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Genetics of sex ratio and other life history traits in the two-spotted spider mite( Tetranychus urticae Koch)
Hales, Lavinia Anne, Ph.D.
The Ohio State University, 1994
UMI 300 N. ZeebRd. Ann Arbor, MI 48106
GENETICS OF SEX RATIO AND OTHER LIFE HISTORY TRAITS IN THE
TWO-SPOTTED SPIDER MITE (TETRANYCHUS URTICAE KOCH)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Lavinia Anne Hales, B. S., M. S.
The Ohio State University
1994
Dissertation Committee: Approved by
D. L. Wrensch
D. E. Johnston A dviser B. Smith Department of Entomology To my parents.
i i ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my adviser, Dr.
Dana Wrensch, for her guidance through this difficult project and support when graduate school got to be overwhelming. I would like to express my gratitude to Don Yehling for his invaluable help with the laboaratory equipment. My thanks also go to the members of my reading committee, Drs. Don Johnston and Brian Smith, who were patient and understanding through last minute corrections and scheduling changes. I also wish to thank the Department of
Entomology for providing much needed financial support. VITA
November 23, 1961 ...... Born - Smithville, Missouri
198 4...... B. S., Portland State University, Portland, Oregon
1987 ...... M. S., Oregon State University, Corvallis, Oregon
1987-1989 ...... Teaching A ssistant in Molecular Genetics at The Ohio State University, Columbus, Ohio
1989-1993...... Teaching Assistant in General Biology at The Ohio State University, Columbus, Ohio
PUBLICATIONS
Hales, L.A., T. F. Savage, and J. A. Harper (1989). Heritability of semen ejaculate volume in medium white turkeys. Poultry Science, 68: 460-463.
PRESENTATIONS 1986, "Heritability of semen quality in medium white turkeys " Presented at PEPA (Pacific Egg and Poultry Association) in a competition.
1991, "Heritability of sex ratio, fecundity and developmental rate in the two-spotted spider mite." Poster, Presented at the ESA (Entomological Society of America) National Conference in Reno, Nevada. FIELDS OF STUDY
Major Field: Entomology Studies in Acarology and Quantitative Genetics.
v TABLE OF CONTENTS
ACKNOWLEDGMENTS...... iii
VITA ...... iv
LIST OF TABLES ...... ix
INTRODUCTION ...... 1
Life History Traits ...... 1 Sex Ratio Evolution...... 3 Reproductive Biology ...... 10 Mating Systems in Acari ...... 14 Rationale and Significance of Experiments...... 20
GENERAL METHODOLOGY...... 24
Mite Rearing ...... 24 Statistical Analysis ...... 28 Heritability Estimations ...... 29
CHAPTER PAGE
I. SCREENING FOR THE POSSIBLE INCIDENCE OF SPONTANEOUS THELYTOKY IN LABORATORY POPULATIONS OF THE TWO-SPOTTED SPIDER MITE (Tetranychus urticae Koch) ...... 37
Introduction...... 37 Materials and Methods...... 40 Results ...... 41 Discussion ...... 44 List of References ...... 49
vi 11. EVIDENCE THAT MALES DO NOT INFLUENCE THEIR MATE’S FECUNDITY OR PROGENY SEX RATIO UNDER NORMAL LABORATORY CONDITIONS ...... 50
Introduction...... 50 Materials and M ethods...... 52 Results ...... 55 Discussion...... 57 List of References ...... 60
III. HERITABILITY OF DEVELOPMENT RATE, FECUNDITY AND SEX RATIO IN A HAPLODIPLOID MITE ...... 62
Introduction...... 62 Materials and Methods...... 68 Results ...... 73 Discussion ...... 76 List of References ...... 86
IV. HERITABILITY OF FECUNDITY, DEVELOPMENT RATE AND SEX RATIO IN TWO GENERATIONS OF MITES SUBJECTED TO DIFFERENT TEMPERATURE REGIMES (25oCand32oC) ...... 87
Introduction...... 87 Materials and Methods...... 92 Results ...... 97 Discussion ...... 102 List of References ...... 108
DISCUSSION ...... i...... 113
vii APPENDICES ...... 118
A. Analyses of Variance of Fecundity and Sex Ratio by Sire ...... 118
B. Analyses of Variance of Development Rate, Fecundity and Sex Ratio by Replicate for the Control Population (25<>C) ...... 121
C. Analyses of Variance of Development Rate, Fecundity and Sex Ratio by Replicate for the Hot Population (32°C) ...... 125
BIBLIOGRAPHY ...... 129
4
viii LIST OF TABLES
TABLE PAGE
1. Number of replicates (families) with all-female progenies. Number of daughters here represents the total number of adult offspring ...... 42
2. Progeny tests of the daughters in all-female families shown in Table 1. Tray and replicate (Rep), presence of sons (S) and daughters (D). Female progeny from each leaf disk were either left unmated or provided with a male mate from another leaf disk ...... 43
3. Number of all-daughter families observed among the various experiments using the Columbus ‘89 stock culture. In each case, only those females that failed to oviposit were deleted from the total sample. Sample size of total families in each experiment, the largest all-female progeny number in the sample, and the percentage (%) of the sample with all-female families are reported for ten different experiments...... 47
4. Sample sizes (N), m eans and starndard errors (SE) for fecundity and sex ratio. Both experimental trials are reported separately ...... 56
5. The results of a one-way analysis of variance (ANOVA) of fecundity and sex ratio between different sires. P-values are reported separately for each experimental trial. Complete ANOVAs in Appendix A...... 56 6. Number of all-son families observed among the various experiments using the Columbus ‘89 stock culture. In each case, only those females that failed to oviposit were deleted from the total sample. Sample size of total families in each experiment and the percentage (%) of the sample with all-female families are reported for ten different experiments...... 59
7. Means and standard errors (SE) for fecundity, development rate (DR: egg to adult in days) and sex ratio (SR: proportion female) in the progenies of P and O generations. Two-sample, unpaired t-tests were used to test for differences between generations. The same letter in a row indicates that the means are not significantly different between parent (P) and offspring (O) generations at the a=.05 level. Different letters in a row incicate that the means are significantly different at a = 0.01. Sex ratio was transformed for calculating the t-tests ...... 74
8. Heritabilities (h2) and standard errors (SE) for fecundity, development rate and sex ratio using four different methods of calculation: full sib correlation, regression of parent (P) on individual offspring (O), regression of parents on mean offspring and regression of parents on weighted mean offspring...... 75
9. Correlations between fecundity (F), development rate (DR) and sex ratio (SR) within parent and offspring generations...... 77
10. Number of families (N), means and standard errors (SE) of fecundity, developement rate (DR) and sex ratio (SR) for mites that developed and oviposited at control (25°C) or treatment (32oC) temperatures for both parent (P) and offspring (O) generations. Different letters in same row designate that means are significantly different at the ot=0.05 level ...... 98
x 11. Summaries of a one-way analyses of variance for fecundity (F), development rate (DR) and sex ratio among replications within control (25oC) and treatment (32oC) temperatures. Complete analyses found in Appendices B and C......
12. Heritability estimates in both treatment (32°C) and control (25°C) environments. Methods of calculating the estimates are: full sib correlation (Full Sib), parent-mean offspring regression (Par-MnOff), and a weighted parent-mean offspring regression (Par-WtMnOff). N = The sample size, h2 = The heritability estimate, SE = The standard error of the heritability estimate. If the ANOVA or slope of the regression was not significantly different from zero, NS (not significant) is denoted instead of a heritability estim ate...... INTRODUCTION
Life History Traits in Tetranychus urticae The two-spotted spider mite (Tetranychus urticae Koch) is an important pest of many major crop species worldwide. They are opportunistic colonizers that efficiently exploit patchy environments. Their high intrinsic rate of increase (rm) contributes greatly to their pest status, because this allows for sudden outbreaks of previously uninfested areas. Dense, growing mite populations cause severe plant damage and economic injury. Some life history factors which influence a mite population's ability to increase are fecundity, hatchability, duration of oviposition, longevity, rate of development, survivorship, and sex ratio (Wrensch, 1979). Local environmental factors also greatly influence the rate of increase of Tetranychus species. Important factors include host plant characteristics (such as variety, age, nutrition and water balance of the leaves), crowding, incidence of predators and pathogens, temperature, and humidity (Gutierrez and Helle, 1985; Wrensch, 1985). Among fitness traits, development rate (DR) has been shown to have the greatest individual impact on the intrinsic rate of increase (Wrensch and Young, 1975). Temperature affects all rate-dependent life history traits, but is especially influential on the developmental rate and thus has a profound effect on a population’s ability to
1 2
increase rapidly (Wrensch, 1985). A curvilinear relationship was found between temperature and development rate Tetranychus in mcdanieli (Crooker, 1985). Development rate decreased rapidly as the temperature increased in the 10-20°C range, decreased gradually in the 20-33°C range, but increased sharply in the 33-38°C range (Crooker, 1985). The best temperatures for the most rapid growth of most spider mite species have been shown to be between 24-29°C (Boudreaux, 1963), with the minimum temperature required for development of 10°C (Herbert, 1981). Wrensch and Young (1978) have shown that development rate is also sensitive to host quality at constant temperatures of 22°C. Fecundity has been shown to contribute substantially to individual net fitness of spider mites (Young and Wrensch, 1983; Wrensch and Young, 1975) and to have an extremely important affect on a population's ability to increase (Wrensch, 1985). In fact, fecundity was second only to developmental rate in determining a population’s rm and was the most important factor influencing individual fitness (Wrensch and Young, 1975). It may be highly sensitive to environmental conditions (Wrensch and Young, 1975). Daily fecundity has also been positively correlated with temperature (Hazan et al., 1973). Resource quality and crowding also have played an important role in influencing the number of eggs a female lays (Wrensch, 1979; Wrensch and Young, 1975; Wrensch and Young, 1978). Lifetime fecundity is also influenced by duration of oviposition and longevity of the female (Wrensch, 1979). Although these two traits are highly correlated, there is some indication that longevity may be controlled by the length of oviposition, with 3
females dying soon after all their eggs have been laid. Therefore, length of oviposition may not have an independent influence on the lifetime fecundity, relative to other factors, and therefore should have a low influence on rm and fitness (Wrensch and Young, 1975). Survivorship is also sensitive to the environmental conditions in which the mites develop and oviposit (Wrensch, 1979). Hazan et al. (1973) showed that mortality of immature mites increased with extreme temperatures and high humidities. Crowding has drastically decreased survivorship (Wrensch and Young, 1978; Davis, 1952). Another major environmental influence is resource quality. Wrensch and Young (1978) found that survivorship was much better when mites were raised with good leaf quality. Even the quality of resource during maternal maturity influences her offsprings’ mortality rates (Wrensch and Young, 1978; Wrensch, 1983). it is well known that different host plants generate differences in fecundity, development rate, and longevity (Crooker, 1985). Host plant resistance, to mite damage, has been utilized in some agricultural systems (de Ponti, 1985; Rodriguez and Rodriguez, 1987). Host plant preference by T. urticae has been demonstrated to involve odor, taste (Rodriguez and Rodriguez, 1987) and phototaxis to dark green (McEnroe and Dronka, 1971).
