Mechanisms of Desiccation Resistance in
Natural Populations of Drosophila suboscura
Darci Desourdy
May 2, 2002
Introduction
Environmental stresses may have an impact on the evolution of Drosophila. These
stresses include anoxia, starvation, exposure to high concentrations of ethanol, and desiccation.
(Hoffmann and Parsons, 1989).
Extreme stresses impose strong natural selection and may cause rapid phenotypic
changes (Hoffmann and Parsons, 1989). Stress resistance is thought to influence the distribution
of species as well as individual fitness in these environments as organisms develop physiological
adaptations that eliminate or mitigate stress (Bradley et al., 1999). If Drosophila are subjected to
a continuous stress, subsequent generations should evolve a resistance towards that type of stress.
If they are subjected to the stress of desiccation, they should develop a mechanism that allows
them to survive longer in dry environments.
Resistance to desiccation can involve several adaptations. First, one may develop a
reduction in the rate of water that is lost. Second, one may hold more water, either in the form of
bulk water or as metabolic water stores. Third, one may be able to tolerate a greater loss of water.
(Gibbs et al, 1997; Bradley et al, 1999).
Adaptive mechanisms for desiccation resistance may include a reduction in cuticular
permeability due to the quantity and physical properties of the cuticular lipids. Cuticular lipids are
extremely diverse; over 100 different compounds have been identified. Epicuticular lipids provide
the primary passive barrier to evaporative water loss in insects and other arthropods (Gibbs,
1995). Other mechanisms include the reduction of respiratory water loss, which is achieved by
decreasing the metabolic rate or the reduction in the amount of excretory water that is lost. (Gibbs
et al, 1997). Graves et al (1992) also suggested that an increased lipid content and an increased
glycogen content may influence desiccation resistance. Folk et al (2001) argued that an increased
amount of hemolymph may have an effect on desiccation resistance.
In this experiment Drosophila subobscura will be studied to determine the mechanisms that have
evolved to increase desiccation resistance. I propose that flies originally from drier environments
will have evolved a stronger resistance to desiccation. They have built this resistance by either
reducing their metabolic or respiratory rate, by obtaining a higher lipid content to prevent loss of
water through the cuticles, or by possessing a larger amount of water, including the water present
in glycogen, than those flies found in more humid climates.
Background
Drosophila subobscura are small insects native to Europe. They have recently been
introduced to parts of North and South America. Because of their small size and large surface to
volume ratio, they are highly vulnerable to dehydration. (Gibbs et al, 1997). Flies that have
qualities that allow them to have a higher desiccation resistance will survive longer in dry
conditions than those that do not possess the mechanism. Flies with an increased desiccation
resistance, inhabiting a dry climate, with live longer than those without the increased resistance.
Because they have a longer lifespan, they will have more chances to reproduce and pass genes
responsible for the increased desiccation resistance onto their offspring.
Adult Drosophila are sexually dimorphic. Females are generally larger than males
(Ashburner, 1989). Because of their increased size, females have the ability to possess a greater
amount of water and other resources, which may contribute for a higher desiccation resistance for
females than for males.
Comparative studies have shown that species of Drosophila from dry environments lose
water slower than those from damper environments. They also survive desiccation longer than
species from damper environments. (Gibbs et al, 1997). The desiccation resistance of Drosophila
melanogaster responds to artificial selection. Gibbs et al (1997) exposed flies to dry air and then
allowed the individuals who survived the longest to propagate for several generations. This
resulted in the desiccation-selected flies being able to survive dry air conditions twice as long as
controls.
It has been shown that populations of Drosophila subobscura from South America
survive desiccating conditions longer than their European counterparts. The earliest deaths in
South America occurred after the deaths of the longest surviving European flies, for both sexes.
Only European females showed a significant decrease in desiccation resistance with increasing
latitude (Dietrich, unpublished data). This increase in desiccation resistance in Chilean
populations may be explained by the colonization of South America by European flies from the
Mediterranean region, based on the chromosomal patterns exhibited (Ayala et al, 1989; Balanya
et al, 1994; Beckenbach et al, 1986).
While many experiments have been done with populations selected for desiccation
resistance in the laboratories, little has been done with the study of mechanisms of desiccation
resistance in natural populations. I will be working with natural populations of Drosophila
subobscura from Europe andSouth America in attempts to determine the mechanisms of
desiccation resistance in these populations. By comparing the desiccation resistance of the natural
populations to other experiments involving laboratory selection, it will be determined if a similar
mechanism is present or if a different mechanism is used in these populations.
Proposed Research
In this experiment, eggs from 6 populations of Drosophila subobscura in Europe, and from 6
populations in South America were tested. Populations were selected based on latitudes. Eggs
were collected from the extreme latitudes for each continent and from four latitudes between the
extremes (Table 1).
