The Journal of Heredity 2002:93(3)
© 2002 The American Genetic Association 93:165-169
Sexual Reproduction in the White Pine Weevil (Pissodes strobi [Peck] [Coleoptera: Curculionidae]): Implications for Population Genetic Diversity
From the Northern Forestry Center, Canadian Forest Service, Edmonton, Alberta, Canada (Lewis), Department of Forest Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (Liewlaksaneeyanawin, Alfaro, C. Ritland, K. Ritland, and El-Kassaby), and Pacific Forestry Center, Canadian Forest Service, Victoria, British Columbia, Canada (Alfaro).
Address correspondence to Yousry A. El-Kassaby at the address above or e-mail: whli{at}uchicago.edu.
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Abstract |
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Controlled mating experiments in the white pine weevil (Pissodes strobi [Peck]) indicated that female weevils either stored sperm or fertilized eggs from one season to the next, and were able to colonize Sitka spruce (Picea sitchensis [Bong.] Carr.) trees without additional mating events. This was interpreted as being beneficial for the insect, in that population establishment in a new habitat could be initiated by dispersing previously mated females without participation of the male. This makes colonization and population/outbreak development more likely as it reduces the need for mate searching in the second season. Paternity identification, based on microsatellite molecular markers, established that the progeny produced in year 2 by females mated only in year 1, were often fathered by more than one male. Multiple paternity, coupled with a lack of parthenogenesis, which was also demonstrated herein, may help to account for the high degree of genetic diversity evidenced in this species.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The white pine weevil (Pissodes strobi [Peck]) is the most damaging pest of young spruce and pines in North America (Figure 1). The main hosts in British Columbia include Sitka spruce (Picea sitchensis [Bong.] Carr.), white spruce (P. glauca [Moench] Voss), and Engelmann spruce (P. engelmanii Parry). Although the white pine weevil has been under intense investigation since the beginning of the 20th century, many questions related to its reproduction remain unanswered. The dynamics of weevil reproduction in this univoltine insect are of interest particularly since McMullen and Condrashoff (1973) and McIntosh (1997) determined that P. strobi can live up to 4 years.
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In British Columbia, its life cycle starts in early spring (late March, April) when adult white pine weevils emerge from overwintering and, after mating, females oviposit in the upper section of the previous year's leader. The larvae mine downward, consuming the phloem, girdling and killing the leader. Pupation occurs in chambers excavated in the xylem. New adults emerge from the leaders in late summer, and when temperatures drop and photoperiod shortens, they go into hibernation in the duff (Silver 1968). During the following season, the new adults and those surviving from previous seasons repeat this cycle. Depending on infestation levels, weevil attack can reduce host tree growth by as much as 40% (Alfaro et al. 1997). Moreover, leader destruction results in stem defects, such as crooks and forks, that reduce wood quality (Alfaro 1989, 1994). The damage of white pine weevil results in losses of up to $500 million/year (Forestry Canada 1993).
Molecular markers such as isozymes, random amplified polymorphic DNA (RAPD), and microsatellites allow us to explore a broad range of questions about population structure and mating systems in many insect species. Isozyme markers were used to study population structure in white pine weevil (Lewis et al. 2000). However, the mean allelic diversity of isozymes was low (2.1 alleles per locus) (Lewis et al. 2000) compared to microsatellites (10 alleles per locus) (Liewlaksaneeyanawin et al. 2001b) in white pine weevil. The applications of microsatellites in insect species have increased over the past few years. At present, according to codominance and high polymorphism, microsatellite markers have proven to be a reliable method for estimating parentage and reproductive success in insects compared with other molecular markers (Simmons and Achmann 2000).
In this article we describe a number of controlled breeding experiments to address questions related to reproduction in P. strobi. Specifically, we wanted to determine if females could produce offspring in two consecutive years with mating restricted to only the first year (i.e., by either storing sperm or fertilized eggs which would ultimately result in the production of a brood). We also want to investigate if there is an effect of female age and sperm and/or fertilized egg storage on offspring sex ratio. Moreover, using microsatellite markers, we wanted to determine if females could produce progeny fathered by more than one male and if parthenogenesis was possible in this species. These questions are important to the population dynamics of the weevil in terms of outbreak development and the maintenance of genetic variation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Since multiple males and females are found per leader in nature (Overhulser and Gara 1975a; McIntosh 1997), we had to set up specific crosses and make observations of individual weevils to discern between possible parthenogenesis, storing sperm or fertilized eggs, and multiple paternity using breeding experiments. Breeding experiments were conducted over a period of 3 years starting in 1997. Virgin female and male weevils, used in the spring of each year, were obtained the previous fall by clipping weevil-infested leaders from sites on the east coast of Vancouver Island. After clipping, the leaders were placed in rearing cages and the callow adult were collected after emergence. Immediately after being captured, the gender of each weevil was determined by the Harman and Kulmann (1966) method. Separate virgin female and male colonies were maintained throughout each winter. In all experiments, weevils were caged on weevil-susceptible Sitka spruce approximately 2 m high using nylon mesh: the entire tree was encased so that the weevils could freely wander up and down the tree as they do under natural conditions.