Sex Ratio Evolution The proportion of daughters in a female's progeny (sex ratio) is a key trait in the population biology of spider mites. Spider mites are colonizing species and only the teneral, inseminated females disperse (Wrensch, 1979). Sex ratio is typically strongly female- biased. The standard explanation for female bias has been that it behooves the female mite to produce as many daughters as possible while having enough sons to ensure insemination of those daughters. That way, the probability of successful colonization increases. Progeny sex ratio is considered to be at some ecological maximum. However, the explanation is more complex because spider mites regulate the proportion of females in response to environmental cues, such as leaf quality (Young et al., 1986) and temperature (Wrensch, 1993), changing both the mean and the variance of sex ratios. This regulation is achieved by a daily control of fertilization by ovipositing females, such that a fixed fraction of eggs is fertilized each day (Young et al., 1986) Females will produce comparatively more sons in deviant environments (differing from optimal rm). This is a highly adaptive character because it allows for more haploid recombinants and therefore a more rapid adaptation to the new environment (Wrensch et al., 1994). Theoretically, haploid males allow for an increased rate of evolution (Hartl, 1971; Griffing, 1982). Young et al. (1986) found differences in the sex ratios, as a response to poor leaf quality, and among four different geographic strains of spider mites. The differences among strains were consistent for three consecutive generations. These results indicate the possibility of a genetic basis for the ability to control sex ratio. Local mate competition (LMC), proposed by Hamilton (1967), predicts a female biased sex ratio not only to increase the probability of successful colonization, but also because brothers compete for mates. Maynard Smith (1976) introduced the idea that 5 sex ratio can be thought of in terms of an evolutionary stable strategy (ESS) and thus provided a theoretical framework to study this life history trait (Wrensch, 1993). An important prediction of the LMC hypothesis is that females should be able to produce relatively more males when the opportunity for outcrossing with non-related individuals increases (Parker and Orzack, 1985). This ESS model has been interpreted as a method of maximizing rm within the local population (Wrensch, 1993; Charlesworth and Williamson, 1975). No single strategy will be optimal since the relative fitness of each sex must be maximized over continually changing resource quality (Charlesworth, 1980; Charlesworth and Williamson, 1975). After all, the colonized patch will continue to deteriorate over time since mites do not have density dependent reproduction and therefore will eventually overrun their resource (Wrensch, 1993). An extensive review of Hamilton’s (1967) theory, including a discussion on its shortcomings in both theory and predictions, has been recently published by Wrensch (1993). The first major prediction of this theory is that the primary sex ratio should be female biased. Reproduction should be haplodiploid (arrhenotokous, pseudoarrhenotokous = parental genome loss). There should be at least one male in each progeny set. Siblings should remain gregarious throughout development (egg to adult). Also the males should be both protandrous (develop faster or emerge first) and polygynous (mate multiple times). A fifth prediction is that the females should mate soon after eclosion and before dispersal. Also, males should not migrate to other patches. The final major prediction of Hamilton’s (1967) model is that females must be able 6 to store enough sperm to fertilize her entire brood with only one mating (Wrensch, 1993; Hamilton, 1967). These predictions can usually be easily tested with spider mites, and shortcomings of Hamilton’s (1967) model elucidated. Wrensch (1993) brought up several challenges to Hamilton’s ESS model. One such problem is that the ESS does not explain the great heterogeneity of individual progeny sex ratios (Young et al., 1986; Young et al., 1985). The model would predict that progeny sex ratios within a population should be similar. Another problem with the ESS model is that fatal injuries are quite common as male mites fight over quiescent deutonymphs, including closely related males such as full brothers (Wrensch, 1993). Hamilton’s model also does not consider the control by females on the sperm allocation and hence the control of fertilization rates. Females fertilize a previously ‘imprinted’ fixed fraction of their eggs each day, depending on their ovipositional and developmental environments. This allocation method maximizes the number of daughters rather than the fraction of daughters produced (Young et al., 1986) thus daily sex ratio control does not follow either the assumptions nor the predictions of optimality theory (Wrensch, 1993; Orzack et al., 1991). The new ESSnet (Wrensch, 1993) model proposes that the progeny sex ratio of a female haplodiploid will be a trade off between maximizing the intrinsic rate of increase, rm (by increasing the proportion of females) and maximizing the selection coefficient Sm (by increasing the proportion of males). Whether the female will 7
lean towards the rm or the Sm strategy will depend upon the environment in which she has matured as well as her current environment. Sex ratio regulation may now be thought of in terms of an optimization strategy over the colonizing episode of a mite population (Wrensch, 1993). The rm part of the new ESSnet model is similar to the original ESS model proposed by Hamilton (1967). Since the mite is a colonizing species and only the females disperse (Wrensch, 1993), females will increase the probability of successful colonization by increasing the number of fertilized daughters within their progeny. Therefore, in a relatively good environment, females will maximize their rm by having relatively more daughters, following the predictions made by Hamilton (1967), Charnov (1982) and others. The Sm part of the ESSnet model differs from the earlier ESS models. Since mites are colonizing species, and good environments enabling maximizing their rm are transitory, these opportunistic
colonizers must exploit a succession of hosts or substandard patches (Wrensch, 1993). Because of predictable environmental heterogeneity, they must regulate the proportion of females in response to environmental cues, such as leaf quality (Young et al., 1986) and temperature (Margolies, 1987). Regulation will result in changes in both the mean and the variance of sex ratios and consistently shifts sex ratio in only one direction (Wrensch, 1993). Females will produce comparatively more sons in deviant environments (Young and Wrensch, 1981; Wrensch and Young, 1975; among others). As stated earlier, this would be a highly adaptive 8 character since it would allow for relatively more haploid recombinants in each progeny. The increase in the proportion of males would correspond to a direct exposure of products of meiotic Prophase I. Sons, then, would serve to screen haploid genomes and also to expose recombinants to selection. Therefore, a more rapid adaptation to the new environment would be assured (Wrensch, 1993). Theoretically, haploid males would allow for a more rapid rate of evolution (Hartl, 1971; Griffing, 1982). Males play a very important part in this new theory. They are not just viewed as the “cheaper” sex as argued by cost-of-sex theorists (Fisher, 1930; Charnov, 1982), but rather as a highly adaptive and indispensable component in the arrhenotokous mites’ ability to colonize and utilize patchy and deteriorating environments. Since males are haploid, their entire genome is exposed to the environment, especially during early development, and thus males are subjected to more intense selective pressures because all alleles are hemizygous (Wrensch, 1993). Furthermore, males remain on the natal patch and mate repeatedly. Two major predictions follow from this reasoning. Because males from previous generations well still be available for inseminating females later in colonization, the functional sex ratio should decrease (proportion of males within the population increases) with colony age (Potter et al., 1976). The second important prediction is that there will be very intense selective pressures placed on the available males. As the size of the colony increases with age, the number of females available for fertilization increases arithmetically. Females are not receptive to males after they have 9 been inseminated (Potter and Wrensch, 1978) and most will disperse as tenerals. This contrasts sharply with the geometric increase in the number of males, and their heightened numbers will result in very fierce competition for the limited resource of quiescent deutonymphs, thus they are under intense selective pressures by the end of a colonizing episode. Therefore, males can be seen as serving two major functions. The first is for haploid screening (fixing and eliminating alleles), and the second is as units of recombination of their mother’s genotype and therefore a mechanism for facultative genetic recombination of females. By altering their sex ratio to produce more males in deviant environments, females may be viewed as increasing the additive genetic variance exposed to selection and thus producing higher quality of mates for their daughters at the cost of a small portion of their rm. Since colonizers, such as spider mites, ultimately use up their hosts, sex ratio control can be viewed as an integral part of a colonizing episode. Therefore, sex ratio regulation may be viewed as a form of adaptive plasticity in which haploid males are a key element for increasing the amount of additive genetic variance exposed under deteriorating host quality (Wrensch, 1993). Little is known about the genetics of sex ratio of any organism (Parker and Orzack, 1985). Overmeer and Harrison (1969) concluded that progeny sex ratio was based on female control rather than sperm load and speculated that the differences in the progeny sex ratios of different females were due to genetic polymorphisms. Mitchell (1972) showed stability in extreme sex ratio phenotypes taken from the wild populations, indicating that genetic 10
determination of sex ratio was likely. Young et al. (1986) found differences in progeny sex ratios in response to four leaf quality regimes and among four different geographic strains of spider mites. Within strains, sex ratio was consistent for three consecutive generations. These findings suggest the possibility of a genetic basis for the ability to control sex ratio. Previous attempts to estimate the degree of the genetic contribution of sex ratio in haplodiploids have given mixed results. Ram and Sharma (1977) were not able to select for increased proportions of females in Trichogramma fasciatum. Others have been successful in selecting for changes in the progeny sex ratio (Parker and Orzack, 1985). Parker and Orzack (1985) successfully selected for an increased proportion of males in the wasp Nasonia vitripennis, and obtained realized heritability estimates for sex ratio of 0.11 and 0.13 in different lines. Of these few experimental studies on heritability or sex ratio selection, it is of great significance that only decreases in the proportion females were possible even when efforts were made to select for increased proportion of females. Takafuji and Ishii (1989) obtained a heritability estimate of 0.85 using mother-daughter regressions in Tetranychus kanzawai. The following research examines the genetics of sex ratio and provided important empirical for understanding allocations in haplodiploids.
Reproductive Biology in Spider Mites To understand sex ratio as a fitness trait it would be helpful to understand the mechanisms of gametogenesis and fertilization. The true mechanism for fertilization and sex ratio control remains unknown (Helle and Pijnacker, 1985) although some information does exist and theories abound. It is relatively easy to explain why the first eggs of a progeny set tend to be male. This may be due to these eggs being too mature for sperm penetration at the time of copulation (Feiertag-Koppen and Pijnacker, 1985). One possibility for the control of sex ratio is that there is less sperm in the ventral region of the ovary so some oocytes in the medio-ventral area might escape sperm penetration (Helle and Pijnacker, 1985). Feiertag- Koppen and Pijnacker (1985) have noticed that a single spermatozoan appears to enter the oocyte by way of the nutritive chord near the end of the yolk accumulation stage of oogenesis. At this point, the spermatozoan becomes torpedo-shaped and then remains near the oocyte until oviposition of this egg (Feiertag- Koppen and Pijnacker, 1985). Therefore, females might be able to control sex ratio by regulating the membrane permeability of the nutritive chord; with a more permeable membrane, the spermatozoan might more readily enter the chord and fertilize the egg. Another possibility is for the female to control sperm entry into the egg by altering the permeability of the oocyte’s plasma membrane. The female reproductive system is the second to largest organ system found in spider mites. It comprises most of the mite’s ventrum and consists of both germ and somatic cells (Alberti and Crooker, 1985). This large organ system consists of a single meroistic ovary, a short vagina, the oviduct and the seminal receptacle (Feiertag-Koppen and Pijnacker, 1985). The ovary is comprised of both somatic and germ cells (Alberti and Crooker, 1985; Feiertag-Koppen and Pijnacker, 1985). The somatic portion of 12
the ovary is the ovary wall, the nutritive cells and the support cells. The nutritive cells provide the developing oocytes with yolk and glycogen during vitellogenesis (Alberti and Crooker, 1985). The lumen of the oviduct is lined with a single layer of epithelial cells. Although some secretions occur within the oviduct, that produce a lubricant in the anterior portion, their function is largely unknown (Alberti and Crooker, 1985). The vagina is short with a thin chitinous lining and a genital flap opening to the posterior oviduct (Alberti and Crooker, 1985). The seminal receptacle lies along the most posterior portion of the female’s reproductive system. It is not directly connected to the oviduct nor the vagina (Alberti and Crooker, 1985), yet sperm migrate to the ovary despite the lack of a direct link. The copulatory pore (bursa copulatrix) leads directly to the seminal receptacle (Alberti and Crooker, 1985). Sperm are stored in the lumen of the sac-like portion of the seminal receptacle. The epithelium in this part of the receptacle consists of the simple columnar variety. Many small vacuoles are present in the epithelium, with a denser distribution being found more basally. Apically, electron dense droplets are present. If sperm are present, they tend to be ovoid and with numerous fingerlike projections and filaments or tubules (Alberti and Crooker, 1985). In T. urticae, male gametogenesis occurs by a single, equational division of the haploid spermatocyte. Spermatogenesis occurs in the sac-like testes of male mites. Mature sperm contains highly condensed DNA with a small amount of dark granular cytoplasm. This DNA-cytoplasmic mixture is tightly enclosed within two membranes, resulting in a small spherical spermatid 13
lacking acrosomes, flagella and organelles. The seminal vesicles, located anteriorly to the testes, hold the mature sperm within a glandular matrix of cells. The mature sperm become amoeboid around the time they leave the vesicles. Sperm is moved through the vas deferens and into the sperm pump by contraction of the muscles lining the vesicles. During copulation, sperm is transferred, one at a time, to the copulatory pore of the female via the adeagus (Pijnacker, 1985). All sperm produced by any individual male are genetically identical (Wrensch et al., 1994). Approximately 250 sperm (242±28, n=10) make their way to the female’s seminal receptacle immediately after copulation, a sufficient number to fertilize an entire brood given the normal fecundity ofT. urticae fem ales (Pijnacker, 1985). The sperm quickly associate themselves with the columnar epithelial cells within the seminal receptacle and pass through these cells within 24 hours (Feiertag-Koppen and Pijnacker, 1985). The next day, most sperm are individually scattered near the ovary. They have also returned to a spherical shape. Sperm distribution becomes more dense in the dorsal region of the ovarian cavity, and many lie near nutritive chords. A single spermatozoan will enter the nutritive chord and then penetrate the egg toward the end of yolk formation if fertilization will occur. They become torpedo-shaped as they remain close to the oocyte until oviposition (Feiertag-Koppen and Pijnacker, 1985). Meiotic arrest at Prom etaphase or M etaphase I in the oocyte is not broken until approximately 2.5 hours after oviposition. Syngamy in fertilized eggs ensues shortly after germinal vesicle breakdown and formation 14 of the female pronucleus (Feiertag-Koppen and Pijnacker, 1985). An inseminated female typically carries the sperm of just one male (Potter and Wrensch, 1978) and this is sufficient for lifetime fecundity of fertilized eggs (Pijnacker, 1985).
