variation in natural populations of Drosophila buzzatii. Evolution 30:213-233. Barker, J.F.S., and Starmer, W.T. (eds) (1982). Ecological Genetics and Evolution: The Cactus-Yeast-DrosopAi/a Model System. (New York: Academic Press). Barrio, E., Latorre, A., Moya, A., and Ayala, F.J. (1992). Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA. Molecular Biology and Evolution 9: 621-635. Barrio, E., and Ayala, F.J. (1997). Evolution of the Drosophila obscura species group inferred from the Gpdh and Sod genes. Molecular Phylogenetics and Evolution 7: 79-93. 62 Baust, J.G., and Rojas, R.R. (1985). Review - insect cold hardiness: facts and fancy. Journal of Insect Physiology 31: 755-759. Block, W. 1982. Cold hardiness in invertebrate poikilotherms. Comparative Biochemistry and Physiology 73A: 581-593. Chen, C.-P., Denlinger, D.L., and Lee Jr., R.E. (1987). Cold-shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiological Zoology 60:297-304. Chen, C.-P., and Walker, V.K. (1993). Increase in cold shock tolerance by selection of cold resistant lines of Drosophila melanogaster. Ecological Entomology 18: 184-190. Chen, C.-P., and Walker, V.K. (1994), Cold-shock and chilling tolerance in Drosophila. Journal of Insect Physiology 40: 661-669. Chiang, H.C., Benoit, D., and Maki, J. (1962). Tolerance of Drosophila melanogaster to sub-freezing temperatures. Canadian Entomologist 94: 722-727. Cohet, Y., Vouidibio, J. and David, J.R. (1980). Thermal tolerance and geographic distribution: a comparison of cosmopolitan and tropical endemic Drosophila species. Journal of Thermal Biology 5: 69-74. Collett, J.I., and Jarman, M.G. (2001). Adult female Drosophila pseudoobscura survive and carry fertile sperm through long periods in the cold: populations are unlikely to suffer substantial bottlenecks in overwintering. Evolution 55: 840-845. Coyne, J.A., Boussy, I.A., Prout, T., Bryant, S.H., Jones, J.S., and Moore, J.A. (1982). Long-distance migration of Drosophila. American Naturalist 119: 589-595. Coyne, J. A., Bundgaard, J., and Prout, T. (1983). Geographic variation of tolerance to environmental stress in Drosophila pseudoobscura. American Naturalist 122:474-488. Crumpacker, D.W., and Marinkovic, D. (1967). Preliminary evidence of cold temperature resistance in Drosophila pseudoobscura. American Natiiralist 101: 505-515. 63 Czajka, M.C., and Lee, R. E. (1990). A rapid cold-hardening response protecting against cold shock injury in Drosophila melanogaster. Journal of Experimental Biology 148: 245-254. Danks, H.V. (1978). Modes of seasonal adaptation in insects. Canadian Entomologist 110: 1167-1205. Danks, H.V. 1991. Winter habitats and ecological adaptations for winter survival. In: Insects at Low Temperature, pp. 231-259, Lee, R.E., and Denlinger, D.L. (eds), (New York: Chapman and Hall). David, R.J., Gibert, P., Pla, E., Petavy, G., Karan, and D., Moreteau, B. (1998). Cold stress tolerance in Drosophila'. Analysis of chill coma recovery in D. melanogaster. Journal of Thermal Biology 23:291-299. Denlinger, D.L., Joplin, K.H., Chen, C.-P., and Lee, R.E. (1991). Cold and heat shock. In: Insects at Low Temperature, pp. 131-148, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Dobzhansky, Th. (1935). Fecundity in Drosophila pseudoobscura at different temperatures. Journal of Experimental Zoology 71: 449-464. Dobzhansky, T., and Epling, C. (1944). Contributions to the genetics, taxonomy, and ecology of Drosophila pseudoobscura and its relatives. Carnegie Institute Washington Publication 55-^: 1-183. Dobzhansky, T., (1948). Genetics of natural populations: altitudinal and seasonal changes produced by natural selection in certain populations of Drosophila pseudoobscura and Drosophila persimilis. Genetics i J: 158-176. Dubinin, N.P., and Tiniakov, G.G. (1945). Seasonal cycles and the concentration of inversions in populations of Drosophila Junebris. American Naturalist 79: 570-572. Dubinin, N.P., and Tiniakov, G.G. (1946). Natural selection and chromosomal variability in populations of D.funebris. Heredity 37: 39-44. 