male from the 1 Female : 4 Males mating. ACKNOWLEDGEMENTS. Parent. Offspring. M F we 2-19. M F we 2-7.2. F A B B A B B B B A B B B B B A ...
ANALYSIS OF MULTIPLE PATERNITY IN WHITE PINE WEEVIL (Pissodes strobi) USING MICROSATELLITE DNA MARKERS
INTRODUCTION
2. Microsatellite DNA analysis
White pine weevil is the most destructive and economically important pest of young Sitka spruce trees in British Columbia. The weevil attack resulting in stem deformation that dramatically reduce growth rate as much as 40% (Alfaro et al., 1997). Knowledge of the weevil biology is important for implementing effective pest management.
Mendelian Inheritance. Inheritance of the microsatellite markers is a prerequisite for their use in mating studies. The mode of inheritance for the four microsatellite markers used was determined using offspring from the 1 Female : 1 Male mating. An example of the female and male parents as well as offspring genotypes is presented in Figure 2. Segregation analyses of the four loci confirmed their Mendelian segregation fashion.
This study utilized DNA microsatellite markers to investigate if the insect practice multiple paternity in its reproduction. Experimental material consisted of offspring from matings experiment between different female(s) : male(s) combinations.
MATERIALS AND METHODS
Multiple paternity. Replications of the 1 Female : 2 Males and the 1 Female : 4 Males matings were used to examine if multiple paternity is practiced. Paternity assignment was first done by visual comparison of offspring and putative parental bands (Fig. 3). In case that some offspring were indistinguishable, the likelihood based paternity inference program CERVUS (Marshall et al., 1998) was used for paternity assignment.
1. Breeding experiment Virgin white pine weevil females and males were mated in a breeding experiment that simulated the insect natural mating and oviposition conditions. Mating was conducted on Sitka spruce trees that were individually covered with nylon mesh (Fig. 1). The breeding experiment involved three different female : male combinations. These were: 1- 1 Female : 1 Male
Parent
M
we 2-19
Offspring
F
we 2-7.2
Multiple paternity. The majority of the studied replications from the multiple male matings provided evidence that offspring were sired by more than one male. These are illustrated in the following three figures (Fig. 3,4, and 5).
Male A Male C
80
Male B Male D
60 40 20 0 1
2
3 4 5 6 Replication number
7
8
Fig. 5 The percentage of offspring sired by each male from the 1 Female : 4 Males mating.
CONCLUSIONS
RESULTS Mendelian Inheritance. Chi-square analyses showed no significant differences between observed and expected ratio of offspring (based on the Mendelian law of inheritance). An example for the four microsatellite loci studied is illustrated in Table 1.
2. Female weevils could mate with up to four different males. 3. The incidence of multiple paternity would make the development of resistance trees a difficult task. F
A
B B
A
B
B
B
B
Parent
Table 1. Inheritance of four microsatellite markers in Pissodes strobi from four controlled crosses with 1F:1M mating. Primer we 2-7.2 we 2-19
we 3-16
we 3-18
Male
Genotype Female
234 / 234
230 / 234
null / null
289 / null
89 / 99
180 / 194
null / null
97 / 126
F1
No. of offspring observed expected
A
B B
B B
B A B
A
A
Offspring
Fig. 3 Paternity assignment in the 1 Female : 2 Males mating. The first three lanes are parents (F = Female adult, A and B = male adults) and followed by their offspring. The banding segregation revealed that two different males (A and B) sired the offspring.
P
230 / 234 234 / 234
27 25
26 26
0.782
180 / null
27
26
0.782
194 / null
25
26
289 / null
20
26
null / null
32
26
89 / 97
13
13
89 / 126
11
13
97 / 99
15
13
99 / 126
13
13
0.096
0.893
100
Male A
Male B
REFERENCES 1. Alfaro, R.I., R. Brown, K. Mitchell, K Polsson, and
R. McDonald. 1997. In: Decision Support Systems for Forest Pest Management , eds (Shore TL, and McLean DA). Can For Serv, Victoria, BC, FRDA Rep. 260, pp 31-41. 2. Marshall, T.C., J. Slate, L.E.B. Kruuk, and J.M. Pemberton. 1998. Mol. Ecol. 7: 639-655.
80 60 40
ACKNOWLEDGEMENTS
20
This study was funded by a grant from the Forest Renewal B.C. Thank are due to Dr. C.H. Newton (B.C. Research) for the development of microsatellite markers.
0 1
2
3
4
5
6
7
8
9
10 11
Replication number
Fig. 1 White pine weevil breeding experiment conducted on Sitka spruce trees.
100
1. The study demonstrated that multiple paternity is common in white pine weevil.
Percent of offspring sired
Adults and offspring of each treatment were collected and frozen at -80ºC for DNA analysis.
*
Fig. 2 An example of segregation at two microsatellite loci (we 2-7.2 and we 2-19) from the 1 Female : 1 Male mating (M=male and F=female). The first two lanes represent the two parents and followed by their offspring (* = null alleles).
2- 1 Female : 2 Males 3- 1 Female : 4 Males
*
M F
Percent of offspring sired
C. Liewlaksaneeyanawin, Y.A. El-Kassaby, K.G. Lewis, R.I. Alfaro, C. Ritland, and K. Ritland Dept. of Forest Sciences, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4
Fig. 4 The percentage of offspring sired by each male from the 1 Female : 2 Males mating.