Mating Systems in. Acari There are three major modes of reproduction found within Acari. These are diplodiploid sexual reproduction, haplodiploid arrhenotoky and thelytoky (Norton et al., 1993). Diplodiploid sexual reproduction occurs when both males and females are of the 2n genotype and females cannot produce offspring parthenogenetically (without fertilization). Males are usually the heterogametic sex in Acari. With haplodiploid arrhenotoky, males are produced parthenogenetically from unfertilized eggs and are haploid, and females are produced from fertilized eggs and are diploid. Thelytokous reproduction occurs when female progeny are produced parthenogenically and males are not needed within the population. Occasionally, spanadric (rare) males appear in thelytokous lineages, but these males appear to be largely nonfunctional. Within Tetranychidae arrhenotoky dominates, but diplodiploid sexual reproduction is common within other families of Prostigmata. Thelytoky is relatively rare with only one representative (Oligonychus thelytokous) within the subfamily Tetranychinae (Norton et al., 1993). Thelytoky is more common within Bryobiinae, a more distantly related subfamily of Tetranichidae (Norton et al., 1993). 1 5
The hypothetical ancestor for Acari is believed to have been diplodiploid with a sexually reproducing mating system (Norton et al., 1993). Also, multiple derivations of arrhenotoky and thelytoky are believed to have occurred within Acari (Norton et al., 1993). Given the repeated evolution of these mating systems, it is important to understand possible mechanisms of their evolution as well as the relative advantages and disadvantages of these systems in mites. Arrhenotoky is widespread in Acari (Havron et al., 1987; Norton et al., 1993), Hymenoptera (where it is the dominant form of reproduction) and Thysanoptera, with scattered representation in the insect orders Homoptera, Coleoptera and Diptera (Havron et al., 1987). There are several advantages of arrhenotoky and other haplodiploid modes of reproduction. In fact, arrhenotoky has the greatest potential, of all the possible mating systems, for a polyphagous colonizing species to rapidly adjust to host conditions (Kaliszewski and Wrensch, 1993). It may even be necessary for the maintenance of variability through genetic recombination and thus the ability to migrate from deteriorating to new food sources (Kaliszewski and Wrensch, 1993). One of the main advantages is that the haploid males act as a screening device for recessive deleterious and lethal alleles, and thus theoretically increases the rate of evolution (Griffing, 1982; Hartl, 1971; Havron et al., 1987) as well as allows the population to tolerate much higher levels of inbreeding (Norton et al., 1993). If selection on hemizygous males yields more rapid adaptation than family or mass selection methods, additional credence must be given 16 to the theoretical idea that the rate of evolution is accelerated by arrhenotokous reproduction. Javier et al (1991) showed more rapid evolution of pesticide resistance with male selection as opposed to mass selection in the Hymenopteran parasitoidAphytis linanesis, but were unable to substantially increase the degree of resistance in Aphytis holoxanthus with either selection method. The authors concluded that the difficulty in selecting for resistance A. in holoxanthus was due to a recent bottleneck which substantially decreased the amount of additive genetic variation available for selection (Javier et al., 1991). Ba (1993) found that selection on male Phytoseiulus persimilus reached the same endpoint as family selection and selection on females, only at a much faster rate. Another advantage of arrhenotoky is that males tend to be the first eggs oviposited (Wrensch, 1993) and are protandrous in development (Norton et al., 1993). Only when haploids develop parthenogenetically into males can life cycles evolve predictable protandric development. Males emerge as adults before the first females and thus guard and mate the first females to emerge - usually sisters (Potter et al., 1976). The result is a tendency for a local surplus of males, from early in colonization, available to mate with predispersal females. In the unlikely case that a female is missed and did not get mated, she is capable of mating with one of her sons and quickly ovipositing a group of female biased progeny (Wrensch, 1993). Potter (1976) demonstrated that greater than 95% of dispersors are inseminated. Even with the highly biased sex ratios found in some species of acarines, the small number of males available are usually adequate to inseminate most of the females 17 before dispersal (Wrensch and Bruce, 1990). This provides a significant advantage for colonization of patchy environments since any female that can utilize the patch should be able to start a new colony irrespective of her natal conditions. There are two major mechanisms by which thelytoky could be derived from sexually reproducing organisms: apomixis and automixis. In apomixis, there is lack of cytokinesis following the first mitotic division of the unfertilized egg. If this occurs, the siblings are not identical clones of their mother, and each sib will be homozygous for all loci. This mode of thelytoky might be advantageous with interdemic selection within unstable environments, but is an inefficient long term strategy because the genomic evolution of these organisms will probably be slower than the environmental changes they are trying to adapt to. Also, this mode of thelytoky is not likely to be efficient in stable environments since there will be a gradual accumulation of mutations due to the lack of repair synthesis in meiosis (Kaliszewski and Wrensch, 1993). Automictic thelytoky is the second possible mechanism. With automixis, the haploid pronucleus fuses with the second (nonsister) polar body during meiosis. Early meiotic synapsis, crossing over and dsDNA repair synthesis does occur. This mechanism should be effective in a stable environment since the maternal genotype is usually faithfully transmitted to her progeny. Automictic thelytoky also provides a possible method of switching from arrhenotoky to thelytoky. An outside agent or other environmental shock might prevent synapsis during Prophase I of meiosis. If meiosis continues 18 without synapsis, the resulting nucleus would be a diploid clone of its mother and all fertilized eggs would be triploid and not develop normally. Such environmental stresses include products produced by endosymbionts, mutagens as well as other agents (Kaliszewski and Wrensch, 1993). Fain-Thornton (1993) found that females subjected to colchicine produced all-female progeny sets in much greater frequency than the control or those groups subjected to other chemical agents. This could have been due to spontaneous thelytoky as a result of meiotic failure. Some of the advantages of thelytoky over sexual reproduction, especially in diplodiploid systems, center around the fact that only female progeny are produced. By producing only daughters, energy and time are not wasted in non-egg laying males nor in mate seeking and mating, and reproduction can occur without males (Norton et al., 1993). Thus, more grandchildren should be produced and they will be related to the foundress by 100% (Norton et al., 1993). It also means that a foundress can easily establish a new population without any males being available (Norton et al., 1993). A successful genotype will be transmitted faithfully, without breaking up successful gene groups through recombination and reconstitution with a parental genotype or by adding new alleles through mate choice (Norton et al., 1993). One of the major disadvantages that authors tend to associate with thelytokous reproduction is that it quickly moves the population towards homozygosity for it is the strongest form of inbreeding. Although this may be true with some species, the holokinetic chromosomes of most mites and all spider mites 19
provides the solution. Because inverted meiosis allows the maternal genome to be faithfully reconstructed every generation, and DNA to be repaired (Wrensch, 1994), increased homozygosity is not a disadvantage of thelytoky in these species. There will only be a minor increase in homozygosity as a result of thelytokous reproduction in this genetic system, and then only as a result of recombination. But there is a cost to thelytoky, if one is to assume that amphimixis results in variability for adaptation, especially since many of the advantages of thelytoky also exist with arrhenotokous reproduction. By controlling their sex ratio, most arrhenotokes only invest in the number of males they require for fertilization or rapid microevolution to a deteriorating environment (Wrensch et al., 1994), and thus reproductive effort is not squandered on superfluous males (Norton et al., 1993; Wrensch et al., 1994). Facultative incest, by mating with a son, mitigates against reproductive failure from unmated dispersers and allows many arrhenotokous mites to establish new colonies (Norton et al., 1993). Also, arrhenotokous organisms have the advantages of sexual reproduction and sex ratio plasticity in unstable and declining resources as well as increased population densities (Norton et al., 1993; Wrensch, 1993) found during each full colonization episode by these plant predators (Wrensch, 1993). Given the apparent advantages of arrhenotoky over thelytoky, it is interesting to see where thelytoky occurs within Acari. Thelytoky is much less common and appears to be quite scattered compared to arrhenotoky (Norton et al., 1993). Thelytoky is also even less common within taxa which contain a lot of arrhenotokous 20 species compared to where diplodiploidy dominates (Norton et al., 1993). The higher Oribatida are largely thelytokous. Thelytoky only seems to dominate in taxa where selection on exact replicas of entire “general purpose” genomes is more efficient than selection on individual alleles or gene groups (Norton et al., 1993). Predictability of the environment, difficulty in mate finding and slower ground, or underground, dispersal are common themes in thelytokous populations (Norton et al., 1993). Thelytoky is rare among Tetranychids (Norton et al., 1993) except for those taxa adapted to perennials. Thelytokous prostigmatids (.ie. Tenuipalpidae) are associated with perennial host plants and are largely thelytokous (Wrensch, pers. comm.). Spider mites, however are aerial disperses that colonize patchy ephemeral habitats (Kennedy and Smitley, 1985; Wrensch, 1985). They rapidly overcome their annual plant hosts and thus are in continually declining resources (Wrensch, 1985). Under these conditions, arrhenotoky is common and is far more genetically flexible and advantageous (Wrensch, 1993).
Rationale and Significance of Experiments Gould (1991) states that one of the major problems facing agriculture is the lack of information about pest adaptation and the resulting rapid failure of pesticides. He suggests the additional information on pest biology might help in the design of new methods of pest control. Spider mites are serious pests to at least 150 agriculturally important crops (Helle and Sabelis, 1985). They achieve this pest status in part because of their extremely rapid 21
intrinsic rate of increase and polyphagous nature (Wrensch, 1985). Tetranychus urticae Koch is also known for its rapid resistance to pesticides. Therefore, population control of this and other spider mite species are quite difficult, especially since many populations are now multiply resistant to pesticides (Helle and Sabelis, 1985). A better understanding of how haplodiploidy, and its resultant potential for sex ratio regulation, enables rapid responses to selective forces is vital to successful control of these pests. This information is especially important given that current pest management systems have largely ignored the role of sex ratio regulation on rapid pesticide resistance. These studies help explain some of this acarine’s ability to evolve so rapidly as well as describing some of the life history traits that make it such a pest. Many important beneficial insects and mites also reproduce by arrhenotoky (Havron et al., 1987). Several of these are predators or parasites of arrhenotokous pests species (Wrensch, 1993). Local outbreaks of pest problems frequently follow the use of pesticides (Van de Vrie et al., 1972). Therefore, genetic improvement of beneficial species, especially selection for pesticide resistance, becomes an important concern of integrated pest management (IPM) programs (Havron et al., 1991). This is especially true since good pesticide resistance is rarely found in field populations of either parasitic Hymenoptera or predatory mites (Havron et al., 1991). The domestic honeybee (Apis melifera) is also a haplodiploid Hymenopteran of great interest. It is used to pollinate much of our agricultural crops as well as providing honey for sale. Honeybees also face the increase in pesticide use as farmers spray crops for 22 other pests. Although the genetics of an acarine pest may not directly pertain to selection in beneficial insects and other mites, knowledge of sex ratio genetics could provide valuable insight to other arrhenotokous species and thus be quite useful. All-female progeny sets have been known to occur in essentially all experim ents usingTetranychus urticae. Chapter I discusses possible reasons why all-female progeny sets could arise under normal laboratory conditions as well as testing for the possibility of spontaneous thelytoky in a population of spider mites. Since females are the most commonly studied gender, the results of genetic studies could be biased if males influenced their progeny’s developmental rate, sex ratio or fecundity, it is important to rule out such influence or understand its magnitude if present. Chapter II presents important work on this topic which confirms that the experimental designs used in later chapters are valid. Little genetic work has been done using arrhenotokous arthropods, and most of that is with solitary or social Hymenoptera in Insecta. Acari has been largely ignored for these studies despite the relative ease of culturing and short generation intervals. These studies are among the first to explore the genetics of sex ratio in any arrhenotokous organism. Under environmental stress, the mean progeny sex ratio (proportion of females) declines and its variance increases (Young et al., 1986). This shift in the progeny sex ratio increases the number of hemizygous males exposed to the stressful environment and thus increases the exposure to selection of these recombinant haploid genotypes (Wrensch, 1993). Therefore, increasing the number of 23
males as a response to environmental stress is functionally equivalent to an increase in the effective genetic recombination. This genetic system is far more effective than diplodiploidy because hemizygous males have their entire genome exposed to the environment. Given the sex ratio adjustment resulting from environmental cues, genetics of this fitness trait is important to a mites ability to rapidly adjust to novel environments. Falconer (1989) states that a trait measured in two different environments can be considered as two different traits for the purposes of quantifying the genotype-environmental interactions. The heritabilities within the different environments can then be estimated and compared to each other. Information gleaned from comparing these estimates should give some insight into the genetics of the observed plasticity of this trait. GENERAL METHODOLOGY
Mite Rearing The experiments reported in the four chapters following this section utilize slightly different methodology in some respects, but the stock culture, mite rearing, laboratory equipment and use of leaf disks remain constant for all experiments. The following section describes the general methods used for the majority of these experiments. Specific methodologies will be found in the appropriate chapters. Two-spotted spider mites, Tetranychus urticae Koch, were obtained from a lab culture originating from mites collected in a Columbus, Ohio garden in August, 1989 (Columbus '89 stock culture). Cultures were maintained at 25<>C, in a constant temperature, chamber on detached kidney bean(Phaseolus vulgaris L.) leaves. The leaves were obtained from plants grown in a “plant mobile” (Jewel Industries, Inc.) within the laboratory to preclude other insect or mite cohabitants. Mites in the stock culture were transferred to new leaves about every two weeks in order to minimize stress due to mold, crowding or declining leaf quality. Fresh colonies were initiated on detached leaves that had been pressed onto tap-water-soaked cotton in 9.5 X 19.5 cm (inside dimensions) rectangular plastic trays. Three of these smaller trays, with two or
24 25
three 1.5 cm holes cut into their bases, were placed into a 25 X 32 cm plastic crisper lid which was then loosely covered with a clear plastic crisper. Water to the cotton was replenished, through the holes on the bottom of the small trays, by maintaining a shallow pool of water in the crisper lid in which the smaller trays rested. Experiments were carried out in either the same environmental chamber which supported the mass stock culture, or a smaller incubator set at 32°C. Stock cultures were maintained in a 141:1 Od light cycle and 25<>C. Several criteria were used when choosing females at the start of each experiment. A conscious effort to choose only healthy looking, robust females was made in all cases. Also, females were taken from throughout the colony in order to assure an accurate sampling of the variation within the Columbus ‘89 stock culture. All experiments were conducted using second generation mites reared from individual leaf disks. The experiments which assessed the effects of males on fitness traits of their offspring and possibility of thelytoky (female parthenogenesis) employed quiescent female deutonymphs. Teneral females were used for most of the heritability work. In some experimental phases, older adult females were taken only if there were no newly emerged females available (approximately 10% of the time) and only young-looking, probably early-ovipositional females were chosen. Such females were used to establish families. Several studies in one lab have demonstrated that lifetime fecundity is 85-95% predicted by fecundities summing across the first 11 days of oviposition, with peak egg production on 26
approximately the 6th day (Wrensch and Young, 1975). The experiments described in the following chapters take advantage of this fact and oviposition is permitted until the first larva emerges and therefore comparisons of female longevity and oviposition time should not confound comparisons between treatments or experiments preformed at different times. Since hatchability tends to be uniformly high (Wrensch and Young, 1975), it also should not overly influence the rm of individual mite populations. All females, tenerals or older, were assumed to have been inseminated before transfer from the stock cultures, and were left to oviposit undisturbed for 3-7 days, depending on the protocol of the individual experiment. Early removal of ovipositing females was chosen because the rate of oviposition is temperature dependent. Removing females at the emergence of the first larva enabled a physiologically repeatable standard across experiments, useful for pooling data when limited resources necessitated completion of an experiment in multiple replicates. Leaf disk technique enabled study of single families. Progeny were permitted to develop in place. Individual females or a male- female pair of mites were placed on leaf disks 2 cm in diameter that had been cut with a cork borer from a plant's first trifoliate leaves. Disks were pressed firmly onto water-soaked cotton, 'bottom-side' up, to prevent water seeping along the leaf veins. Between 24 and 32 leaf disks were placed onto wetted cotton in each small tray. In order to compensate for the “edge” effect caused by differential rates of drying at the cotton margins, smaller trays were occasionally rotated within the larger tray. Also, progeny families 27
were not all placed in the same position as their sisters, but rather put into different locations whenever possible. Environmental influences resulting from the location of the larger tray in the environmental chamber were not noticed, but trays positions were changed every day in the incubator (32°C) because differences were observed. Female mites were transferred from the stock culture one generation before the start of the experiment in order to eliminate the effects of variable crowding in the stock cultures on the first generation. Because crowding affects fitness in several ways, development of parents on crowded stock cultures could confound the heritability estimates by introducing an uncontrolled environmental variable. When females are grown on crowded stock cultures, both fecundity and sex ratio are reduced as compared to those growing up on healthy leaf disks (Hales, unpublished data). The first generation, the parents of the individuals used in experiments, served to standardize the rearing environment and to decrease crowding effects. This did not completely control for crowding since offspring from larger families were more crowded than those from smaller families on the 2 cm disks, but it did control for other differences between stock culture leaves and leaf disks. Once moved to individual leaf disks, the females were permitted to oviposit undisturbed. Following the general protocol, both parents were removed from the leaf disk when the first larva within a progeny emerged. All eggs produced by females were left in place to develop to adulthood. 28
Adult offspring were scored by sex and removed daily, as they emerged. These data were collected by calendar date, but transformed for computer entry by physiological age of the mother at the time the egg was laid. This was done by using the day of emergence of the first adult offspring (male or female) being assigned Day 1 of progeny emergence. Development rate was determined by subtracting the calendar dates of the day the first egg was observed from the day of the first adult emergence for each leaf disk.
