Lab Lunch!

Rinse, lather, rep()

In some of our assays we sort flies by size, and record the number of individuals found in each sieve. When the data is entered, it might look like this (ht Conor Delar)

> deLHM2
     Assay2 Size.um. Male.Unmated. Female.Unmated. Male.Mated. Female.Mated.
1  LHm (L2)     1420             1               3           1             3
2  LHm (L2)     1365             4               1           2             3
3  LHm (L2)     1313             1               2           1             3
4  LHm (L2)     1262             2               4           0             1
5  LHm (L2)     1214             6               2           3             0
6  LHm (L2)     1167             3               8           3             3
7  LHm (L2)     1122             7              21           0             3
8  LHm (L2)     1079             4              10           3             0
9  LHm (L2)     1038             2               7           0             2
10 LHm (L2)      998             4               8           0             0 

11 LHm (L2) 948 3 6 0 0
 But how can we actually compare (in R), for instance, the weight of mated and unmated females from the sample? We could do contingency tables to see if the frequency of counts in the two groups, but what if we wanted to compare the mean "girth" of these two groups? You could tediously enter the size of each individual (e.g. for unmated females, 1420,1420,1420,1356,1313,1313 etc...), but there must be a better way

Here, I will show how we can use the rep() function
Where I will specifiy in the first parameter what values I want repeated, and then for the second how many times I want it repeated.

> deLHmFU <-rep(c(deLHM2$Size.um.),c(deLHM2$Female.Unmated.))
> deLHmFM <-rep(c(deLHM2$Size.um.),c(deLHM2$Female.Mated.))
> deLHmFU
 [1] 1420 1420 1420 1365 1313 1313 1262 1262 1262 1262 1214 1214 1167 1167 1167 1167 1167
[18] 1167 1167 1167 1122 1122 1122 1122 1122 1122 1122 1122 1122 1122 1122 1122 1122 1122
[35] 1122 1122 1122 1122 1122 1122 1122 1079 1079 1079 1079 1079 1079 1079 1079 1079 1079
[52] 1038 1038 1038 1038 1038 1038 1038  998  998  998  998  998  998  998  998  948  948
[69]  948  948  948  948
> deLHmFM
 [1] 1420 1420 1420 1365 1365 1365 1313 1313 1313 1262 1167 1167 1167 1122 1122 1122 1038
[18] 1038
> 

Now I could proceed with the correct analyses (in this case due to non-normality of distributions, a Mann-Whitney test and a Cliff's delta) to compare the two groups.

Male genotype influences female reproductive investment in Drosophila melanogaster

Pischedda, A., Stewart, A.D., Little, M.K. & Rice, W. 2011. Male genotype influences female reproductive investment in Drosophila melanogaster. Proc Biol Sci 278:2165-72.

This study is the first form of direct evidence that males vary genetically in their influences on female fecundity, egg sizes and overall female investment in reproduction. Female Drosophila melanogasters were mated with males from 10 different ‘worldwide lines’ (to account for genetic variation) for 2 hours. After this time, females were placed into individual oviposition vials for 22 hours, followed by transfer into a new oviposition vial for another 22 hours. After that, all of the females who mated with males from the same worldwide line were put into the same egg laying chamber and allowed 4 hours to oviposition. Eggs from the chamber were then photographed and measured. Results found that a male’s population of origin did not affect egg size, but did affect females' fecundity in the first 22 hours after mating. The genotype of the males within a population did however account for some of the variation seen in egg size. This study is very useful to me, as it relates directly to the question at the forefront of my research. It does not however address the mechanisms causing the variations seen, such as female cryptic choice or manipulations by males, so leaves room for further investigation and analysis. 

Females increase egg deposition in favour of large males...

Evans, J.P., Box, T.M., Brooshooft, P., Tatler, J.R. & Fitzpatrick, J.L. 2010. Females increase egg deposition in favour of large males in the rainbowfish, Melanotaenia australis. Behavioural Ecology 21:465-469.

Through this study, the researchers address how sexual selection favours flexibility in maternal investment, using rainbowfish as a model species. Females were individually placed into large tanks, with both a small and a large male that were confined to containers within the tank. First, they observed the amount of time the female spent within one body length of the containers containing the males. After four days, either the large or the small male was released into the tank with the female, and they were allowed 4 more days to interact. The eggs produced by the female during this time were collected, counted and photographed. Results showed that during the initial 4 days, females spent 70% of their time within one body length of the large male, and they produced two times as many eggs when they mated with the large male (large males are phenotypically preferred). The variation in maternal investment defined within this study is important for understanding the effect of a mate’s phenotype on maternal investment, but it fails to address the effect of a mate’s genetic identity.

