Name: assessing differences in sperm competitive ability in drosophila
Uploaded: 2013-08-22T13:00:00
Description: Watch this Scientific Journal Video about Assessing Differences in Sperm Competitive Ability in Drosophila at JoVE.com

The authors thank NSF (MCB-1157876) for funding. We also thank Alberto Civetta, John Roote, and two anonymous referees for their comments.

Small-scale experiments should be performed to become familiar with the whole procedure.
1. Collecting Virgin Females and Na&iuml;ve Males
The simplest version of the outlined experiment consists of four types of initial crosses, which involve the following combination of adults: a) w1118 individuals in order to collect virgin females; b) Oregon-R individuals in order to collect na&iuml;ve reference males; c) P{sw} homozygous males and vir...

<ol>
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We have described the experimental design to assess differences in the relative contribution of genetically distinct D. melanogaster males to the progeny in controlled double-mating experiments 7,8. This has been done in the context of a genetic factor hypothesized to influence sperm competitive ability and it has been illustrated within the offense assay although a similar procedure applies to the defense assay (Figure 1). This experimental design can be modified to test other...

Since Geoff Parker noted the prevalence of sperm competition in insects and its evolutionary implications 2, a surge of studies in Drosophila and other species have tried to shed some light on this phenomenon at many different levels. Some examples of areas of interest have been the survey of its variation in natural populations 9,10, its genetic architecture and relevance of underlying genetic factors 11-14, and its role in driving coevolution between the sexes 15,16. In D. melanogaster females, the limited capacity of the specialized sperm-storage organs, a pair of spermatheca and the seminal receptacle 6,17, contributes to the competition of the sperm from different males. Approximately 1,500 sperm are transferred during mating to the female but only ~500 can be accommodated in the mentioned organs 18,19. In the laboratory, controlled double-mating experiments involving a reference male and one or more males of interest have been extensively used for evaluating sperm competitive ability 7,8.	
	Sperm competitive ability is estimated as the proportion of progeny sired by the experimental male in double-mating experiments over the total progeny, i.e. that from both the experimental and reference males. Sperm competitive ability comprises two components, each of them evaluated in a separate assay. In the offense assay, the ability of the experimental male sperm to displace the sperm from the first male, i.e. the reference male, is evaluated. Conversely, in the defense assay, the ability of the experimental male sperm to resist displacement or to reduce the fertilization success of the reference male sperm is evaluated. Depending on the type of assay, defense or offense, sperm competitive ability is estimated through the scores P1 or P2, respectively. P1 and P2 can only take values between 0 and 1. Intermediate values are usually interpreted as indirect evidence of sperm mixing, which suggests a physiological scenario involving direct sperm competition. Following the same rationale, extreme values can be interpreted as evidence for strong differential sperm competitive ability. Early studies showed that P2 in D. melanogaster is over 0.8 increasing as the time elapsed between the two matings lengthens 7. This same experimental design has been used in other Drosophila species, P2 being the commonly used statistic in studies to evaluate sperm competitive ability 20. For most species, the P2 values of the strains tested is higher than 0.6 21. Nevertheless, several other mechanisms unrelated to the direct competition between sperm of different males can yield identical scores (see Discussion).	
	Distinguishing progeny sired by the first or second males is possible through the use of easily identifiable markers. In early studies, one of the males was irradiated at sublethal doses of, for example, X-rays such that virtually all ova fertilized by irradiated sperm failed to hatch 7. Subsequently, mutations altering eye pigmentation or wing shape have been the most commonly used markers. Examples of the former are the mutations bw (brown) 9, cn (cinnabar)22 and w (white) 23, while the mutation Cy (Curly) 24 corresponds to the second type of phenotypes; some of these mutations have been combined in the same individual, e.g. cn bw. To a lesser extent, allozymes 25 and microsatellites 26,27 with known inheritance patterns have also been used.	
	The experimental design to test for differences in sperm competitive ability described here follows essentially that of Clark et al. 9. Results derived from these experiments give information solely about the differential paternity of the experimental male types under scrutiny. Assays that also make allowance for post-fertilization differences in fitness 14,28 and sperm visualization techniques 24 enable differences in P1 (or P2) scores to be interpreted as differences in sperm competence.	
	Figure 1 outlines the rationale of both the offense and the defense assays. To illustrate the logistics of the process, an offense experiment carried out in D. melanogaster 14 will be explained in detail. This particular offense assay was used to test for a measurable effect of the multigene family Sperm-specific dynein intermediate chain (Sdic) on sperm competitive ability. All the members of this multigene family reside in tandem on the X chromosome. Knockout males were generated by deleting the Sdic cluster. Because the deleted segment also included the essential gene short wing (sw) and the purpose of the study was to evaluate the relevance of Sdic, males carrying the Sdic-sw deletion were rescued by a transgenic copy of sw (symbolized as P{sw}, which also carried a mini-white reporter gene) on chromosome 2. Eye color was used as a visible marker for paternity identification. All the flies were in a white mutant background with the exception of those from the strain Oregon-R, which were used as reference males.

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assessing differences in sperm competitive ability in drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Table 2 summarizes some salient features of two offense experiments (Assays 1 and 2) in which D. melanogaster experimental males with and without (Type I and II, respectively) a functional Sdic cluster are compared 14. After taking into account different incidences encountered with some replicates, 58-83% of the females were found to be informative and therefore the paternity counts of their progeny could be used for calculating P2. Results of non-parametric tests ...

Watch this Scientific Journal Video about Assessing Differences in Sperm Competitive Ability in Drosophila at JoVE.com

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

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