Statistical Analysis Not all families provided data adequate for analysis. Criteria for data elimination were as follows. Those females which did not lay eggs (usually due to damage during transfer) were deleted from the sample set. Mites with fecundities less than 5 were also deleted because the first few eggs laid are male biased and progeny sets that low may indicate that the female was a poor competitor or injured at transfer. Records with only a single day of emergences were also deleted for similar reasons. Small numbers of eggs that are usually male (Helle, 1967) are characteristic of Day 1, therefore inclusion of families with just Day 1 records would bias results by decreasing the mean progeny sex ratio compared to those families with records from more than one day of oviposition. If families consisted of one-sex offspring, such as if only males or only females were in the progeny set, their records were also deleted. It was assumed that if there were only males in the progeny set this was due to insemination failure of the mother. One-sex progenies 29 are infrequent in families with normal fecundities, so inclusion of these extreme sex ratios would tend to artificially increase the variance. Single sex progenies were also deleted because the two extremes (0 and 1) will bias the centralizing tendencies of proportion data and thus could skew the results. All statistical analyses involving sex ratio were performed on arcsine squareroot transformations of the proportion of females. Presentation of descriptive statistics always retains or back- transforms to original units of measurement. Several statistical packages, even different machines, were used for the statistical analysis due to replacements and upgrades of the systems and software. A Macintosh llci was used for the descriptive statistics, t-tests and regressions. Statview II, Minitab 8.2, or SuperAnova were the packages used for these analyses. Heritability analyses were performed on an IBM 3081, mainframe using Minitab 7.1 for the heritability study (Chapter III), and an IBM 3090 600 J using SAS 6.07 for the temperature experiment (Chapter IV).
Heritability Estimations Heritability estimates are presented in Chapters III and IV. Chapter III discusses the genetics of these three reproductive traits, developmental rate, fecundity, and sex ratio, while Chapter IV examines possible phenotypic plasticity. In all cases, narrow sense heritability estimates are given. One of the methods used to estimate the narrow sense heritabilities was the full sib correlation method, using the methods described by Becker (1985). Intraclass correlations were computed 30 from information in computer generated ANOVA (analysis of variance) tables. This method could not be used to obtain a heritability estimate if the differences between families was not significantly different from zero. The coefficient of the variance component being measured (k), or the number of individuals per family (Becker, 1985), had to be adjusted for unequal sample size (ki). The number of individuals in each family was squared then summed over all the families using Statview II on a Macintosh II ci.
X(Number per family)2 k = ki N - 1 N ( 1)
Where: k = The number of individuals per family, adjusted for unequal family sizes. S = The number of families. N = The total number of leaf disks.
The next step was to obtain the within and between variances from the ANOVA tables and calculate the covariance of relatives (c2s): 31
Gyjf = MSE, (2)
q2 , MST - MSE S ki (3)
Where: MSE = Mean squared error (from the ANOVA). MST = Mean squared treatment, where the treatment was the family, o2W = The variance within families.
Given the covariance of full sisters, the intraclass correlation (t) was then calculated (Becker, 1985): o f + «w (4) The heritability estimate (h2) was directly derived from the intraclass correlation by multiplying it by the coefficient of relationship. For diploid full sisters in a haplodiploid mating system, the coefficient of relationship is 4/3 (Grossman and Eisen, 1989); therefore, 32 h2 = 4.t . 3 (5) The standard error (SE) of the heritability estimate was calculated according to Becker (1985): (6) If the standard error (SE) was larger than the heritability estimate (h2), the estimate was considered not significantly different from zero and could not be used for further calculations. Three different regression methods were used to provide heritability estimates within Chapters III and IV. All were variations on mother-daughter regressions. Thus the heritability, for each estimate was given by: h2 = 2b, (7) where b was the slope of the regression line. The slope of the regression line was multiplied by 2 because mother’s and daughters are related to each other by one-half (Falconer, 1989, Table 10.2). 33 The standard error of the heritability estimates were calculated using the methods described by Falconer (1989). The variance of the slope was calculated using (Falconer, 1989): (8) Where: o2b = The variance of the slope of the regression. N = The number of mother-daughter pairs used in the analysis. o 2 y = The daughter’s variance for this trait (Y axis). o2x = The mother’s variance for this trait (X axis), b = The slope of the regression line. The mother’s and daughter’s variance was calculated using Equation 10.7 presented by Falconer (1989): ( 9) Where: Vp = The phenotypic variance measured for each trait in the mothers, k = The number of parents measured per offspring. 34 The phenotypic variance (Vp) was obtained directly from the computer printout. For all the experiments contained within this document, k=1 because only females were tested for the three estimated fitness traits (Falconer, 1989). the variance was calculated as: ( 10) Where: n = The number of offspring per family, t = The intraclass correlation. Vp = The phenotypic variance for the daughter generation. The number of offspring per family (n) was taken as the mean value for both the weighted and nonweighted parent-mean offspring regressions. The intraclass correlation was obtained from the full sib correlations calculated earlier. Again, the phenotypic variance was taken directly from the computer analysis for the daughter generation. The standard error of the heritability estimate was calculated using formula 10.9 in Falconer (1989): SEh2 = 2ob- (11) 35 In the parent-offspring regression on individual daughters, mothers values were repeated for each daughter, thus biasing the estimates toward those families with more surviving daughters in the parent generation (Falconer, 1989). Two methods using parent-mean offspring regressions were used for heritability estimates. The mean reproductive value of the daughters (Y) was regressed against the mother’s value (X) and the slope and standard error of the regression line was calculated (as discussed above). The weighting method described in Falconer (1989, Equation 10.11) was used for weighting each family, such that the weighted mean values for the daughters were regressed on their mother’s value. This method of weighting compensated for unequal family size, including families that consist of only one offspring per parent. A weight of 1 is assigned to families with only one offspring, with increasing weighting for larger families being dependent upon the intraclass (phenotypic) correlation and the reciprocal of the variance that would exist if all families were of the same size as the family being weighted (Falconer, 1989). The first step of the weighting method was to calculate the value for T: T = (tV) (1 -t) ( 12) 36 Where: t = The intraclass correlation. b = The slope of the regression line with non-weighted means. After T was calculated, enough information had been obtained to set the weights. The weights (oo) assigned to each mother, hence each family were determined as follows (Falconer, 1989, Equation 10.11): (n + nT) (Dr. = )------f . n 1 + nT) (13) The standard errors of the weighted regressions were also calculated using the same formula described for standard errors of the other regressions presented earlier in this chapter. CHAPTER I SCREENING FOR THE POSSIBLE INCIDENCE OF SPONTANEOUS THELYTOKY IN LABORATORY POPULATIONS OF THE TWO- SPOTTED SPIDER MITE (Tetranychus urticae Koch) There are a few ways in which all female progeny sets can be obtained from arrhenotokous mites. The female may be heterozygous for a recessive lethal allele. In this case, half her male progeny will die and there is a finite chance that she will randomly give this allele to all the sons if her fecundity is low. A female may naturally have a highly biased progeny sex ratio, maybe even 100% females. Sex ratio control may be accomplished through differential membrane permeability of the nutritive chords or the individual oocytes (see Introduction section of this document for a more detailed description) and more permeable membranes could result in a higher sex ratio (more females). If a female inherited a extraordinarily permeable membrane, the ability to control her 37 progeny sex ratio would also be lost and she would produce all female progeny sets. However, this would be selected against since it would impede a female’s ability to control the sex ratio of her offspring and thus severely handicap her success as a foundress since males would not be available to mate with her daughters if she founded a new patch. It might also affect fecundity or other fitness traits since a hyperpermeable membrane would probably affect an array of reproductive traits associated with egg development and vitellogenesis. The final possibility is for thelytoky to spontaneously arise within the population. Thelytoky is believed to have independently evolved many times within Acari, most commonly from sexually reproducing diplodiploid, but also from parahaploid and arrhenotokous mating systems (Norton et al., 1993). Thelytoky is rare within Acari, but most frequently found in Orbatida, having representatives within nearly 10% of the families (Norton et al., 1993). It is moderately common in Tetranychidae, compared to other families in Prostigmata, with the subfamily Bryobiinae comprising the majority of the thelytokous species (6 out of 17 listed species within that subfamily). Oligonychus thelytokous is the only 39 thelytokous member of the 109 listed species within the subfamily Tetranychinae (Norton et al., 1993). Thelytokous populations within sexually reproducing species of acarines have been reported; thus, thelytoky appears to be a step within allopatric or sympatric speciation (Norton et al., 1993). Spontaneous thelytoky has occurred at least once in tarsonemids (Heterostigmata), Tarsonemus pallidus, a normally arrhenotokous mite (Garmon, 1917 a,b). Garmon (1917b) managed to maintain these thelytokous mites for more than 5 months without observing a single male. Karl (1965) also isolated a thelytokous reproducing population from a normally arrhenotokous species. He maintained a thelytokous population of Tarsonemus confusus Ewing, another tarsonemid, for more than 30 generations. Spontaneous thelytoky has been known to arise in populations of tetranychid mites (Oliver, 1971), even in Tetranychus urticae (Boudreaux, 1963). If spontaneous thelytoky arises with any regularity, it would have interesting implications for evolutionary theory. 40 Materials and Methods A total of 535 teneral females were taken randomly from the Columbus ‘89 stock culture and plated out on individual leaf disks, following the protocol mentioned earlier in the General Methods section. Of these, 498 produced progenies and the rest died as a result of transfer. Each tray was observed daily for the emergence of a male. If a male was observed within a family, the leaf disk was removed from the tray (if in poor condition) or was carefully turned over in such a manner to kill all immatures and adults on that disk. This was done to facilitate identification of disks which still had all female progeny sets on them. If a family was composed of only daughters, the number of females was scored and then all the females were plated out, individually, onto fresh leaf disks. Half of these second generation females were then mated to a fresh male from a nearby leaf disk. Mated and unmated females were permitted to oviposit until their first larva offspring emerged. At that point, all parents were removed from the disk. Immatures were left in place until maturity. The families that were of mated second generation 41 females were scored for progeny sex ratio. The families of unmated second generation females were observed to see if any daughters emerged. If unmated mothers produced daughters, this would indicate spontaneous thelytoky. R esults Of the 498 families, only 10 (1.81%) had all female progeny sets on their disks. Most were from very small family sizes, with an arithmetic mean of only 6.0 daughters per family. Table 1 shows the distribution of all-female progenies within the experimental population as well as the progeny size of each family. The largest family size was only 16, with one family of 14 and one of 11 offspring. These were very small family sizes given the reproductive capabilities of these mites. The results of progeny testing these all-female families in a second generation are shown in Table 2. Those daughters which remained unmated are shown in the left columns, whereas those that were provided with mates are shown on the right. All females given mates had both sons and daughters in their progenies. None of these families consisted of all-female progeny sets and only one was Table 1. Number of replicates (families) with all-female progenies. Number of daughters here represents the total number of adult offspring. Trav Number Replicate Number Number Of .Daughters 6 2 4 16 19 16 14 4 10 5 14 2 3 3 15 21 3 19 19 3 13 5 2 14 14 2 13 9 1 43 Table 2. Progeny tests of the daughters in all-female families shown in Table 1. Tray and replicate (Rep), presence of sons (S) and daughters (D). Female progeny from each leaf disk were either left unmated or provided with a male mate from another leaf disk. BSC Females Not Mated EemalSS-lMed TraWReol Offspring ItaylBfiPl Offspring 1 6(24) S 6(24) S+D 2 6(24) S 6(24) S+D 3 6(24) s 6(24) S+D 4 13(5) s 13(5) S+D 5 14(10) s 18(22) S+D 6 14(14) s 14(14) S+D 7 14(23) s 14(23) S+D 8 15(21) s 15(21) S+D 9 19(16) s 19(16) S+D 10 19(16) s 19(16) S+D 1 1 19(16) s 19(16) S+D 12 19(16) S+D 18(22) S+D 13 19(16) s 19(16) S+D 14 19(16) s 19(16) S+D 15 19(16) s 19(19) S+D 16 6(24) dead 15(21) S+D 17 6(24) dead 6(24) S+D 18 6(24) dead 6(24) S+D 19 6(24) S 6(24) dead 20 6(24) S 6(24) S+D 21 18(22) S+D 18(22) S+D 22 18(22) S+D 18(22) S+D 23 18(22) S+D 18(22) S+D 24 18(22) S+D 18(22) S+D 44 unusually biased towards females. Of the families not given mates, most produced only sons. There were two instances where progeny of presumably unmated females included daughters. All of the families plated out from disk 18 had both