While I continue researching for my thesis project, I’m realizing more and more not to disregard ‘old’ articles just because they are, well, ‘old’. 3 articles by Partridge and colleagues from the 1980s have proven to be of great use to me in understanding costs associated with mating, and how choice influences all individuals in the equation (i.e. males, females and subsequent offspring.) The following are annotations for these articles: 

Fowler, K. & Partridge, L. 1989. A cost of mating in female fruit flies. Nature 228:760-761.

This article looks at the costs a female endures when mating with a male in the model organism Drosophila melanogaster. This is done through mating experiments where virgin females were subjected to either a group of males who all were capable of mating (high-mating) or a group of males where only one of the males was actually capable of mating (low-mating). Males in the low-mating scenario still displayed courting behaviours towards females, but had genitalia removed so they could not actually mate. Results showed that the females who were exposed to high-mating scenarios had significantly lower life spans then those exposed to low-mating. Although this may be a consequence of injury, parasites, or effects of sperm, it still is related to the actual process of mating. These results therefore have profound effects on the future reproduction success of these females. These conclusions are beneficial for me to understand in order to predict then why a female may alter her investment in the subsequent offspring, depending on the costs she has endured through mating.

 Partridge, L. 1980. Mate choice increases a component of offspring fitness. Nature 283:290-291.

In this experiment, larval survival rates are used as a measure of D. melanogasters’ offspring fitness. Males and females are exposed to treatments in which they have the ability to choose mates (cages with many individuals), or where they are unable to choose their mate (randomly selected female exposed to single randomly selected male).  Results show higher fitness in subsequent offspring (i.e. more offspring when exposed to competing larvae) of parents who did have choice in who they mated with, compared to those who did not. The author discusses possibilities of the genetics of these individuals influencing choice, and how if there are differences in paternal and maternal genes, it can lead to fitter offspring because of heterozygosity. This information is relevant to my research, because it is important to understand the how the identity of the parents can have a significant affect on their offsprings’ fitness, especially since the flies in my experiment are exposed to a no-choice treatment.

 

Partridge, L. & Farquhar, M. 1981. Sexual activity reduces lifespan of male fruit flies. Nature 294:580-581.

Partridge and Farquhar study how mating affects the fruit flies’ lifespan by exposing males (Drosophila melanogaster) to different numbers of receptive females and comparing their longevity with control groups. Males in experimental groups were exposed to 1-8 virgin females a day, while males in the control groups were exposed to females who had already been inseminated and therefore would not remate, or with no females at all. Males exposed to the highest number of receptive females (i.e. 8 virgins per day) were shown to have the lowest longevity compared to males who were exposed to fewer receptive females (slightly higher longevity), and control groups (greatest longevity). Understanding the costs associated with mating can help us to understand differences in parental investment of their offspring.

Although these articles are not exactly directly related to my research, the basis of mate choice and affect on all of the individuals involved is very useful background knowledge to have. It allows me to better understand costs of mating behaviours, and hypothesize therefore why differences in parental investment based on the identity of mates may have adaptive benefits.

Long Lab Group Photo October 2012

L:R Conor Delar, Heather McLeod, Erin Sonser, Hannah Tennant, Adam Lounsbury, Maya Ashoka, Sahsa Thomsen (Not pictured: Justine Kraemer or Tristan Long)

Assessing Direct and Indirect Costs

Orteiza, N., Linder, J., & Rice, W. 2005. Sexy sons from re-mating do not recoup the direct costs of harmful male interactions in the Drosophila melanogaster laboratory model system. Journal of Evolutionary Biology, 18(5), 1315-1323.

