Cold tolerance in Sonoran Desert Drosophilaspecies

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COLD TOLERANCE IN SONORAN DESERT

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, 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 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 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 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 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 , 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

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