sons and daughters in their progenies, as did one of the 7 replicates of 19(16). This particular family had only 2 sons and 24 daughters. D iscussion Given the results of this experiment, spontaneous occurrence of thelytoky does occur with high enough frequency, under these environmental conditions, to be observed in this strain of mites. Thelytoky arising from certain environmental stimuli or stress may still be a possibility, but this aspect was not within the scope of this experiment. Hoy and Cave (1986) screened for spontaneous thelytoky in the parahaploid phytoseiid Metaseiulus occidentalis. They also failed to find spontaneous thelytoky although thelytokous strains have been found in lab colonies and in nature. The possibility of either hyperpermeable membranes or haploid lethals cannot be tested with these results. Either possibility, or both, may be the cause of the observed all-female progeny sets. 45 Further genetic studies of these families (all-female progeny sets) would be needed in order to discount the possibility of haploid iethals. Because only a few progenies, and those with small progeny size, were obtained their occurrence may be due to chance. Two of the families showed the possibility of spontaneous thelytoky, however neither of these families showed complete thelytoky for there were males in all progeny sets. I believe that the greatest likelihood is that all of the replicates from family 18 were mated prior to being moved to the new disks. A male was probably on disk 18 and missed when I was looking for males within the progeny set. Perhaps he was mistaken for an immature and later drowned. In either case, I contend this was probably not an all female progeny set, thus only 9 all female progeny sets were within this experiment. This leaves the unusual case of offspring replicate 12, from parental disk 19(16). Although this one cannot be fully explained, it was probably not the result of spontaneous thelytoky either. The 2 sons and 24 daughters were plated out to fresh leaves and a new subcolony was started. I watched this subcolony over several generations. At first, the sex ratio was very high, but not 46 thelytokous. After a few generations, the sex ratio appeared to normalize a little above the stock colony (Columbus ‘89), but definitely within the normal range of sex ratios. Nothing unusual was noticed during approximately 5 months of observation. The occurrence of all-female progeny sets seen in this experiment was very similar to other experiments using the same stock culture (Table 3). In all such all-female families, the progeny size was much lower than the mean for that experiment, with the largest all-female family size observed consisting of only 20 individuals. The occurrence of all female progenies appears to comprise a very small fraction of the population, probably around 1%. Within the Columbus ‘89 stock culture, the mean percentage of families which were all-female was 0.85±.0.63%. The lack of thelytoky within arrhenotokous mite populations is expected given the life history and mating system Tetranychus of urticae. The only thelytokous species within this subfamily (Tetranychinae) is Oligonychus thelytokous, demonstrating the rarity of this mode of reproduction within spider mites (Norton et al., 1993). This suggests the relative advantages and disadvantages of these two mating systems. The two-spotted spider mite is a 47 Table 3. Number of all-daughter families observed among the various experiments using the Columbus '89 stock culture. In each case, only those females that failed to oviposit were deleted from the total sample. Sample size of total families in each experiment, the largest all-female progeny number in the sample, and the percentage (%) of the sample with all-female families are reported for ten different experiments. Number of Sam ple L argest % of Experim ent Fam ilies Size Progenv Fam ilies Thelytokyi 9 498 16 1.81 Male Influence? 4 922 6 0.43 H2-1, Parent? 0 202 0 0.00 H2-1, Offspring? 7 743 10 0.94 H2-2, Parent?* 2 106 2 1.89 H2-2, Offspring?* 2 639 3 0.31 Temp. 22, Parent4 2 254 2 0.79 Temp. 22, Offspring4 7 1015 10 0.69 Temp. 32, Parent4 4 315 20 1.27 Temp. 32, Offspring4 4 1055 4 0.38 ‘Replicate experiment not discussed within this document. 1) Results of experiment reported in Chapter 1. 2) Results of experiment reported in Chapter 2. 3) Results of experiment reported in Chapter 3. 4) Results of experiment reported in Chapter 4. colonizer with a r-selected type life history (Wrensch, 1979). Thelytokous acarines are typically found in the deeper soil environments, where there might be some advantage of a generalized genome and there is a lower incidence of mutagenic factors such as uv light and intense heat (Norton et al., 1993). Also, it might be more difficult to locate mates in those environments (Norton et al., 1993). Thelytoky also seems to be associated with mites with lower mobility than those that disperse aerially (Norton et al., 1993), thus might not be as good for a Tetranychid to utilize as a mating system. Thelytoky in Tetranychidae is normally associated with perennial plants (Wrensch, pers. comm.), a more stable resource than annuals. Thelytoky may be induced in some natural populations of arthropods. For example high temperatures (30°C) and exposure to antibiotics have been known to permentantly revert some previously thelytokous strains of Trichogramma to arrhenotoky (Stouthamer et al., 1990). This was interpreted as microbe-induced thelytoky reverting back to the original mating system when the microbes were destroyed (Luck et al., 1993; Stouthamer et al., 1990). Not all thelytokous lines were converted back to arrhenotoky by shock 49 induction, 3 lines remained thelytokous (Luck et al., 1993). Thelytoky also may be induced by gynogenesis resulting from males mating with females of sibling species (Luck et al., 1993). List of References Boudreaux, H. B. (1963) Biological aspects of some phytophagous mites. Ann. Rev. Entomol. 8: 137-154. Garmon, P. (1917a) Notes on Tarsonemus pallidus Banks. J. Econ. Entomol. 10: 503. Garmon, P. (1917b) Tarsonemus pallidus Banks, a pest of geraniums. Maryland Agric. Exp. Sta. Bull. 208: 327-342. Hoy, M. A. and F. E. Cave (1986) Screening for thelytoky in the parahaploid phytoseiid, Metaseiulus occidentalis (Nesbitt). Exper. Appl. Acarol. 2: 273-276. Karl, E. (1965) Untersuchungen zur morphologie und okologie von Tarsonemiden garterischer Kulturplanzen. II.Hemitarsonemus latus (Banks), Tarsonemus confusus Ewing, T. talpae Schaarschmidt, T. setifer, Ewing, T. smithi Ewing undTarsonemoides belemmitoides Weis-Fogh. Biol. Zbl. 84: 331-357. Luck, R. F., R. Stouthamer and L. P. Nunney (1993) Sex determination and sex ratio patterns in parasiticHymenoptera. In: Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman and Hall Publishers. New York and London, D.L. Wrensch and M. A. Ebbert (Eds.), pp. 442-476. 50 Norton, R. A., J. B. Kethley, D. E. Johnston and B. M. OConnor (1993) Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman and Hall Publishers. New York and London, D. L. Wrensch and M. A. Ebbert (Eds.) pp. 8-99. Oliver, J. H., Jr. (1971) Parthenogenesis in mites and ticks (Arachnida: Acari). Am. Zool. 11: 283-299. Stouthamer, R., R. F. Luck and W. D. Hamilton (1990) Antibiotics cause parthenogenetic Trichogramma (Hymenoptera: Trichogrammatidae) to revert to sex.Proc. Natl. Acad. Sci. 87: 2424- 2427. Wrensch, D. L. (1979) Components of reproductive success in spider mites. Recent Advances in Acarology 1: 155-164. CHAPTER II EVIDENCE THAT MALES DO NOT INFLUENCE THEIR MATE'S FECUNDITY OR PROGENY SEX RATIO UNDER NORMAL LABORATORY CONDITIONS. Although researchers have long assumed that males do not influence the number of progeny or the sex ratio of these progeny, the limited available data has not supported this hypothesis. Wrensch and Young (1975) found that there was lower fecundity in uninseminated females compared to those that had a full sperm load. The same authors also found that females had a lower sex ratio towards the end of their ovipositional cycle as they ran out of sperm. A second insemination of older females did not produce daughters (Potter and Wrensch, 1978). Sex ratio also decreased if the female had an incomplete sperm load whether due to repetitive matings by the male (Wrensch and Young, 1975). or as a result of copulation being disturbed (Helle and Pijnacker, 1985). Males have been found to potentiate egg laying inPyemotes tritici (Wrensch and 51 52 Bruce, 1991). Previous studies in haplodiploid mites provide no additional information on the possibility of differences between genetically different males. When studying reproductive parameters in arrhenotokous arthropods, it was normally assumed that the male would not influence the fitness components of his mate. Therefore, only the effects of the female were usually studied and the possibility of sire effects were largely ignored. This would result in an important bias if the male did indeed affect the fecundity or sex ratio of his progeny, especially when trying to ascertain the quantitative genetics of these traits for it would tend to negatively bias the heritability estimates by creating an ignored variable. This experiment tests this common assumption in spider mites. Materials and Methods Several adult females were transferred from the stock culture to individual leaves one generation prior to beginning of the experiment for three reasons. First, this ensured that the males would be approximately the same age. Second, raising the experimental animals under as similar conditions as possible 53 limited the environmental effects on both genders. Differential crowding normally occurs within the stock cultures as well as varying microenvironments, and may effect both the fecundity and progeny sex ratio. Third obtaining quiescent deutonymphs and males was facilitated. Synchronized cultures yield a large batch of newly emerged males and quiescent deutonymph females within a short period of time, frequently within a day or two of each other. Also, familial relationships in both males and females can be ascertained by recording disk number from which the mite was taken leading to tighter control of breeding strategy. The females chosen to start the experimental cultures were allowed to oviposit until the first larva emerged. At this point, they were removed from the leaf disk and all offspring were permitted to develop in place. The cultures were watched closely for the emergence of the first adults. One male was taken from each leaf disk once sufficient numbers of similar ages had emerged. These males were given several quiescent deutonymph females (5-10) each day for three days. Females were haphazardly assigned to a male, based on a block design, such that the males were given as many genetically different females as possible. Therefore, all teneral females should had been mated and were transferred to fresh leaf disks for oviposition daily. The males were provided with additional quiescent deutonymphs so that there was up to ten quiescent deutonymphs per male each day. The newly transferred females were permitted to oviposit on their separate leaf disk until the first larva emerged, at which point the female was removed from her leaf disk. Daily fecundity and sex ratio were scored for each individual disk. This experiment was repeated once, so that two trials were examined. Approximately ten days were allowed to pass between the last tray of the first replicate and the first tray of the second. Each tray of males was started approximately 3 days after the previous tray. There was a total of 6 trays of males in two trials of three. The standardized protocol for deletion of unusable families from the analysis was described in the General Methods section of this document. A total of 457 families were analyzed for the first trial and 338 for the second trial. Descriptive statistics and ANOVAs (Analyses of Variance) were calculated. An additional two families were deleted from the ANOVA because they contained only 55 one representative per family. The ANOVAs were used to decide if there was a sire effect on either fecundity or sex ratio in either replicate. Differences in the means were tested using a standard t- test, transformed values of sex ratio were used for this test. All-male progeny sets within various experiments using the Columbus '89 stock culture were tabulated to examine the incidence of fertilization failure within this strain of mites (Columbus '89) using the same deletion method discussed above except that single sex progenies were included in the analyses. R esults Means and standard deviations of fecundity and sex ratio are shown on Table 4. Mean fecundities were significantly different between the two trials, with the first trial having a substantially lower fecundity than the second. Mean sex ratios were not significantly different between replicates. 