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 virgin females from a control line that carries the wild-type organization of Sdic in order to collect na&iuml;ve experimental males (Type I); and d) P{sw} homozygous males and Sdic-sw-deletion-carrying virgin females in order to collect na&iuml;ve experimental males which carry the Sdic-sw deficiency (Type II).
<ol>
	<li>Set up multiple vials containing 8-10 females and 5 males each. Allow the females to lay eggs and transfer the adults to new vials every 3-5 days. Use bottles rather than vials if necessary and always use fresh food. The number of vials required will depend on the number of experimental male types under study and the number of individuals estimated to be necessary to detect differences (Table 1). More than 10 females per vial might result in over-crowding during larval growth, which may cause variation in the fertility of the progeny. Store vials at 25 &deg;C in a temperature-controlled chamber.</li>
	<li>Start collecting virgin females and na&iuml;ve males on days 11th and 12th after setting up the initial crosses. The waiting period varies according to the temperature; lower temperatures result in longer developmental time delaying the collection of individuals. Another factor affecting the timing to eclosion is the type of medium. Nutritious food, such as cornmeal-yeast medium 29, assures proper development of the adult reproductive system, which facilitates mating. Collect unmated flies every 4-6 hr. When collecting, anesthetize flies by introducing CO2 into the vial, tap the flies down, and sex them under stereomicroscope (Figure 2). Place the desired flies into different vials by sex and phenotype and label the vials appropriately.</li>
</ol>
Note 1. Collection routine starts in the morning by discarding the adults that emerged during the previous night. Collect virgin females and na&iuml;ve males once or twice during the day. Typically, D. melanogaster males become sexually mature 8 hr after eclosion at 25 &deg;C 30. If flies are maintained under 12:12 hr light/dark cycles, two peaks of eclosion are expected: during the first 1-2 hr after the light is turned on, and during the 2 hr before the light is turned off. Eclosion occurs within 24 hr after the pupa darkens.
Note 2. No more than 10 females should be put into the same vial to prevent over-crowding. This also limits the loss of females in the case they have to be discarded because at least one of them is not virgin.
Note 3. In order to collect the appropriate number of virgin females and na&iuml;ve males in a short time period, the following measures can be adopted. Set up 15-20 vials of w1118 for experiments involving two types of experimental males (Table 1). Lightly sprinkle the surface of the media and add a few dried active yeast pellets to facilitate oviposition. Transfer parents at least 4 times on consecutive days. To increase the available surface for pupation in each vial, insert multi-folded paper (7 x 5 cm) during the 4th or 5th day after the parents are transferred into another vial (Figure 3). If the number of emerged adults from the initial crosses is not enough, wait for a few more days and collect individuals from vials set up at different dates. Plan to carry out the experiments over several consecutive days to even out the workload.
2. Double-mating Experiments
Figure 4 shows the main steps involved in double-mating experiments performed in 14.
<ol>
	<li>On the morning of Day 1, set up the first mating. The Oregon-R males are the first to mate in the offense assay.
	<ol>
		<li>Using the aspirator, set up vials containing 10 4-5-day-old white-eyed w1118 virgin females and 10 red-eyed Oregon-R na&iuml;ve males each. Two to three vials are set up daily for 5 consecutive days. The number of vials to be set up might vary depending on the number of available individuals. Allow the flies to mate for 2 hr.</li>
		<li>Discard Oregon-R males and place each female into a new vial using an aspirator (Figure 5). Sex identification can be achieved by visual inspection of a few morphological differences (Figure 2). Label each vial appropriately and leave the female in the vial for 2 days (hereafter "v1").</li>
	</ol>
	</li>
	<li>On Day 3, set up the second mating. Selection of males and cross set up should be performed at random in order to minimize any potential bias towards any of the experimental male types.
	<ol>
		<li>Two hours before the light is turned off, transfer again the female into a new vial with an aspirator. Label the new vial appropriately (hereafter "v2").</li>
		<li>Introduce three 5-6-day-old experimental males of the same genotype into v2.</li>
		<li>Repeat 2.2.1 and 2.2.2 for each female.</li>
		<li>On Day 4, two hours right before the light is turned on (i.e. no more than 12 hr after 2.2.1), discard males by using the aspirator.</li>
	</ol>
	</li>
	<li>On Day 6, transfer each female again into another new vial. Label the new vials appropriately (hereafter "v3").</li>
	<li>On Day 10, discard the w1118 females.</li>
	<li>Examine the progenies in v2 and v3. The first inspection is performed after 13-15 days after the female is introduced into v2 (or v3), e.g. on Day 17 after the female first started ovipositing in v2. The second inspection is performed exactly after 17 days. This time frame poses a safe upper temporal boundary that guarantees no second generation in the same vial. Two inspections prevent over-crowding while facilitating progeny counting.
	<ol>
		<li>On Day 17, inspect v1 and ensure that there are red-eyed progeny present; if not mark the vial accordingly. Presence of white-eyed progeny indicates that the female was not virgin at the time of initial mating while no progeny indicates that mating with the reference or the experimental males did not occur.</li>
		<li>On Day 17, perform the first inspection of v2. Anesthetize emerged progeny in v2 with CO2 and sort them by eye color and sex. Record progeny numbers fathered by the first and second males. Figure 6 shows the expected phenotypes in the progeny of each mating scheme. In this particular experiment 14, only female progeny can be unambiguously assigned as sired by the reference or the experimental males and therefore only the information from daughters can be used to calculate P2. If with other markers the paternity of male offspring can be unambiguously assigned, progeny counts of both sexes can be used in downstream calculations. Discard the progeny but keep v2 for second inspection.</li>
		<li>On Day 20, proceed as in 2.5.2 with the newly emerged progeny in v2 and then discard the vial.</li>
		<li>On Day 20, inspect the emerged progeny in v3 for the first time and follow step 2.5.2.</li>
		<li>On Day 23, proceed as in 2.5.3 with the newly emerged progeny in v3.</li>
	</ol>
	</li>
</ol>
Note 1: Due to the potential inflating effect on P scores that multiple mating might have (2.2.2 and 2.2.3), mating can be limited to a particular time frame. The time frame will depend on the remating frequency associated with the genotype of the female and males used. The probability of multiple mating is specially reduced during night time 11.
Note 2: Do not add live yeast pellets because this might cause problems due to potential overgrowth of the yeast when the number of adult flies is low.
3. Data Analysis
<ol>
	<li>Recorded progeny counts should be organized appropriately for easy visual inspection and efficient analysis with a suitable statistical package (e.g. JMP from SAS Institute) or free web-based tools (e.g. <a href="http://www.vassarstats.net/" target="_blank">http://vassarstats.net/</a>).</li>
	<li>Sperm competitive ability. Add counts from v2 and v3. Female flies that have given rise to no or very limited progeny (e.g. &lt;10), died during the procedure, or were only successfully inseminated by one of the two males are considered as non-informative and are excluded from any downstream statistical analysis (2.5.1 and Table 2). Calculate the P2 score for each informative female.</li>
	<li>Test whether there are statistically significant differences in P2 values among the experimental males compared. This can be done using parametric (e.g. Tukey's HSD) or non-parametric (e.g. Steel-Dwass) tests depending on several factors including the skewness of the distribution of P2 values and the dependency between the variance and the mean. The angular transformation is commonly applied to proportions such as P2 prior to the use of parametric tests 31.</li>
	<li>Mating rate differences (optional). For each experimental male type, calculate the number of doubly mated females and those that only mated with the first male (the reference male in the offense assay and the experimental male in the defense assay). Using a two-tailed Fisher's exact test (available at <a href="http://www.langsrud.com/fisher.htm" target="_blank">http://www.langsrud.com/fisher.htm</a>), determine whether there are statistically significant differences between the two types of experimental males.</li>
	<li>Sex ratio (optional). For each experimental male, use the chi-square test to determine whether the sex ratio in the progeny from each female deviates from the expected 1:1 ratio.</li>
</ol>

