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Building a Bridge Between Laboratory and Field Studies: Allelic Variation at a Drosophila melanogaster Male Accessory Gland Protein Gene (Acp 36DE)

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Presentation on theme: "Building a Bridge Between Laboratory and Field Studies: Allelic Variation at a Drosophila melanogaster Male Accessory Gland Protein Gene (Acp 36DE)"— Presentation transcript:

1 Building a Bridge Between Laboratory and Field Studies: Allelic Variation at a Drosophila melanogaster Male Accessory Gland Protein Gene (Acp 36DE)

2 Research Goal and Motivation The goal is to investigate natural genetic variation at the 36DE gene in the laboratory and the field. –Are there discernible phenotypes associated with the natural genetic variation? –Can we understand what maintains polymorphism in this gene? –Can we use the natural genetic variation for insight into the function of the gene? “natural mutants”

3 Mating success Fecundity Longevity Egg viability Larval viability Pupation success Developmental rate Life History Traits

4 Drosophila Life History Characters Juvenile (Larval) Viability Female Fecundity Female Longevity Male Mating Success Male Longevity Sperm Competition, Cryptic Choice of Sperm

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6 Characteristics of D. melanogaster Seminal Proteins (largely Acps) Wolfner (Chapman, Partridge) –Acp26Aa, Acp36DE Acp62F, Acp76A and others –oocyte release by the ovary and increased egg production, efficient sperm storage, antibacterial activity, female sexual attractiveness, decreased female survival, male proteins similar to lipases, a mating plug constituent, and protease inhibitors Kubli (Chen, Applebaum) –sex peptide (Acp70) and DUP99B –increased egg production and female refractoriness to remating

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9 Characteristics of Acp 36DE Females mated to a 36DE null mutation male exhibit poor sperm storage (Nuebaum and Wolfner 1999, Chapman & Partridge 2000) – Acp36DE facilitates the storage of sperm in females, but how the protein functions is not known sperm s 1997)

10 Characteristics of Acp36DE The protein is transferred to females at the time of mating and is tightly associated with sperm (Neubaum and Wolfner 1997) Acp36DE localizes to a position at the top of the oviduct, in both types of sperm storage structures and in the mating plug (Wolfner laboratory) The protein is probably highly glycosylated and it has numerous serines, glutamines and glutamic acids –Similarity to some “structural proteins”

11 Characteristics of Acp36DE - Genetic Variation in Populations Allelic variation at the gene is associated with P1 (sperm competition “defence”) in a laboratory study –Clark et al. 1995 Population resequencing of the gene indicates that it has undergone rapid evolution (Begun and Clark 2000) –The pattern of sequence polymorphism and divergence is compatible with adaptive protein divergence –Rapid evolution suggests strong selection

12 Getting Started (the plan)

13 General Plan: Study Female Remating Incidence &Sperm Competition in Relationship to Acp 36DE Allelic Variation Allelic variation is the “stuff of evolution”, but it has been frustratingly difficult to document phenotype associated fitness effects of alleles in an meaningful evolutionary context. –The plan was to combine female remating (phenotype) with an investigation of alleles at a gene undergoing strong selection

14 Specific Plan – One Source of Flies for Field and Lab Studies Ravenswood –A winery in Sonoma (N. CA) –The idea was to focus on one population site for “field” and laboratory studies for informative correspondence between lab and field Field: females collected in the field, offspring collected in the laboratory –Genotypes of mothers and progeny are used to estimate female remating incidence and sperm competition Laboratory: artificial selection –Selected for female remating refractoriness and first male sperm representation (select to increase P1, sperm competition defense for which 36DE alleles were previously implicated Clark et al. 1995)

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16 Laying the Field Foundation: Joint Estimate of Second Male Sperm Precedence (P2) and Female Remating Incidence (CMP) Griffiths, McKechnie and McKenzie (1982) –Collected female D. melanogaster from a winery population and harvested their progeny in the laboratory –Genotyped mothers and progeny to jointly infer P2 and CMP (concurrent multiple paternity) a few Adh alleles, hundreds of families maximum likelihood analysis

17 Estimates of CMP and a Sperm Competition Parameter (P2) Griffiths et al. (1982) estimated: –P2 = 0.83, CMP = 21% Problem…

18 D. melanogaster Microsatellite Loci Locus Repeat # Alleles Diversity nanos TA 6 0.88 ula TA 8 0.94

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20 Ravenswood #1: Use Microsatellites to Estimate CMP and P2 Harshman and Clark (1998) Ravenswood sample (females and progeny) Microsatellite genotypes of field females and laboratory progeny (19 families, average 13 progeny per family) Direct enumeration of male gametes among the progeny suggested that the female concurrent remating rate (CMP) was 0.84

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23 The Other Foundation

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25 Selection Experiment Rationale Correlated responses to selection can be informative –Correlated (indirect) responses to selections can reveal trait associations with the direct response …genetic correlations provide a hint about mechanism Do 36DE alleles change in frequency as a result of selection for P1 (female refractoriness to remating and first male sperm representation among progeny)?