64 Durando, C.M., Baker, R.H., Etges, W.J., Heed, W.B., Wasserman, M., and DeSaile, R. (2000). Phylogenetic analysis of the repleta species group of the genus Drosophila. Molecular Phylogenetics and Evolution 16: 296-307. Ehrman, L. (1960). The genetics of hybrid sterility in Drosophila paulistorum. Evolution 14: 212-223. Ehrman, L., and Powell, J.R. (1982). The Drosophila willistoni species group. In: Genetics and Biology of Drosophila, Volume 3B, pp. 193-225, Thompson, J., Carson, H. (eds). (London: Academic Press). Feder, M.E., and Hofman, G.E. (1999). Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual Reviews Physiology 61:243-282. Gibbs, A.G., and Matzkin, L.M. (2001). Evolution of water balance in genus Drosophila. Journal of Experimental Biology 204:2331-2338. Gibert, P., Moreteau, B., Petavy, G., Karan, D., and David, J.R. (2001). Chill-coma tolerance, a major climatic adaptation among Drosophila species. Evolution 55: 1063- 1068. Gibert, P., and Huey, R.B. (2001). Chill-coma temperature in Drosophila: effects of developmental temperature, latitude, and phylogeny. Physiological and Biochemical Zoology 74: 429-434. Goto, S.G., and Kimura, M.T. (1998). Heat and cold-shock responses and temperature adaptations in sub-tropical and temperate species of Drosophila. Journal of Insect Physiology1233-1239. Heed, W.B. (1978). Ecology and genetics of Sonoran Desert Drosophila. In: Ecological Genetics at the Interface, pp. 109-126, Brussard, P. (ed).( Springer-Verlag). Heed, W.B. (1982). The origin of Drosophila in the Sonoran Desert, In: Ecological Genetics and Evolution: The Cactus-Yeast-Drosophila Model System, pp. 65-80, Barker, J.S.F., Starmer, W.T. (eds). (New York: Academic Press). 65 Heinrich, B. (1993). The Hot-Blooded Insect. (Cambridge, MA: Harvard University Press). Hoffinan, A.A., and Watson, M. (1993). Geographical variation in the acclimation responses Drosophila to temperature extremes. American Naturalist 142: S93-S113. Hori, Y., and Kimura, M.T. (1998). Relationship between cold stupor and cold tolerance in Drosophila. Physiological and Chemical Ecology 27: 1297-1302. Huey, R.B., Wakefield, T., Crill, W.D., and Gilchrist, G.W. (1995). Within-and between- generation effects of temperature on early fecundity of Drosophila melanogaster. Heredity 74: 216-223. Izquierdo, J.I. (1991). How does Drosophila melanogaster overwinter? Entomologia Experimentalis et Applicata 59: 51-58. Kelty, J.D., and Lee, R.E. (1999). Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster. Journal of Insect Physiology 45: 719-726. Kelty, J.D., and Lee, R.E. (2001). Rapid cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 20-/: 1659-1666. Kerkis, J. (1941). The effect of low temperature on a mutation frequency in Drosophila melanogaster with consideration about the causes of mutation in nature. Drosophila Information Service No. 15: 25. Kimura, M.T. (1988). Adaptations to temperature climates and evolution of overwintering strategies in the Drosophila melanogaster species group. Evolution 42: 1288-1297. Kimura, M.T., and Beppu, K. (1993). Climatic adaptations in the Drosophila immigrans species group: seasonal migration and thermal tolerance. Ecological Entomology 18: 141-149. 66 Kimura, M.T., Ohtsu, T., Yoshida, T., Awasaki, T. and Lin, F.-J. (1994). Climatic adaptations and distributions in the Drosophila takahashii species group. Journal of Natural History 28\ 401-409. Knipling, E.B., and Sullivan, W.N. (1957). Insect mortality at low temperatures. Journal of ^onomic Entomology 50\ 368-369. Kohler, R.E. (1994). Lords of the Fly: Drosophila Genetics and the Experimental Life. (Chicago: University of Chicago Press). Krebs, R.A. (1999). A comparison of Hsp70 expression and thermotolerance in adults and larvae of three Drosophila species. Cell Stress and Chaperones 4:243-249. Lakovaara, S., and Saura, A. (1982). Evolution and speciation of the Drosophila obscura group. In: The Genetics and Biology of Drosophila, pp. 1-59, (Ashbumer, M., Carson, H.L., Thompson, J.N. (eds). (New York; Academic Press). Lee Jr., R.E., Chen, C.