To address the possibility of parthenogenesis in this insect, an experiment was performed in 1998. In the spring, solitary, virgin female weevils (10 replicates) and solitary, virgin male weevils (10 replicates) were caged on trees. In late July, close to the time the larvae should have completed development, the caged trees were cut down and brought into the laboratory, where the adult weevils were retrieved and the leaders of each tree cut. If oviposition punctures were present, the leader was placed in a rearing tube for retrieval of the offspring.
To investigate the production of offspring in the second season and the effect of sperm and/or fertilized egg storage on offspring sex ratio by females that had successfully produced a brood during their first season, an experiment conducted over a 2-year period was performed. Starting in the spring of 1998, 50 replicates of single virgin female (collected in the fall of 1997) were caged on trees with two virgin male weevils. After mating and egg laying, and close to the time the brood would be emerging, the trees were cut down and brought to the laboratory and treated similarly to the parthenogenesis experiment. Because of summer mortality (and possible escape), only 35 of the 50 female-parent weevils, along with their putative male mates, were captured alive. The retrieved female parents from these 35 replicates were placed in separate containers containing spruce branches for food and moisture. During the fall of 1998, the 35 surviving, solitary female parents overwintered in a shadehouse. Food (lateral branches from Sitka spruce) was periodically changed throughout the winter to ensure a fresh supply of nutrients and moisture.
Eighteen of the 35 one-year-old females survived the winter (i.e., 49% overwintering mortality). However, only 11 of these had successfully produced a brood in the previous year (1998). Thus, in the spring of 1999, these 11 surviving females were again caged on Sitka spruce trees. Eight of the surviving 11 females were caged alone, without potential mates (treatment 1; Table 1); the remaining 3 were each caged with two virgin males (treatment 2; Table 1).
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To address the effect of female age on offspring sex ratio, two additional treatments were included in 1999. In the first treatment (four replications), virgin females, which had emerged from leaders in September 1998 and were maintained throughout the winter, were caged with two virgin males (treatment 3). In the second treatment (four replications), virgin females, which had emerged from leaders in September 1997 and were maintained as virgins throughout two winters, were caged with two virgin males (treatment 4). All the males used during the 1999 breeding experiments were obtained in the fall of 1998. As in previous years, in the late summer of 1999 the trees were cut down and brought into the laboratory, where the adults were retrieved and the leaders placed in rearing tubes for collection of offspring. All parent weevils were subsequently placed in individual sterile plastic Eppendorph tubes, quick frozen in liquid nitrogen, and maintained at -80°C. Offspring of each treatment were collected from rearing tubes daily as they emerged.
Five replications from treatment 1 (females mated in 1998 with no males in 1999), which had produced offspring in 1999, were selected to examine paternity in the white pine weevil using microsatellite molecular markers (Table 2). After the gender of each 1999 offspring was determined, the weevil was treated identically to that of the female parent, in that they were placed individually in sterile plastic Eppendorph tubes, quick frozen in liquid nitrogen, and maintained at -80°C for microsatellite DNA analysis. Total genomic DNA was extracted from the weevil using the procedure described in Boyce et al. (1989), with slight modification. Three microsatellite markers (we2-7.2, we2-19, and we3-18) along with the polymerase chain reaction (PCR) conditions were used in this study (Liewlaksaneeyanawin et al. 2001). The Mendelian segregation of the microsatellite markers in accordance with the hypothesis of null alleles were confirmed (Liewlaksaneeyanawin et al. 2002).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Each of the four treatments in the 1999 experiment resulted in the production of weevil offspring (Table 1). In 1998, the eight females from treatment 1 produced offspring showing a mean sex ratio of 1.19 (male:female). More importantly, six of the eight replicates in treatment number 1, wherein mating was restricted to the previous season, produced offspring in 1999, with a mean sex ratio of 1.01. The remaining two females did not produce any offspring (Table 1). These findings support the observations of Overhulser and Gara (1975a,b), who also observed that female P. strobi, once successfully mated, do not have to mate again to lay fertile eggs the following spring. Two possible mechanisms could explain these results: (1) fully fertilized eggs are stored over the winter, and/or (2) sperm are stored in the weevil's spermatheca, with fertilization occurring sometime prior to oviposition during the following spring. Furthermore, our work expands upon Overhulser and Gara's (1975b) study in definitively establishing that the fertile eggs laid in the second season can successfully develop into adults.