56 Table 4. Sample sizes (N), means and standard errors (SE) for fecundity and sex ratio. Both experimental trials are reported separately. Trial.. J. TdflL2 U Mean £ to Mean. & Fecundity 4 5 7 38.05 0.834 338 43.59 0.90 Sex Ratio 4 5 7 0.616 0.007 338 0.611 0.008 ANOVA s are shown in Table 5. Males did not have a significant effect on their mate's fecundity in the first experiment, whereas there was a significant (p = 0.007) effect on fecundity in the second. No sire effect was found for sex ratio in either trial of this experiment. Table 5. The results of a one-way analysis of variance (ANOVA) of fecundity and sex ratio between different sires. P-values are reported separately for each experimental trial. Complete ANOVAs in Appendix A. Fecunditv N £ D^y.al.we trial 1 4 5 7 1.208 0.116 trial 2 33 8 1.589 0.007 Sex Ratio trial 1 4 5 7 0.900 0.724 trial 2 3 3 8 0.086 0.757 57 Males have no affect on the sex ratio of their progeny. This makes sense when considering the morphology of the sperm. Mite sperm are ameboid, lacking flagella or other effective locomotion (Feiertag-Koppen and Pijnacker, 1985; Pijnacker, 1985). Also, they have no acrosome (Pijnacker, 1985) so they cannot break into a mature egg. Therefore, the female should have complete control of sex ratio. The only time or place the sperm seems to be able to penetrate the egg is toward the end of vitellogenesis, by way of the nutritive chord (Feiertag-Koppen and Pijnacker, 1985) and this is likely to be heavily influenced by the membrane permeability of the nutritive chords. Several studies with arthropods have reported that males might affect their m ate's fecundity (Wrensch and Bruce, 1991). Although this does indicate some effects on a male's progeny size, almost all of these cases have shown the result of sperm depletion, not an effect of individual males. Care was taken to limit the number of mates provided to a male, so that he would not be likely to suffer from sperm depletion. Males, of both T. urticae and P. 58 tritici, are capable of serially mating several times before sperm depletion becomes a problem (Bruce and Wrensch, 1991; Helle, 1967; Wrensch and Bruce, 1990). The males used for this experiment were given a maximum of 10 females per 24 hour period, but only 2-5 emerged each day so males should have been able to regenerate sperm during the guarding of quiescent deutonymphs. These experiments yielded mixed results a male's effect on his mate's fecundity. One group showed no significant difference between males, but the other group showed a significant difference. Given these results, there might be a small effect due to the choice of male. Thus, we cannot completely rule out a sire effect on fecundity. Males do transfer some materials in addition to sperm, so there may be a slight nutritive effect of the male. Also, a female may limit her early fecundity if she did not get a full load. There was no difference in sex ratio so this possibility would indicate that females use up the sperm early in oviposition and then run out if they were permitted to live their full lifespan. The fecundity in the second trial was significantly larger than the first. All-male progeny sets observed in the various experiments using Columbus the '89 stock culture were tabulated to provide a 59 Table 6. Number of all-son families observed among the various experiments using the Columbus 89 stock culture. In each case, only those females that failed to oviposit were deleted from the total sample. Sample size of total families in each experiment and the percentage (%) of the sample with all-female families are reported for ten different experiments. Number of Sam ple % of ExDeriment Families Size SamDle Male Influence^ 74 873 8.48 H2-1, Parents 6 194 3.09 H2-1, Offsprings 63 736 8.56 H2-2, Parents* 4 100 4.00 H2-2, Offsprings* 46 603 7.63 Temp. 22, Parents 4 247 1.62 Temp. 22, Offsprings 8 979 0.82 Temp. 32, Parents 17 293 5.80 Temp. 32, Offsprings 20 1021 1.96 ‘Replicate experiment not discussed within this document. 1) Results of experiment reported in Chapter 2. 2) Results of experiment reported in Chapter 3. 3) Results of experiment reported in Chapter 4. general overview of lack of fertilization in this strain of mites (Table 6). The fertilization rate for this stock culture was adequate under the conditions of the various experiments, indicating that males normally fertilize the teneral females without any problem. The mean failure rate is only 4.66±3.04%, providing a success rate of over 95%. Reasons for fertilization failure include infertile or dam aged males due to transfer. By eliminating families with progeny sizes of less than 5 or only one day of emergence, any bias resulting from male excess during the first day of oviposition should have been eliminated. List of References Bruce, W. A. and D. L. Wrensch (1990) Reproductive potential, sex ratio and mating efficiency of the straw itch mite (Acari: Pyemotidae). J. Econ. Entomol. 83: 384-391. Feiertag-Koppen, C. C. M. and L. P. Pijnacker (1985) Reproduction and Development. Oogenesis. In: World Crop Pests. Spider Mites: Their Biology. Natural Enemies and Control. Vol. 1A. W. Helle and M. W. Sabelis (Eds.). Elsevier. Amsterdam, pp. 117-127. Helle, W. (1967) Fertilization in the two-spotted spider mite (Tetranychus urticae: Acari). Entomol. Exp. Appl. 10: 103-110. 61 Helle, W. and L. P. Pijnacker (1985) Parthenogenesis, chromosomes and sex. In: World Crop Pests. Spider Mites: Their Biology. Natural Enemies and Control. Vol. 1A. W. Helle and M. W. Sabelis (Eds.). Elsevier. Amsterdam, pp. 129-139. Luck, R. F., R. Stouthamer and L. P. Nunney (1993) Sex determination and sex ratio patterns in parasiticHymenoptera. In: Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman and Hall Publishers. New York and London, D. L. Wrensch and M. A. Ebbert (Eds.), pp. 442-476. Pijnacker, L. P. (1985) Reproduction and development, spermatogenesis. In: World Crop Pests. Spider Mites. Their Biology. Natural Enemies and Control—YoH A. W. Helle and M. W. Sabelis (Eds.), Elsevier Publishers, Amsterdam, pp. 109-116. Potter, D. A. and D. L. Wrensch (1978) Interrupted matings and the effectiveness of second inseminations in the twospotted spider mite. Ann. Entomol. Soc. Am. 7: 499-501. Wrensch, D. L. and W. A. Bruce (1991) Sex ratio, fitness and capacity for population increase in Pyemotes tritici (L. - F. & M.) (Pyemotidae). In: The Acari. Reproduction. Development and Life- History Strategies. R. Schuster and P. W. Murphy (Eds.). Chapman & Hall Publishers. London, New York, Melbourne and Madras, pp. 209- 2 2 1. Wrensch, D. L. and S. S. Y. Young (1975) Effects of quality of resource and fertilization status on some traits in the two-spotted spider mite, Tetranychus urticae Koch. Oecologia 18: 259-267. CHAPTER III HERITABILITY OF DEVELOPMENT RATE, FECUNDITY AND SEX RATIO IN A HAPLODIPLOID MITE. Introduction An understanding of the inheritance of a trait provides vital insight to its evolution as well as predicting the selective pressures and assessing the change in the trait over time (Crow, 1986; Falconer, 1989; Margolies and Cox, 1993). This knowledge can then be applied to breeding programs of beneficial species (Margolies and Cox, 1993) or to understanding and predicting the biology of pest species. Most important life history traits are continuous in phenotype. This necessitates the use of quantitative genetic methods if one is to study their pattern of inheritance and potential for natural selection to operate. These methods have provided important information in studies of arthropod evolution of life history traits (Mousseau and Roff, 1987), including sex ratio (Orzack et al., 1991), behaviors (Mousseau and Roff, 1987), host interactions 62 63 (Margolies and Cox, 1993) and pesticide resistance (Roush and Tabashnik, 1990). Understanding genetics of continuous traits centers around analyzing the variance of said trait, especially the magnitude of the variation in addition to the degree to which relatives resemble each other (Falconer, 1989). The phenotypic variance (Vp) of any character can be partitioned into individual variance components to be studied separately. In the most general partitioning, the phenotypic variance is considered to be composed of the genetic variance(V g) of that trait, and variance due to environmental factors(V e) and the interactions of genes with the environment(V qe)-' Vp = Vg + Ve + Vg e - (14) This formula is frequently further subdivided since the genetic component of the phenotypic variance is of prime concern to many of those studying quantitative genetics (Falconer, 1989). Genetic variation is important in that it reflects the variation in the genotypes of the individuals within the population. It is this 64 variation which is passed down to the next generation irrespective of the environment. The genetic variance, V q, can be further subdivided into its components, which are the additive genetic variance(V a), the variance due to differential dominance of the loci(V d) and the variance which results from the interaction of the genetic com ponents (V|), also known a s epistasis: VG = VA + VD + V|. ( 15) Of these components, the additive genetic component is the most important since it is the primary cause of resemblance between relatives and thus the most easily measured by genetic studies (Falconer, 1989). Geneticists quantify the degree to which a trait is heritable through the calculation of heritability estimates. Heritability is the most common way to quantify the degree to which resemblance between relatives is due to heredity. There are two major types of heritability, broad sense and narrow sense. Heritability in the broad se n se (H 2), also known as the degree of genetic determination, is 65 defined as the amount of phenotypic variance (Vp) due to the variance in the genotypes: h2 = ¥£-. Vp (16) Narrow sense heritability (h2) is the degree to which the phenotypic variance observed within a population is due to allelic differences which are additive in nature. Narrow sense heritabilities are generally considered to be more useful than broad sense estimates for a several reasons. The most important of these reasons is its predictive role in expressing the reliability of an individual's phenotype in terms of a breeding value (Falconer, 1989): h2 Vr (17) Additive genes are the chief determinants of observable differences within a population as well as the source of genetic variance that is acted upon by selection (Falconer, 1989). It thus 66 regulates the rate of genetic change (Roff and Mousseau, 1987). This makes additive variance more important in understanding the evolution of a trait and how it will respond to selective pressures. As recently as 1982, there was no conclusive proof as to whether sex ratio was heritable, nor how heritability estimates could be applied to predicting this very important life history trait (Bull et al., 1982). Ample phenotypic variation was noted in haplodiploid organisms (Bull et al., 1982; Mitchell, 1972), but few attempts were made to predict the additive genetic variance for this trait. Even now, little is known about the genetics of sex ratio of any organism (Parker and Orzack, 1985) and even less is known about sex ratio within Acari, a very important group of pest and beneficial arthropods. Mitchell (1972) was one of the early authors to report that progeny sex ratio was a trait under female control rather than caused by variation in sperm load. He also concluded that differences in progeny sex ratios of different females were due to genetic polymorphisms. Overmeer and Harrison (1969) also believed sex ratio to be under genetic control. Young et al. (1986) found differences in progeny sex ratios in response to poor leaf quality 67 among four different geographic strains of spider mites. These findings suggest that there may be a genetic basis for the ability to control sex ratio, but offer no conclusive proof. Previous attempts to estimate the degree of the genetic contribution of sex ratio in haplodiploids have given mixed results. Ram and Sharma (1977) were not able to select for increased proportions of females in Trichogramma fasciatum. But, their design would tend to drastically reduce the genetic variation in the population and thus obtaining a realized heritability estimate almost impossible. Parker and Orzack (1985) were able to select for an increased proportion of males in the waspNasonia vitripennis, and obtained realized heritability estimates for sex ratio of 0.11 and 0.13 in different lines. One of the very few heritability estimates obtained from mites was the work of Takafuji and Ishii (1989), who calculated a heritability estimate for progeny sex ratio of 0.85 using mother-daughter regressions inTetranychus kanzawai. The following research examines genetics of sex ratio and provides important empirical work for understanding sex ratio allocation in haplodiploids. 68 Materials and_M_eth_ods Two-spotted spider mites (Tetranychus urticae Koch) were maintained under laboratory culturing conditions (see General Methodology for specifics) for approximately 50 generations prior to the start of the first heritability experiment. Experiments were carried out in the environmental chamber which maintained a constant temperature of 25±.1°C and 141:1 Od light cycle. Female mites were transferred from the stock culture one generation before the start of the experiment in order to prevent the effects of crowding the first generation. Development on crowded stock cultures could confound the heritability estimates by introducing an uncontrolled environmental variable. When females were grown on crowded stock cultures there was a significant decrease in both fecundity and sex ratio compared to those growing on healthy leaf disks. The first generation, used to standardize the rearing environment and to decrease crowding effects, was called the "grandparent" generation. This effort to standardize did not completely control for crowding since offspring from larger families were more crowded than those from smaller families, but it did control for differences between stock culture leaves and leaf 69 disks. Recently emerged adult females (tenerals less than 36 hours old) were isolated on 2 cm diameter leaf disks to establish the grandparent generation. Older adult females were used only if newly emerged females were not available (approximately 10% of the time). All females, teneral or older, were assumed to have been inseminated before transfer from the stock cultures, and were left to oviposit undisturbed for 7 days. Progeny produced were permitted to develop in place. These progeny were the source of the parent (P) generation, which was generated using one adult male and one quiescent deutonymph female (QDN) for each family. Since it was difficult to transfer quiescent deutonymphs without mortality, 2-3 QDNs, always sisters, were transferred to each leaf disk. Adult males were transferred in the same manner, and were from a different family than the QDN's with which they were paired. These matings allowed for random association of genomes in the daughter generation. Males were checked after transfer, and dead ones were replaced, providing at least, initially, a healthy malefor insemination; Excess males and females were killed one day later, before the first egg was laid, thus only one male and female 70 remained on the leaf disk to produce offspring. P generation adults were killed 7 days after the first egg was laid. Their progeny constituted of the offspring (O) generation. The O generation was produced by placing a teneral female (presumably mated) on a fresh leaf disk and adding a brother to assure insemination. If a brother was not available, a male was taken from another family, of unknown relation, and placed with the female. From each family in the P generation, several daughters (4- 8) were transferred to separate leaf disks in this manner. Each pair of the 0-generation adults was removed and killed 7 days after the first egg was laid and immatures were allowed to complete development undisturbed. Adult offspring from the O generation matings were scored and removed daily. Most O generation adults were counted every other day once two or three days of emergences were tabulated. These data were collected by calendar date, but transformed for computer entry by physiological age of the mother at the time the egg was laid. This was done by using the day of emergence of the first adult offspring (male or female) being assigned Day 1 of progeny emergence. Development rate was determined by subtracting the 71 calendar dates of the day the first egg was observed from the day of the first adult emergence for each leaf disk. A total of 256 P generation records and 864 O generation records were obtained. Not all families provided data adequate for analysis. Criteria for data elimination are described in the General Methods section of