Building a Bridge Between Laboratory and Field Studies:

Allelic Variation at a Drosophila melanogaster Male Accessory

Gland Protein Gene (Acp 36DE)

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”

Mating successFecundityLongevity

Egg viability

Larval viabilityPupation success

Developmentalrate

Life History Traits

Drosophila Life History Characters

• Juvenile (Larval) Viability

• Female Fecundity• Female Longevity

• Male Mating Success• Male Longevity

• Sperm Competition, Cryptic Choice of Sperm

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

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)

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”

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

Getting Started (the plan)

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

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)

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

Estimates of CMP and a Sperm Competition Parameter (P2)

• Griffiths et al. (1982) estimated:– P2 = 0.83 , CMP = 21%

• Problem…

D. melanogaster Microsatellite Loci

• Locus Repeat # Alleles Diversity

nanos TA 6 0.88

ula TA 8 0.94

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

The Other Foundation

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)?

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)

X

24 hours

Rse - a set of three selected lines

X

13 - 20 days

(+)

(+)

(+)

(bwD)

• 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

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

Cr - a set of three control lines

X24 hours

X

13 - 20 days ***(+ males are not replaced w/ bwD)

Cr Lines

• Control lines are treated the same way as Rse, except that the wild type males are removed and added back (no bwD 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

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

% Females Once-Mated

G0 G5 G10 G15 G19

Rse 1 5.44 15.4 55.6 72.2 69.5

Rse 2 18.9 53.9 52.8 62.6 66.7

Rse 3 14.2 14.4 53.4 60.7 70.0

Days with the bwD male: 13……………………………………..20

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

Per

cent

Ref

ract

ory

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

Number of eggs Number of progeny Rse 1 756.79 (36.7) 476.48 (25.0) Cr 1 719.88 (31.5) 417.31 (25.9)

Rse 2 725.15 (45.1) 451.68 (29.5) Cr 2 567.28 (35.0) 344.09 (27.8)

Rse 3 906 (61.1) 504.51 (30.4) Cr 3 966.14 (62.14) 421.73 (35.3)

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

a b c ladderAcp36DE SSCP Alleles

20

30

40

50

60

70

80

90

100

0 8 14

Generation

Alle

le F

req

ue

nc

yRse1 Rse2 Rse3

Cr1 Cr2 Cr3

Conclusion

• 36DE allele frequencies appear to have indirectly responded to selection– One allele, “a”, is possibly associated with

female refractoriness to remating

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

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

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 • CMP57% multiple mating

(direct enumeration of paternal alleles)

• Male Genotype and CMP Paternal Female Mating

36DE Once > Once aa 8 6 other 4 10

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

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

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)

Ravenswood #2 Ravenswood #3

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

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?

SNP

LINEAcp26s72

5Acp26s11

87Acp26s11

88Acp26s11

91Acp26s11

96Acp26s15

52Acp26s21

93Acp26s22

01Acp26s22

02Acp26s26

00

01A 3 1 2 4 2 2 2 4 1 3

01B 3 2 2 1 2 2 4 3 2 1

01C 4 2 2 1 2 1 4 3 2 1

01D 4 1 2 4 2 1 4 3 2 1

01E 4 1 2 4 2 1 4 3 2 1

01F 3 2 1 4 3 2 2 3 2 1

01G 3 2 2 1 2 1 4 4 1 3

01H 4 2 2 1 2 2 4 3 2 1

02A 3 1 2 4 2 2 2 4 1 3

02B 4 1 2 4 2 1 4 3 2 3

02C 4 2 1 4 3 1 4 3 2 1

02D 3 1 2 4 2 2 4 3 2 3

02E 3 2 2 1 2 1 4 3 2 3

02F 4 2 2 4 2 1 4 3 2 3

02G 3 1 2 4 2 2 4 3 2 1

02H 3 1 2 4 2 2 4 3 2 1

03A 3 1 2 4 2 1 4 3 2 1

03B 4 1 2 4 2 1 4 3 2 1

03C 3 2 1 4 3 2 4 4 1 1

03D 3 2 1 4 3 1 4 4 1 1

03E 4 1 2 4 2 1 4 3 2 1

03F 3 1 2 4 2 2 4 3 2 1

03G 4 1 2 4 2 2 4 3 2 3

03H 3 1 2 4 2 2 4 3 2 1

LINE Acp26s725Acp26s1187Acp26s1188 Acp26s1187Acp26s1188Acp26s1191 Acp26s1188Acp26s1191Acp26s1196

01A 312 124 242

01B 322 221 212

01C 422 221 212

01D 412 124 242

01E 412 124 242

01F 321 214 143

01G 322 221 212

01H 422 221 212

02A 312 124 242

02B 412 124 242

02C 421 214 143

02D 312 124 242

02E 322 221 212

02F 422 224 242

02G 312 124 242

02H 312 124 242

03A 312 124 242

03B 412 124 242

03C 321 214 143

03D 321 214 143

03E 412 124 242

Haplotype