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No conflicts of interest declared.

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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		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Amber latex tubing</td>
			<td>VWR</td>
			<td>62996-473</td>
			<td>1/4 inch ID, 1/6 inch Wall</td>
		 </tr>
		 <tr>
			<td>1 ml graduated XL filter tips</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td>Any 1 ml pipette tip can be used but semi-transparent and long tips are better as a fly receiver. If filter tips are used, poke out and discard the filter using a needle after the tip end is trimmed</td>
		 </tr>
		 <tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P-7793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mesh Fabric</td>
			<td></td>
			<td></td>
			<td>Thin and smooth</td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CO2 Tank</td>
			<td>Airgas</td>
			<td>CD-50</td>
			<td>Use FlyNap (Carolina Biological Supply Company) as an alternative if CO2 is not available</td>
		 </tr>
		 <tr>
			<td>FlyStuff Foot Valve, complete system</td>
			<td>Genesee Scientific</td>
			<td>59-121C</td>
			<td>Not necessary if FlyNap is used</td>
		 </tr>
		 <tr>
			<td>Plastic vials</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td>Come with carboard trays that can be reused for holding vials</td>
		 </tr>
		 <tr>
			<td>Cotton balls</td>
			<td>Fisher Scientific</td>
			<td>AS-212</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Active dry yeast</td>
			<td>Red Star</td>
			<td></td>
			<td>Found in general grocery store</td>
		 </tr>
		 <tr>
			<td>Sharpie markers</td>
			<td></td>
			<td></td>
			<td>Different colors may be used for marking different genotypes</td>
		 </tr>
		</tbody>
 </table>

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 ...

assessing-differences-in-sperm-competitive-ability-in-drosophila

Research

JoVE Journal

Biology

14.5K Views.  University of California, Irvine. 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.