this document. After necessary eliminations were completed, a total of 194 families in the P generation and 667 O families were used in the following analysis. For regression of O on P generations, 663 daughter records were used. The remaining 4 were deleted because information on the mother was not available. Since daughter families were not always available for each mother, P-0 regressions were performed on 179 families. All statistical analyses involving sex ratio were performed on arcsine squareroot transformations of proportions female in individual progenies. T-tests were used to determine if there were significant differences between the P and O generations. Heritability estimates were obtained by four different methods: (1) full sib correlation, (2) parent-offspring regression on individual daughters, (3) parent-offspring regression on mean of the daughters' individual values, and (4) parent-offspring regression on 72 weighted mean values for daughters. The exact methodology and formulas are provided in the General Methods section of this document. The intraclass correlations, used to calculate heritability by full sib analysis, were calculated using the formulas provided by Becker (1985). Intraclass correlations used for the full sib heritability estimates, were obtained from computer generated ANOVA tables. The heritability estim ate w as adjusted for haplodiploid genetic system by multiplying by times the intraclass correlation of 4/3. The operand of 4/3 was used because the coefficient of relationship between haplodiploid full sisters is 3/4 (Margolies and Cox, 1993). Heritability estimates were also derived from parent- offspring regressions. Heritabilities for the three methods using regression were calculated by multiplying the slope of the regression line by 2 since daughters are related to their mothers by 1/2. The weighting method of Falconer (1989) was adopted for the parent-weighted mean daughter regressions. The standard errors for all parent-offspring regressions were calculated using formula 10.6 73 in Falconer (1989), which are described in greater detail in the General Methods section of this document. Phenotypic correlations between the various fitness traits were calculated on the Macintosh llci using Statview II statistical package. The critical values for the sample correlation coefficient were obtained from a statistical table (Sincich, 1985). R esults Table 7 contains the descriptive statistics for fecundity, developmental time and sex ratio. Mean total fecundity was significantly higher (P < 0.05) in the mother's generation compared to the daughter's. Average developmental time was significantly faster (P < 0.05) in the mother's generation. The mean sex ratios were not significantly different between the families in parent and the offspring generations. The intraclass correlations and regression coefficients were significantly different from zero (P < 0.05) in all cases. Heritability estimates for development rate, fecundity and sex ratio are shown in Table 8. The heritability estimates for fecundity were between 0.16 and 0.23. Moderate heritability estimates were obtained for 74 Table 7. Means and standard errors (SE) for fecundity, development rate (DR: egg to adult in days) and sex ratio (SR: proportion female) in the progenies of P and O generations. Two-sample, unpaired t-tests were used to test for differences between generations. The same letter in a row indicates that the means are not significantly different between P and O generations at the a = 0.05 level. The use of different letters in a row indicates that the means are significantly different at a = 0.01. Sex ratio was transformed for calculating the t-tests. G eneration T ra it Eaifiat Offspring a MssdLSB N MeanfSD Fecundity 179 53.96(1.78)a 667 44.62(0.78)b DR 177 9.86(0.06)a 661 10.47(0.03)b SR 179 0.62(0.01 )a 6 6 7 0.60(0.01 )a Table 8. Heritabilities (h2) and standard errors (SE) for fecundity, development rate and sex ratio using four different methods of calculation: full sib correlation, regression of parent (P) on individual offspring (O), regression of parents on mean offspring and regression of parents on weighted mean offspring. Eaau.ndi.ty Development Rate Sex Ratio Method U h2(SE^ U h2(SE) H h2(SE) Full Sib Corr. 667 0.23(0.08) 661 0.24(0.08) 667 0.48(0.08) Regressions of P on: Individual O 663 0.16(0.03) 659 0.37(0.04) 663 0.61(0.05) Mean Offspring 179 0.19(0.01) 177 0.40(0.01) 179 0.56(0.01) Weighted Mn. O 179 0.17(0.01) 177 0.38(0.01) 179 0.60(0.01) 76 development rate (0.24-0.40). Sex ratio exhibited fairly high heritability estimates in this study (0.48-0.61). Table 9 shows the correlations between pairs of the three fitness traits. Fecundity and development rate were significantly negatively correlated in the O generation (P < 0.01). As fecundity increases, progeny development rate decreases. Sex ratio was not correlated with either fecundity nor development rate at the a = 0.05 level. D iscussion Mean fecundity was slightly higher and mean development time slightly faster in the P generation as compared to the O generation, probably due to there being slightly lower leaf quality in the offspring generation. Although healthy primary leaves were used, the larger number of leaf disks necessary to plot out all of the offspring necessitated that sub-optimal leaf disks be used. Another possibility is that using deutonymphs in the P generation and teneral females in the O generation resulted in one generation of females being handled differently during a critical time, the preovipositional. A delay of oviposition is known to occur after an 77 Table 9. Correlations between fecundity (F), development rate (DR) and sex ratio (SR) within parent and offspring generations. Generation H EADB SR & DR SR&F Parent 179 -0.15 -0.06 -0.05 Offspring 661 -0.21“ 0.07 0.07 * significantly different from zero at the a =0.05 level ** significantly different from zero at the a = 0.01 level 78 adult mite is disturbed (Wrensch, pers. comm.)- This delay could result in a lower fecundity for the offspring generation since they were moved as teneral adults and mites were scored only once a day. The mean fecundity for the first day of oviposition was significantly lower in the O generation. With the exception of fecundity, the heritability estimates obtained from full sib correlation (Table 9) were lower than those estimates obtained by parent-offspring regression. Of the four heritability estimates computed, the estimate obtained from the weighted mean regression is normally considered to be the least biased (Falconer, 1989). Heritability estimates obtained from the full sib correlation tend to be biased upwards due to dominance deviations and similarities in the environment of sibs raised together (Falconer, 1989). Therefore, it is of interest that the regression methods generally yielded higher estimates than those provided by the full sib correlation. When the daughters are kept separate, there will be disproportionate representation by those mothers who had more daughters in their progeny set because the mother's value is replicated for each daughter for the regression. The number of O 79 records, per family, was not controlled for and varied between 1-8. Under these conditions, the regression of individual daughters on their mother inflates the number of degrees of freedom. The regression method using a nonweighted mean of the daughters placed equal weight on mothers regardless of the number of progeny represented. The weighted mean regression method is a compromise between being able to derive more information from using those additional daughters being represented by certain mothers while not placing too much emphasis on mothers with large progeny sets (Falconer, 1989). Falconer (1989) states that regressions on the weighted means generally show less bias than those on non weighted means, although providing approximately the same precision. Kempthorne and Tandon (1953) compared the different methods of parent-offspring regression. They stated that keeping offspring separate and repeating the parents is the appropriate weighting method if the correlation among the offspring was equal to zero, while taking the average of the offspring's value would be more appropriate if the progeny's correlation was equal to 1. They proposed a weighted regression method which would work with intermediate 80 correlations. The weighting method used for this paper is a modified form of the method they proposed (Falconer, 1989). The heritability estimate for fecundity is approximately 0.2. This low estimate is typical for fecundity (Falconer, 1989). Roff and Mousseau (1987) reviewed the literature containing heritability estimates for Drosophila and reported that fecundity estimates tended to be scattered, although low. The estimate reported here is approximately in the middle of the estimates Roff and Mousseau, (1987) found in the literature. It may not be valid to compare heritabilities in diplodiploids with those estimates obtained for haplodiploids. This is because loci in haploid males are hemizygous and expose more additive genetic variance which might result in larger heritability estimates. These larger estimates are valid and reflect the mating system of haplodiploid organisms, not experimental bias. The heritability estimate for developmental rate (0.24-0.40) is in the low to moderate range in general, but is towards the high end of those estimates reported for development rate inDrosophila (Roff and Mousseau, 1987). Mousseau and Roff (1987) report that most heritability estimates for life history traits are below 0.4 81 (median = 0.26). Most of the estimates used by Mousseau and Roff were for diploid organisms, thus creating the possibility for invalid comparisons to haplodiploid species. Real differences would be expected due to niche variation between Tetranychidae and Hymenoptera, the latter one of the few haplodiploid arthropods for which heritability estimates have been reported. Spider mites are small and thus highly susceptible to environmental variation within and among host plants. This contrasts sharply with the larger pupae of hymenoplerous parasitoids, which tend to exploit hosts that vary in size and quality. An even greater contrast arises between mites and social Hymenoptera, which maintain careful environmental control and are thus less susceptible to environmental fluxuation. Less environmental fluxuation allows for selection for developmental rate over time and results decrease in additive genetic variation for developmental rate, and hence lower heritability estimates. The heritability estimate for sex ratio (0.48-0.60) is in the moderate to high range. This value may be due to the role sex ratio serves in providing mites with flexibility for rapid change in stressful environments. Tetranychus urticae is a colonizing species 82 that rapidly overruns its host and, therefore, may be subject to continually decreasing host quality during each colonizing episode. Since these animals experience great environmental variation during a colonizing episode, a flexible sex ratio response would expose more haploid males. Falconer (1989) states that organisms that are in a poor environment would tend to have a larger amount of genetic variation than those in a good environment. The ability to rapidly adjust to variable environmental qualities should also involve the mite having a great deal of phenotypic plasticity in sex ratio so that they may present a wide spectrum of sex ratios when faced with difficult conditions. Therefore, a high heritability for sex ratio may be advantageous and not unexpected despite its importance as a fitness component. Bull (1987) points out that genetic variance (measured as a high heritability estimate) can be maintained by temporal variation in fitness for that trait. Mousseau and Roff (1987) state that additive genetic variance in fitness traits can be maintained in natural populations due to a number of possible causes. Fluctuating environments were one of the given m echanism s likely to occur in mite populations. Other mechanisms, described by Mousseau and Roff (1987), which might be 83 maintaining genetic variation for sex ratio Tetranychus in are: frequency dependent selection, heterozygote advantage and negative correlations with other fitness traits. Frequency dependent selection should be a major factor in sex ratio evolution. Sex ratio was not correlated with either fecundity nor developmental rate in this study. Sex ratio is, therefore, an independent trait. Although our estimates of around 0.6 are high, they are not unreasonable for fitness traits (Mousseau and Roff, 1987). A sufficient amount of genetic variation(V g> was maintained in the Columbus ‘89 stock culture despite its maintenance under laboratory conditions for approximately 50 generations. Retaining a good amount of Vq was a result of maintaining large population sizes throughout the entire time the stock culture remained in the laboratory. The effects of random genetic drift were minimum since the effective population size (Ne) was rarely permitted to drop below 900 individuals, and then only during transfer when fresh leaves were scarce. Takafuji and Ishii (1989) reported a heritability estimate, in Tetranychus kanzawai Kishida, of 0.85 using mother-daughter regression. This estimate was not significantly different from mine given the standard error of their regression line. My heritability estimates, for sex ratio, were higher than those reported by Parker and Orzack (1985) for Nasonia vitripennis. They incorrectly multiplied the slope by 1.5 (instead of 2.0) to obtain realized heritability estimates for sex ratio. Correcting for this results in their estimates being 0.15 and 0.17, still much lower than the estimates that we obtained. There are a few possible causes for the differences between the estimates. First, realized heritabilities tend to be lower than other methods, partially due to a decrease in additive genetic variance because of selection. This is especially true for highly heritable traits (Falconer, 1989). If the population sizes used in Parker and Orzack's (1985) study were much smaller, there may be drift effects (Falconer, 1989). Also, there may be real differences in the genetic variances for this trait, especially given the large difference in biology between a wasp and a mite. Wasps are much larger and utilize more stable hosts, such as insect eggs and pupae, that exhibit a smaller degree of environmental fluxuation. Mites, however are smaller and very much at the mercy of very small changes in the environment. Given these differences, it is quite 85 likely that the genetics of sex ratio regulation is under different constraints in acarines compared with hymenopterons. There was a significant, negative, correlation between fecundity and developmental rate. Crowding appeared to decrease the rate of development. This