Transcript Sperm Precedence and Remating Propensity in Female D

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”
Life History Traits
Mating success
Fecundity
Longevity
Egg viability
Developmental
rate
Pupation success
Larval viability
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
nanos
ula
Repeat
TA
TA
# Alleles
6
8
Diversity
0.88
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)
Rse - a set of three selected lines
X
(+)
(+)
24 hours
X
(+)
(bwD)
13 - 20 days
• females placed singly
into vials for 4 days to
lay eggs
• 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
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
X
24 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 - ancestral generation time control lines
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
% Females Once-Mated
G0
Rse 1 5.44
G5
15.4
G10
55.6
G15
72.2
G19
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)
Percent Refractory
80
70
60
50
40
30
20
10
0
Rse1
Rse2
Rse3
Cr1
Cr2
Cr3
NC1
NC2
NC3
Mean (S.E.) Lifetime Egg and Progeny
Produced Per ONCE-MATED Female
Rse 1
Cr 1
Number of eggs
756.79 (36.7)
719.88 (31.5)
Number of progeny
476.48 (25.0)
417.31 (25.9)
Rse 2
Cr 2
725.15 (45.1)
567.28 (35.0)
451.68 (29.5)
344.09 (27.8)
Rse 3
Cr 3
906 (61.1)
966.14 (62.14)
504.51 (30.4)
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
Acp36DE SSCP Alleles
a
b
c
ladder
Rse1
Cr1
Rse2
Cr2
100
Allele Frequency
90
80
70
60
50
40
30
20
0
8
Generation
14
Rse3
Cr3
Conclusion
• 36DE allele frequencies appear to have
indirectly responded to selection
– One allele, “a”, is possibly associated with
female refractoriness to remating
SSCP sequence base number
Acp36DE position
nucleotide
Corresponding amino acid
SSCP sequence base number
Acp36DE position
nucleotide
Corresponding amino acid
SSCP sequence base number
Acp36DE position
nucleotide
Corresponding amino acid
SSCP sequence base number
Acp36DE position
nucleotide
No amino acid difference
a
b
c
37
1867
G
serine
36
1862
T
isoleucine
37
1862
G
serine
a
b
c
183
2013
C
proline
182
2008
C
proline
183
2008
G
alanine
a
b
c
185
2015
T
proline
184
2010
T
proline
185
2010
A
alanine
a
b
c
206
2036
C
205
2031
C
206
2031
G
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
• 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
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?
Acp26s72
5
Acp26s11
87
Acp26s11
88
Acp26s11
91
Acp26s11
96
Acp26s15
52
Acp26s21
93
Acp26s22
01
Acp26s22
02
Acp26s26
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
SNP
LINE
Haplotype
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