As in treatment 1, the group of females mated in the first season and again in the second season (treatment 2) also produced offspring in each year, with mean sex ratios of 0.91 and 1.38, respectively (Table 1). Females that were maintained as virgins for one or two seasons before mating in 1999 (treatments 3 and 4) produced offspring with sex ratios of 1.27 and 1.32, respectively (Table 1). Thus these experiments indicate that the offspring sex ratio from individual P. strobi is independent of female age at the time of mating and that sperm and/or fertilized egg storage did not significantly affect sex ratio. While we report the offspring sex ratio of individual female weevils under controlled conditions, these ratios are within the range of those obtained from natural populations consisting of many females. For instance, Overhulser and Gara (1975a) reported the male:female sex ratio of newly emerged field-collected weevils as 1.56 in 1971 (total of 347 weevils) and 0.86 in 1972 (total of 389 weevils).
Parthenogenesis seems unlikely since not one of the replicates in 1998 containing either solitary, virgin female or male weevils produced offspring. Oviposition punctures were entirely absent as well, indicating that unfertilized eggs were not produced or laid. This suggests that parthenogenesis is highly unlikely in P. strobi and that mating between males and females must occur for offspring to be produced.
The analysis of the three microsatellite loci from the five replications (treatment 1—females mated in 1998, no males in 1999; Table 1) also confirmed that there was no parthenogenesis in this insect (Table 2). Microsatellite markers have been successfully used in detecting parthenogenesis in Sitobin aphids (Sitobion miscanthi) (Sunnucks et al. 1996). Since males which previously mated to females in 1998 were not available, we used microsatellites to infer male genotypes and assign paternity. With the known mother-offspring genotypes, and the combinations of the three microsatellite loci, the possible male genotypes were inferred and paternity was assigned to each individual offspring (see example in Figure 2 and Table 2). Three of the five studied replications (1, 4, and 7) indicated that only one male succeeded in siring all the offspring, while the remaining two replications (3 and 6) provided evidence that offspring were sired by two males (Table 2). The use of microsatellite markers was effective in demonstrating the lack of parthenogenesis and the presence of multiple paternity. The three microsatellite loci used were sufficient to successfully assign paternity to the 213 offspring analyzed.
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Observations of weevil activity over the season (Alfaro et al. 2000) indicate that most P. strobi mating events on Sitka spruce occur during a relatively short period (narrow biological window). This is initiated when heat sum accumulations reaches approximately 200 degree-days, and ends at approximately 350 degree-days (accumulated from April 1, with a threshold of 5°C). In coastal British Columbia, these heat sum accumulation limits are commonly reached between the first and last week of May, respectively. Occasionally, however, mating pairs were observed well into mid-June (
500 degree-days). Thus P. strobi females seem to be able to store sperm and/or eggs fertilized from one or more males from midsummer through fall and winter, and are able to lay viable eggs in the following spring without additional mating. This has interesting implications for population outbreak development, as it implies that dispersing fertilized females, carrying a sperm load from various males, could initiate colonies without the participation of males. Thus a female arriving in a new habitat would have no need to "call in" a male, thus reducing the need for pheromone communication. This is reinforced by the fact that several researchers have attempted to demonstrate sexual attraction in P. strobi, with limited success. It is likely that mating in this insect involves only random encounters in the limited space of the leader, where most mating events take place. Weevils may simply move to the top-most shoot (leader) and search for new mates by random encounters. If the leader is empty, they may search for mates in other trees. The fact that female weevils need not mate in each year to produce offspring if they mated with one or more males during the previous season, along with the lack of parthenogenesis, could help to explain the high degree of genetic variation seen within populations of this insect (Lewis et al. 2000). Immigrating females into new habitats or ongoing outbreaks bring a foreign gene pool, thus providing a mechanism for effective gene flow. These findings shed new light on the population dynamics and genetic diversity of this important pest of spruce and pines in North America. Also, this study provided the evidence that there is no effect of female age and sperm and/or fertilized egg storage on offspring sex ratio. The offspring sex ratio is important for an understanding of population expansion and for predicting the number of sterile males needed for control whenever the sterile insect technique is recommended.
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Acknowledgments |
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We sincerely thank D. Brescia, D. Andrucko, P. Blake, A. Shand, D. Levesque, G. Brown, and L. Van Akker for assistance throughout the caging experiments. This work was partially funded through Forest Renewal BC grants HQ96062-RE and HQ96251-RE (to Y.A.E., K.G.L., and R.I.A.).
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Footnotes |
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Corresponding Editor: Ross MacIntyre
Received November 6, 2000
Accepted November 27, 2001
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References |
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