Eggs were collected in a petri dish over a 24-hour time period. Eggs were collected in
sets of 50 and placed in vials containing 10 mL of a cornmeal/molasses/agar medium. Within 24
hours of eclosing, the flies were sexed and placed in vials with fresh food.
One set of flies consisting of 20 males and 20 females from each population was used to
determine the water loss rates of Drosophila subobscura. Five to six days post-eclosion, flies
were transferred individually into microcentrifuge tubes and weighed. The microcentrifuge tubes
were pierced with a 21 gauge, 1½ inch hypodermic needle to allow for air circulation before the
flies were placed in them. After the initial weighing, the flies were placed in a desiccator with
Drierite desiccant. The flies were weighed every four hours for a period of 48 hours. At each
weighing, the flies were recorded as being dead or alive. They were then replaced in the
desiccator and allowed to dry for an additional 24 hours. They were reweighed to determine their
dry weight.
A second set of flies consisting of 60 males and 60 females from each population were
used to determine the amount of cuticular lipids that are present in an individual from a given
population of Drosophila subobscura. Five to six days post-eclosion, flies were killed with ether.
They were weighed in groups of five and placed in micro-centrifuge tubeswith 1.5 mL of hexane.
The solution was heated at 60 degrees Celsius. After 20 minutes, the flies were transferred to
clean centrifuge tubes to dry .The groups of flies were reweighed. The mass of the epicuticular
lipids was determined for each group by subtracting the final mass from the initial mass. Lipid
content was determined for each fly and for each gram of weight.
A third set of Drosophila subobscura consisting of 60 males and 60 females from each
population were used to determine the glycogen content. Five to six days post-eclosion, flies were
killed with ether. They were weighed in groups of five and placed in microcentrifuge tubes. The
flies were digested for 30 minutes in 1.5 mL of a 30% solution of potassiurn hydroxide at 80
degrees Celsius. The flies were removed from the solution. The glycogen was precipitated from
the solution with 1 milliliter of cold 95% ethanol and centrifuged to pellet the glycogen. The
pellet was washed with 1 milliliter of distilled water, re-centrifuged, and resuspended in 1
milliliter of distilled water.
A sulfuric acid-anthrone reagent was made to cause a colorized reaction with the
glycogen. 760 mL of sulfuric acid was mixed with 300 mL of distilled water and cooled.
Immediately before use, 150 mg of anthrone was mixed with the diluted sulfuric acid (Van
Handel, 1965).
100 L of the extracted solutions were placed in glass test tubes. Three mL of the sulfuric
acid-anthrone reagent were added to each test tube. The solution was heated at 90 degrees Celsius
for 20 minutes. The samples were cooled and their absorbencies were read in a spectrophotometer
set at 620 m. Absorbencies were compared to those of known standards containing 10 to 50 g
of glycogen to determine the amount of glycogen in each sample. Glycogen content was
determined per fly and per gram of weight.
Another set of flies consisting of 15 males and 15 females from each population was used to
determine hemolymph content. Five to six days post-eclosion, flies were killed with ether and
individually weighed. The abdomen of each fly was tom open with forceps and blotted with a
Kim-wipe to remove the hemolymph. Each fly was reweighed. The hemolymph content was
determined by subtracting the final weight from the initial weight. The percent of hemolymph per
gram of fly was determined.
My preliminary data has shown a significant decrease in water content with increasing
latitude that is present only in European females. In Chilean males, there is a significant decrease
in lipid content with increasing latitude. There is a significant decrease in glycogen content in
Chilean males and European males and females. With respect to hemolymph content, only
European males showed a significant trend in that the amount of hemolymph increased with
latitude.
I will be continuing to process the data that I have recently collected and will combine it
with my preliminary data to determine if similar trends are present in other populations of
Drosophila subobscura. The data that I have already collected will be compared with previous
studies on the desiccation resistance of natural populations in Europe and Chile to determine if
there is a positive or negative correlation between the mechanisms and desiccation resistance of
populations at different latitudes. If there is a strong correlation between the water content and
evolved resistance, it can be assumed that the an increased resistance is the result of flies holding
more total water. If there is a strong association between the desiccation resistance and the
amount of hemolymph or glycogen, it can be assumed that the resistance is a result of water
stores. If there is a strong relationship between the presence of lipids and the desiccation
resistance of populations, it can be assumed that the resistance to desiccation is a result of the
flies being able to build up a waxy cuticle. If there is a strong association between the desiccation
resistance and the amount of hemolymph or glycogen, it can be assumed that the resistance is a
result of water stores.
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