-P., and Denlinger, D.L. (1987). A rapid cold-hardening process in insects. Science 255: 1415-1417. Lee Jr., R.E. (1989). Insect cold hardiness: to freeze or not to freeze? Bioscience 39: 308- 313. Lee, R.E., and Denlinger, D.L. (1991). Insects at Low Temperature. (New York: Chapman and Hall). Lee Jr., R.E. (1991). Principles of insect low temperature tolerance. In: Insects at Low Temperature, pp. 17-46, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Lee Jr., R.E., Lee, M.R., and Strong-Gunderson, J.M. (1993). Insect cold-hardiness and ice nucleating microorganisms including their potential use as biological control. Journal of Insect Physiology J9: 1-12. 67 Lee Jr., R.E., Costanzo, J.P., and Mugnano, J. A. (1996). Regulation of supercooling and ice-nucleation in insects. European Journal of Entomology 93:40S-418. Leopold, R.A. (1991). Cryopreservation of insect germplasm. In; Insects at Low Temperature, pp. 379-407, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Levins, R. (1969). Thermal acclimation and heat resistance in Drosophila species. American Naturalist 103:483-499. Levitan, M. (1951). Response of the chromosomal variability in Drosophila robusta to seasonal factors in a southwest Virginia woods. Genetics 36: 561-562. Loomis, S.H. (1991). Comparative invertebrate cold hardiness. In: Insects at Low Temperature, pp. 301-317, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Lowe, C.H., Heed, W.B., and Halpem, E.A. (1967). Supercooling of the saguaro species Drosophila nigrospiracuta in the Sonoran Desert. Ecology 48: 984-985. Marinkovich, D., Crumpacker, D.W., and Salceda, V.M. (1969). Genetic loads and cold temperature resistance in Drosophila pseudoobscura. American Naturalist 103:235-246. Markow, T.A., and O'Grady, P. (2001). Drosophila Species Workshop manual. Center for Insect Science, University of Arizona, Tucson, AZ. McKenzie, J.A. (1975). The influence of low temperature on survival and reproduction in populations of Drosophila melanogaster. Australian Journal of Zoology 23: Til-lAl. Mohn, N., and Speiss, E.B. (1962). Cold resistance of karyotypes in Drosophila persimilis from timberline of California. Evolution 17: 548-563. 68 Morin, J.P., Moreteau, B., Petavy, G., Parkash, R., and David, J.R. (1997). Reaction norms of morphological traits in Drosophila: adaptive shape changes in stenotherm circumtropical species. Evolution 51: 1140-1148. Morris, G.J. (1987). Direct chilling injury. In: The Effects of Low Temperature on Biological Systems, pp. 120-146, Grout, B.W.W., and Morris, G.J. (eds). (London: Edward Arnold). Noor, M.A. (1995). Speciation driven by natural selection in Drosophila. Nature 375: eiA^lS. Novitski, E., and Rush, G. (1949). Viability and fertility of Drosophila exposed to sub zero temperatures. Biological Bulletin 97: 150-157. O'Grady, P. (1999). Reevaluation of phylogeny in the Drosophila obscura species group based on combined analysis of nucleotide sequences. Molecular Phylogenetics and Evolution 12: 124-139. Parsons, P. A. (1973). Genetics of resistance to environmental stresses in Drosophila populations. Annual Review of Genetics 7:239-263. Parsons, P.A. (1977). Resistance to cold temperature stress in populations of D. melanogaster and D. simulans. Australian Journal of Zoology 25: 693-698. Parsons, P.A. (1978). Boundary conditions for Drosophila resource utilization in temperate regions, especially at low temperatures. American Naturalist 112: 1063-1074. Patterson, J.T., and Stone, W.S. (1952). Evolution in the Genus Drosophila. (New York: MacMillan). Payne, N. M. (1926). Freezing and survival of insects at low temperatures. Quarterly Review of Biology I: 270-282. 