This article explores how female interactions with males can reduce lifetime survival and fecundity, and assesses any indirect benefits as a result. Virgin females were introduced into two conditions simulating constant male exposure and minimal male interaction. Wild-type males and virgin females are housed and allowed to mate, and then half the females are given the chance to re-mate again with brown eyed males. To determine any direct costs to females by re-mating and constant male exposure females were housed with brown-eyed females and would compete for limited yeast. The costs would then be calculated by recording the eye colour and number offspring each female produced. To assess any indirect benefits virgin females were allowed to mate once with a red-eyed male, and then given the chance to re-mate with a brown-eyed male. The male offspring were then separated according to whether their father was an initially mated or re-mated male and cultured to test for paternity. The study found that the grand-offspring of the re-mated male had slightly lower fecundity than the initially mated male and did not make up for the direct costs involved in re-mating caused by increased male exposure. It is estimated that about 10% of a female’s fecundity is lost through re-mating while about 3% is gained through indirect benefits.

Sex Ratio in Drosophila melanogaster Populations Effect Male Mating Success

Pavkovic-Lucic, S., Kekic, V., & Cvoro, A. 2009. Larger male mating advantage depends on the sex ratio in Drosophila melanogaster. Ethology Ecology & Evolution, 21(2), 155-160.

In this study the mating success of large and small males was examined under specific sex ratios. The mating status of females and males were held constant. Larger males were more successful than smaller males in conditions where the sex ratio was equal, and when there were more males present than females. When more females were present in the population there was no significant difference in mating success of large and small males. In all cases larger males mated earlier, while there was no significant size difference of mated females across the conditions. This is most likely due to the high tracking speeds and increased courtship of larger males. Larger males may have greater mating success than smaller males in male dominated sex ratios because of increased male-male competition. These findings support male-male competition as being an important factor in determining the mating success of larger males rather than a female preference for larger males.

Effects of Male Size on Mating Success and Female Preference in Drosophila melanogaster

Partridge, L., Ewing, A., Chandler, A. 1987. Male Size and Mating Success in Drosophila melanogaster: The Roles of Male and Female Behavior. Animal Behaviour, 35, 555-562. 

In this article the effects of male size in relation to courtship behavior was examined in a non-competitive environment to determine what factors lead to higher courting success in larger males. Male tracking speeds, song frequency and amplitude, as well as frequency of decamping by females was examined. Larger males exhibited louder and more frequent courtship songs as well as faster tracking speeds, while female decamping did not differ significantly among large and small males. This suggests that mating success by larger males is not due to female preference but rather a result of male-male competition. Although the frequency of decamping by the females did not differ, female preference for louder songs and their increased movement as a result of faster tracking speeds by larger males is an important factor in determining female preference for larger males and increased mating success among larger males in a non-competitive environment.

Food Cook!

Gradate Students Hannah Tennant and Adam Lounsbury mixing up a delicious batch of Drosophila banana media (yum!)

Sieve Sorting

I am sure that ever since you read about the the sieve sorter system mentioned in our paper "A cost of sexual attractiveness to high-fitness females" you have been wondering what you will need to make your own set of sieves? In addition to a set of electo-formed sieves (which we purchased from Precision Electroforming Inc.), and a sieve-shaker (we use a Global Gibson SS-III), you'll need to make some fly-friendly modifications. I have posted some pictures here.

Sieve column on sieve shaker

Practice Makes Perfect

The last couple of weeks I’ve been getting in some practice here and there, taking pictures of eggs laid by backup lab culture flies, trying to figure out how to get the best picture I possibly can for measuring egg size in ObjectJ.

The 'Old' Way

Yesterday I size sorted IV flies, and let them lay on juice cookies over night. After clearing the flies and putting the cookies in the fridge to control for any changes in egg size from development, I worked my way through taking pictures of eggs in each size group. The pictures were looking pretty good, except for the lack of contrast between the red background and the eggs (that pick up a sort of pink colour from the cookie).

The 'New' Way

 It wasn’t until the last cookie that I discovered something that would make the photos much better. Instead of using a white platform underneath the juice cookie as I had been doing previously, I tried  using a black one. This instantly increased the contrast between the eggs and the background. I believe that with this discovery, future picture taking and egg measurements will be much more effective.

Getting in this practice and working out all the little details now will be very helpful in the future when I start working on my thesis project.