26 Ravenswood Winery – Source of the Laboratory Population (R) for Selection Rse - 3 selected lines -selected for female remating refractoriness and first male sperm retention (13-20 days with second males) Cr - 3 control lines - same generation times as Rse NC - 3 control lines - same generation time as during lab adaptation of the R population (before selection)

27 X 24 hours Rse - a set of three selected lines X 13 - 20 days (+) (bw D )

28 paternal proportion of offspring (P1, P2) for each family was determined by scoring progeny eye color in each vial selected families for next gen. if P1 was greater than 50% or if the female did not remate females placed singly into vials for 4 days to lay eggs

29 Family Selection Regime For RSE selected lines –Progeny were collected as virgins (no sibs mating) –Used wild type progeny from approximately 100 families out of approx. 300 families per population (line) for the next generation

30 Cr - a set of three control lines X 24 hours X 13 - 20 days ***(+ males are not replaced w/ bw D )

31 Cr Lines Control lines are treated the same way as Rse, except that the wild type males are removed and added back (no bw D males) –Same generation time (the time males and females were together) –Similar number of families used for the next gen. –Same density in mating bottles –Progeny collected as virgins and among family crosses for the next generation

32 NC females x NC males ( no replacement male) 4 days only! each female placed into a vial for oviposition (4 days) families randomly selected to propagate the next generation NC - ancestral generation time control lines

33 % Females Once-Mated Days with the bw D male: 13……………………………………..20

34 Mean Percentage of Females (±SE) that are Refractory to Remating in Selected (Rse) and Control Lines (Cr, NC) Percent Refractory

35 Mean (S.E.) Lifetime Egg and Progeny Produced Per ONCE-MATED Female

36 36DE SSCP Alleles Four alleles in the Ravenswood population –a (65% in the R#1 base pop.), b, c, d –Similar in relative abundance to the “same” SSCP alleles in Temecula CA and Australian populations

37 abcladder Acp36DE SSCP Alleles

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40 Conclusion 36DE allele frequencies appear to have indirectly responded to selection –One allele, “a”, is possibly associated with female refractoriness to remating

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42 DNA Sequence of the Entire Gene Corresponding to Alleles a, b and c 18 silent substitutions 13 replacement substitutions –4 amino acid changes between “a” compared to “b” and “c” –Duplication of a glutamine and a serine in the “a” allele –A prospective glycosylation site change

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44 Back to the Winery: Second Phase of Field Studies Genotype (36DE SSCP alleles) individual progeny from Ravenswood females to identify paternal 36DE alleles –AND, genotype the mothers and the same progeny for the microsatellite loci to estimate P2 and CMP The Goal: Test for an association between paternal 36DE alleles with CMP (female remating incidence) and P2

45 Preliminary Results of the Microsatellite and 36DE SSCP Allele Survey (Ravenswood #2) Design 28 families, ave. # progeny = 17.1 microsatellite and 36DE SSCP genotype of each individual (mothers and progeny) Data CMP 57% multiple mating (direct enumeration of paternal alleles) Male Genotype and CMP Paternal Female Mating 36DE Once > Once aa 8 6 other 4 10

46 Ravenswood #3 75 families (females and offspring) –Females collected from the field approximately four years after Ravenswood #2 –Mothers and progeny typed by microsatellites –A sub-sample of families were also typed for 36DE SSCP alleles

47 Preliminary Results From Ravenswood #3 Only 9 of 75 females were multiply-mated – A low incidence of multiple mating (compared to 84% in Ravenswood #1 and 57% in Ravenswood #2) The frequency of the “a” allele increased to 0.89 –Within 4 years, the frequency of the “a” allele changed from 65% to 89% in the Ravenswood population 39 families were genotyped for microsatellites and Acp 36DE alleles

48 Female Multiple Mating in Relationship to Male 36DE Genotype (R#3) Paternal Genotype Once-Mated Females Multiply-Mated Females “aa” 19 3 other 12 5 Ravenswood #2 & #3 combined: G = 3.89 (3.84)

49 Ravenswood #2 Ravenswood #3

50 Future Research Finish the full association study of 36DE SSCP alleles in relationship to CMP and P2 (Ravenswood #2, #3) –B. Jones and A. Clark What maintains the Acp 36DE polymorphism in natural populations and in laboratory populations

51 Future Research (Lab) Transform 36DE alleles into a 36DE null background (w/ M. Wolfner) –Measure sperm competition, remating propensity etc. –We have a specific expectation based on association in the field and an indirect response to laboratory selection Prediction: the “a” allele causes remating refractoriness Will natural genetic variation provide insight into how the Acp35DE protein functions?

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53 SNP LINE Acp26s72 5 Acp26s11 87 Acp26s11 88 Acp26s11 91 Acp26s11 96 Acp26s15 52 Acp26s21 93 Acp26s22 01 Acp26s22 02 Acp26s26 00 01A3124222413 01B3221224321 01C4221214321 01D4124214321 01E4124214321 01F3214322321 01G3221214413 01H4221224321 02A3124222413 02B4124214323 02C4214314321 02D3124224323 02E3221214323 02F4224214323 02G3124224321 02H3124224321 03A3124214321 03B4124214321 03C3214324411 03D3214314411 03E4124214321 03F3124224321 03G4124224323 03H3124224321

54 LINEAcp26s725Acp26s1187Acp26s1188Acp26s1187Acp26s1188Acp26s1191Acp26s1188Acp26s1191Acp26s1196 01A312124242 01B322221212 01C422221212 01D412124242 01E412124242 01F321214143 01G322221212 01H422221212 02A312124242 02B412124242 02C421214143 02D312124242 02E322221212 02F422224242 02G312124242 02H312124242 03A312124242 03B412124242 03C321214143 03D321214143 03E412124242 Haplotype

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