69 Perez-Salas, S., Richmond, R.C., Pavlovsky, O., Katstrisis, D., Ehnnan, L., and Dobzhansky, Th. (1970). The interior semi-species of Drosophila paulistorum. Evolution 2^; 519-527. Popadic, A. and Anderson, W. W. (1994). The history of a genetic system. Proceedings of the National Academy of Sciences of the United States of America 91:6819-6823. Powell, J.R. (1992). Inversion polymorphism in Drosophila pseudoobscura and Drosophila persimilis. In: Drosophila Inversion Polymorphism, pp. 73-126, Krimbas, C., and Powell, J.R. (eds). (Boca Raton: CRC Press). Powell, J.R. (1997). Progress and Prospects in Evolutionary Biology: The Drososphila Model. (New York: Oxford University Press). Ring, R.A. (1980). Insects and their cells. In: Low Temperature Preservation in Medicine and Biology, pp. 187-217, Ashwood-Smith, M.J., and Farrant, J. (eds). (Tunbridge Wells, UK: Pitman Medical). Russo, C.A.M., Takezaki, N., and Nei, M. (1995). Molecular phylogeny and divergence times of Drosophilid species. Molecular Biology and Evolution 12: 391-404. Salt, R.W. (1936). Studies on the freezing process in insects. University of Minnesota Agricultural Experimental Station, Technical Bulletin 116. Salt, R.W. (1950). Time as a factor in the freezing of undercooled insects. Canadian Journal of Research 28:285-291. Salt, R.W. (1961). Principles of insect cold hardiness. Annual Review of Entomology 6: 55-74. Schawaroch, V.A. (2000). Molecular phylogeny of the Drosophila melanogaster species group with special emphasis on the montium subgroup biology. (New York: The City University of New York, xii +339). 70 Somme, L. (1982). Supercooling and winter survival in terrestrial arthropods. Comparative Biochemistry and Physiology 73A: 519-543. Somme, L., and Block, W. (1991). Adaptations to alpine and polar environments in insects and other terrestrial arthropods. In: Insects at Low Temperature, pp.318-359, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Spicer, G.S., and Pitnik, S. (1996). Molecular systematics of the Drosophila hydei subgroup as inferred from mitochondrial DNA sequences. Journal of Molecular Evolution 43'. 281-286. Stanley, S.M., Parsons, P. A., Spence, G.E., and Weber, L. (1980). Resistance of species of the Drosophila melanogaster subgroup to environmental extremes. Australian Journal of Zoology 28: 413-421. Steponkus, P.L., Myers, S.P., Lynch, D.V., Pitt, R.E., Lin, T.-T., Maclntyre, R.J., Leibo, S.P., and Rail, W.F. (1991). Cryobiology of Drosophila melanogaster embryos. In: Insects at Low Temperature, pp.408-423, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Storey, K.B., and Storey, J.M. (1988). Freeze tolerance in animals. Physiological Reviews 68: 27-84. Storey, K.B., and Storey, J.M. (1991). Biochemistry of cryoprotectants. In: Insects at Low Temperature, pp.64-94, Lee, R.E., and Denlinger, D.L. (eds). (New York: Chapman and Hall). Stratman, R., and Markow, T.A. (1998). Resistance to thermal stress in desert Drosophila. Functional Ecology 12: 965-970. Timofeeflf-Ressovsky, N.W. (1940). Mutations and geographical variation. In: The New Systematics, Huxley, T.J. (ed). (London: Oxford University Press). Tucic, N. (1979). Genetic capacity for adaptation to cold resistance at different developmental stages of Drosophila melanogaster. Evolution 33:350-358. 71 Wassennan, M. (1982). Evolution of the repleta group. In: The Genetics and Biology of Drosophiia, volume 3B, pp. 61-139, Ashbumer, M., Carson, H.L., and Thompson, J.N. (eds). (London; Academic Press). Wasserman, M. (1992). Cytological evolution of the Drosophiia repleta species group. In: Drosophiia Inversion Polymorphism, pp. 455-552, Krimbas, C.B., and Powell, J.R. (eds). (Boca Raton: CRC Press). Watson, M.J.O., and Hoffman, A.A. (1996). Acclimation, cross-generation effects, and the response to selection for increased cold resistance in Drosophiia. Evolution 50:1182- 1192. Yamamoto, A., and Ohba, S. (1984). Temperature preferences of eleven Drosophiia species from Japan: the relationship between preferred temperature and some ecological characteristics in their natural habitats. Zoological Science /: 631-640. Yamamoto, A., and Ohba, S. (1984). Heat and cold resistances of sixteen Drosophiia species from Japan in relation to their field ecology. Zoological Science I: 641-652. Zachariassen, K.E. (1982). Special section: cold-hardiness in poikilothermic animals. Comparative Biochemistry and Physiology 73A: 517-518. Zachariassen, K.E. (1985). Physiology of cold tolerance in insects. Physiological Reviews 65: 799-832.