Repeatability of mate choice in female red jungle fowl

Johnson, T.S. and Zuk, M. 1995. Behavioural Ecology. 7:245-246

The authors investigated the repeatability of mate choice in female red jungle fowl by examining the heritability of female preference that is an assumption in models explaining the evolution of mate choice. Male morphology was characterized 2 weeks before mate choice trials. For the mate choice trials, males were chosen randomly with the only condition that females never saw a male more than once and males were not paired more than once during the experiment. Females were placed in a small cage in front of two males and left there for 30 min. The female's behaviour was then observed for 20 mins after being released from the small cage. Preference was scored if a female copulated with a male. The results showed that female preference differed with both male trait and the timing of the breeding season. The highest estimate of repeatability was found to be 0.19, which indicates that current heritability may be low. Yet there is still evidence that a heritable component exists in the female red jungle fowl. Females showed repeatability with respect to male combs, but not with respect to hackle feather colour. Male traits can thus evolve through female choice when female preference is genetically determined. and the male trait is heritable.

Positive genetic correlation between female preference and offspring fitness

Hine, E., Lachish, S., Higgie, M., Blows, M.W. 2002. Proc. R. Soc. Lond. 269:2215-2219

These authors showed that female Drosophila serrata prefer extreme male cuticular hydrocarbon (CHs) blends and that this preference affects offspring fitness. Mate choice experiments were preformed using virgin females individually placed with two virgin males. After observed copulation, the males were removed and killed and prepared for gas chromatography analysis of the CHs. From these results, the authors estimated the sexual selection fitness function and linear selection gradient for the more attractive male CH blend. It was then determined if female choice affected offspring fitness with a quantitative genetics experiment. This experiment encompassed female preference, male attractiveness, and offspring fitness all in one. The authors found that female preference is positively correlated with offspring fitness, and that choosing the more attractive male results in genetic benefits. In addition, males with the highest probability of mating conferred intermediate levels of offspring fitness, indicating that female choice in under stabilizing natural selection. Even though male cuticular hydrocarbons experience strong sexual selection, the genes underlying this conferred lower offspring fitness, suggesting a balance between sexual selection and natural selection may have occurred in this population.

Pushing Flies

Effects of Drosophila melanogaster Female Size on male Mating Success

Turiegano, E., Monedero, I., Pita, M., Torroja, L., Canal, I. 2012. Effect of Drosophila melanogaster Female Size on Male Mating Success. J Insect Behav.  

This article examines the importance of female body size on mating success in Drosophila melanogaster. The authors first confirm that larger males do mate more rapidly and more frequently, but stress the importance of examining female size relative to males. Through observation and analysis, the authors were able to conclude that in a non-competitive environment, an increase in female size prolongs copulation latency (i.e. the time between introduction and initiation of copulation), specifically that larger differences in size between a male and a female causes an increase in copulation latency. Larger females were also found to display lengthened avoidance behaviour during courtship. In competitive environments, it was found that the first male to initiate courtship had a much higher probability of mating, and that an increase in female size reduces the likelihood of the larger male initiating courtship.  This study is significant because examines the importance of the relative size of both males and females in determining mating success, and leaves room for further research into this topic.

Effect of Drosophila melanogaster Female Size on Male Mating Success

Turiegano, E., Monedero, I., Pita, M., Torroja, L., & Canal, I. 2012. J Insect Behav.   

The authors examined the effect of female Drosophila melanogaster body size on mating success. Their results confirmed that larger males have a higher mating success. The authors also found that female size with respect to male size also affects mating success; previously the role of female size was unknown. The effect of female size on mating behaviour and dynamics appears to influence male courtship displays. Males show an increased time in courtship initiation towards larger females, larger females are the recipients of more wing vibrations, and larger females tend to avoid males for longer periods of time during courtship attempts. Large males are known to copulate more rapidly in non-competitive experiments. In addition, large males are favoured to initiate courtship in a competitive environment.The analysis of these behaviours confirms that the female is the most important in courtship opportunities and that both male and female body size is important for successful mating.

Annotated Bibliography

Turiegano E, Monedero I, Pita M, Torroja L, Canal I. 2012. Effect of Drosophila melanogaster Female Size on Male Mating Success. J Insect Behav.

This article considers the importance of female size when examining male mating success. Previous studies have revealed that larger males tend to have higher mating success, but did not take female size into account. The study confirmed that male size affects mating success, but stresses that this must always be considered relative to female size. Males show greater latency to courtship toward larger females, larger females receive more wing vibrations from males during courtship, and larger females move around more during courtship. The article discusses possible reasons for observed behaviours, and invites further study into the relation of female and male size variation toward mating behaviour.