was an unexpected result since crowding would be expected to increase the feeding time hence increase the time for development. Falconer (1989) predicted negative correlations between fitness traits. On the surface, this would appear to be the case. Directional selective pressures should generally act for increasing fecundity and decreasing the time necessary for development. This negative correlation may be more a result of experimental procedure rather than a real genetic correlation since the mites were raised on small leaf disks which resulted in crowded conditions directly proportional to their mother's fecundity. In nature, crowding would be the result of the length of time in a colonizing cycle and not directly related to the mother's fecundity. Roff and Mousseau (1987) reviewed the literature on correlations between fitness components and observed that the magnitude and sign of these correlations were variable between different populations ofDrosophila, suggesting that these 86 differences may be due to the choice of traits measured, the amount of inbreeding or the evolutionary history of the stock cultures used. List of R eferences Becker, W. A. (1985) Manual of Quantitative Genetics. Fourth Edition. Academic Enterprises, Pullman, Washington, USA. Bull, J. J. (1987) Evolution of phenotypic variance. Evolution 41(2): 3 0 3 -3 1 5 . Bull, J. J., R. C. Vogt and M. G. Bulmer (1982) Heritability of sex ratio in turtles with environmental sex determination.Evolution 36: 333- 341. Crow, J. F. (1986) Basic Concepts in Population. Quantitative, and Evolutionary Genetics. W. H. Freeman and Company, New York. Falconer, D. S. (1989) Introduction to Quantitative Genetics. Third Edition. Longman Scientific & Technical, Longman House, Burnt Mill, England. Kempthorne, O. and B. Tandon (1953) The estimation of heritability by regression of offspring on parent.Biometrics 9: 90-100. Margolies, D. C. and T. S. Cox (1993) Quantitative genetics applied to haplodiploid insects and mites. In: Evolution and Diversity of Sex Ratio in Insects and Mites. Chapman and Hall Publishers. New York and London, D. L. Wrensch and M. A. Ebbert (Eds.), pp. 548-560. Mitchell, R. (1972) The sex ratio of the spider mite Tetranychus urticae. Entomol. Exp. & Appl. 15: 299-304. 87 Mousseau, T. A. and D. A. Roff (1987) Natural selection and the heritability of fitness components. H eredity 59: 181-197. Orzack, S. H., E. Davis Parker and J. Gladstone (1991) The comparative biology of genetic variation for conditional sex ratio behavior in a parasitic wasp, Nasonia vitripennis. Genetics 127: 5 8 3 -5 9 9 . Overmeer, W. P. J. and R. A. Harrison (1969) Notes on the control of sex ratio in populations of the two-spotted spider mite,Tetranychus urticae Koch (Acarina: Tetranychidae).N. Z. J. Sci. 12: 920-928. Parker, E. Davis and S. H. Orzack (1985) Genetic variation for the sex ratio in Nasonia vitripennis. Genetics 110: 93-105. Ram, A. and A. K. Sharma (1977) Selective breeding for improving the fecundity and sex-ratio ofTrichogramma fasciatum (Perkins) (Trichogrammatidae: Hymenoptera), an egg parasite of lepidopterous hosts. Entomon. 2: 133-137. Roff, D. A. and T. A. Mousseau (1987) Quantitative genetics and fitness: lessons from Drosophila. Heredity 58: 103-118. Roush, R. T. and B. E. Tabashnik (1990) Pesticide Resistance in Arthropods. Chapman and Hall, New York. Sincich, T. (1985) Statistics bv Example. Second Edition. Dellen Publishing Co. San Francisco. Takafuji, A. and T. Ishii (1989) Inheritance of sex ratio in the kanzawai spider mite, Tetranychus kanzawai Kishida. Res. Popul. Ecol. 31: 123-128. Young, S. S. Y., D. L. Wrensch and M. Kongchuhesin (1986) Control of sex ratio by female spider mites.Entomol. Exp. Appl. 40: 53-60. CHAPTER IV HERITABILITY OF FECUNDITY, DEVELOPMENT RATE AND SEX RATIO IN TWO GENERATIONS OF MITES SUBJECTED TO DIFFERENT TEMPERATURE REGIMES (25<>C AND 32<>C). Introduction Female spider mites are capable of controlling their sex ratio (Mitchell, 1972, Young et al., 1986) by some as of yet unknown physiological mechanism (Feiertag-Koppen and Pijnacker, 1985; Helle and Pijnacker, 1985). Although it is commonly believed that sex ratio has a strong genetic component (Overmeer and Harrison, 1969; Mitchell, 1972), there is little empirical evidence to show that there is an additive genetic component and thus the potential for response to selective forces. Chapter III of this document is one of the few studies that demonstrate additive genetic variance for progeny sex ratio. Both the mean and variance of a female's progeny sex ratio change in response to environmental cues (Wrensch, 1993), 88 89 such as temperature (Young et al., 1986), host plant quality (Young and Wrensch, 1981) and population density (Andersen, 1961; Wrensch and Young, 1978). McEnroe (1967) discussed the importance of haploid males in protecting a population from the effects of inbreeding and random genetic drift, as well as stating that the selection of new chromosome combinations would occur more rapidly in the hemizygous males. Bodmer and Parsons (1962) also discussed the importance of males in assorting the genetic variability, which can then be rapidly selected upon since males are hemizygous. Quick changes in response to selective pressures are therefore expected since more genetic variability is exposed in the male (McEnroe, 1967). Thus, arrhenotoky results in an accelerated temporal response which facilitates adaptation to environmental changes (Wrensch, 1993), including the rapid resistance to acaricides observed in agriculture (Havron et al., 1987; Helle, 1965; Helle and Sabelis, 1985). Although similar to what other authors describe as phenotypic plasticity and norm of reaction (Bull, 1987; Falconer, 1989), the environmental sensitivity of sex ratio serves a specific purpose. 90 Bull (1987) described differential responses to varying environmental cues as one way in which genetic variation may be maintained in a highly selected fitness trait. The ability to alter the proportion males, in response to the environment, can be thought of as haploid sons reflecting the recombinant genotypes of their mother (Wrensch et al., 1994). This aspect of males means that the production of a lower sex ratio (higher proportion of males) results in an in increase in the effective recombination of a female s diploid genotype and thus her ability to produce a greater variety of males to mate with her daughters. Increased genetic recombination, as a result of environmental stress is not a new idea. As early as 1917, Plough (1917) reported that recombination increased inDrosophila melanogaster when subjected to temperatures above or below those normally encountered. Darlington (1939) stated that recombination is a generalized response to environmental stress. Therefore, males could serve two major purposes: to reconstitute the original genome and to facilitate rapid adaptation to changing resources. The level to which a trait may be plastic, that is its ability to reflect different phenotypes in different environments, is believed to be under selective pressures because the resultant phenotypic response 91 may assist adaptation to localized environments (Via and Lande, 1985). This is especially true for sex ratio since its regulation is a highly adaptive trait for colonizers of annual plants (Wrensch, 1993). Bull (1987) predicted that an increase in temporal variation for the optimum phenotype would result in an advantage of that underlying genotype to produce a large range of phenotypes and thereby to increase the chance that at least some of the progeny would be close to this optimum phenotype each generation. By decreasing the progeny sex ratio while increasing its variance, a female would produce the greatest range of haploid recombinant phenotypes for the next generation (Wrensch et al., 1994). Not only will sex ratio be adjusted in response to the environmental cue, but so will a range of additional traits because haploid males are hemizygous recombinants of their mothers and open to all selective forces. Therefore, females can efficiently adjust to varying environments by shifting to producing a greater proportion males under deviant, stressful conditions. Temperature is a good environmental cue to use for the study of sex ratio plasticity since it influences several important traits 92 in arthropods (Congdon and Logan, 1983; Crookar, 1985; Wrensch, 1993). This experiment examines sex ratio regulation in terms of changes in both means and variances, as well as assessing how narrow sense heritabilities responded to increased temperature. Since mites used in this experiment were maintained for several years at 25<>C, it was assumed that they had locally adapted to that temperature. Rearing them at 32°C was considered to be a substantial environmental change and thus the deviant environment for this study. Materials and Methods The Columbus '89 stock culture had been maintained under laboratory conditions for approximately 120 generations prior to the start of this experiment in September, 1991 (see General Methods section for maintenance procedures of the stock culture). Two temperature regimes were chosen for this experiment: the treatment group was reared and oviposited under hot conditions (32°C), and the control was reared in the same temperature as the stock population (25°C). 93 Mites grown at 25°C remained on a central table within the environmental control chamber which also housed the stock population. Those mites grown under the experimental treatment of 32°C, were placed within a General Electric incubator and given the same light regime as the controls. Trays were rotated every second day throughout the course of the experiment. This was done to minimize and randomize any microenviromental effect of position within the tray or incubator. As discussed in the General Methods section of this document, a grandparent generation (GP) was initiated by randomly selecting healthy-looking teneral adults from the stock population and placing them on 2 cm diameter leaf disks in either hot (32oC) or control (25°C) temperatures. It was presumed that these females were mated within the stock population and were not provided with an additional male. Two larger trays of GP females, containing 3 smaller trays with 32 leaf disks each, were set up in each temperature. The reason for using only two trays per trial, when starting the GP generation, was that 4 of these trays were the maximum number that could fit into the smaller incubator (32<>C). 94 After transfer, females were left to oviposit in place until their first larva was observed on the leaf disk. At that point, the female was removed. The purpose of interrupting oviposition upon the emergence of the first larvae was to synchronize the physiologies between the two temperatures. Since mites develop much faster at the higher temperatures, removing females after a fixed number of days of oviposition would bias the fecundity estimates upward within the hot line. Lifetime oviposition was not used for the same two reasons which are discussed in the Methods section of Chapter III of this document. The first reason regards the problem of overlapping generations and the second reason regards the necessity of large sample sizes required to obtain valid heritability estimates. The small (2 cm diameter) leaf disks degenerate far too rapidly when large numbers of immature mites are present, especially disks used in the treatment group s incubator (32°C). Immatures on each leaf disk developed in place until they emerged as adults. One teneral female and one adult male were taken from each leaf disk transferred to a fresh one to initiate the parent (P) generation. If an adult male was not available in each 95 family, one was taken from a nearby leaf disk to increase the probability of insemination of each female. Parent females were observed each day after transfer and the day of their first egg was recorded. When the first larva subsequently emerged, both male and female parents were removed from the leaf disk and their progeny were permitted to develop in place. All progeny were counted and removed daily as they emerged as adults. These progeny comprised the parent (P) generation data. Progeny counts were tabulated to provide fecundity and sex ratio for each parent female within the P generation. From each P generation leaf disk, 5 sons and 5 teneral daughters were removed during the daily counts and each pair was transferred to individual leaf disks. These young adult pairs became the parents in the offspring (O) generation. Females were permitted to oviposit in place until the first larva emerged from their progeny, at which point both adult parents were removed. Daily counts of progeny were again made as they emerged as adults. These counts provided the raw data for the O generation. Developmental rate (DR) was scored by subtracting the day of the first egg from the day of first adult progeny emergence for both 96 P and O generations. Fecundity was recorded as the total number of adult offspring from each female. The proportion females in each progeny, or sex ratio (SR), was calculated by dividing the total number of adult females by the total number of adult offspring. Two replications of this experiment were required in order to obtain a sufficient sample size for the heritability calculations. The population size of each replication was the maximum that would fit into the smaller incubator (32°C). Since mites develop much faster at 32°C, two trials were possible, while still overlapping the 'first replication at 25°C. This resulted in 3 trials of the treatment population overlapping with the 2 replications of control animals. Temperature readings were taken daily at both temperatures so as to assure consistency within environments. Mean temperatures were not significantly different by replication or trial within run when t-tests were used to compare the means. The mean temperatures were 25.4±2.4oC for the control, and 32.0±0.5°C for the heat treatment. Data were entered into Statview II on a Macintosh llci. Thea priori deletion methodology discussed in the General Methods section of this document was used for each generation and 97 temperature in order to provide a standard data set for further analysis. For all calculations, the proportions female were transformed using arcsine squareroot transformation. Heritability estimates were estimated by three methods: full sib correlation, mother-mean daughter regression, and mother- weighted mean daughter regression. Methodology used for these calculations is described in further detail in the General Methods section of this document. The Analyses of Variance (ANOVA) used for calculating intraclass correlations were performed on the IBM 3090 600 J using SAS 6.07. All other calculations were performed with Minitab 8.2 on the Macintosh llci. R esults Descriptive statistics and mean comparisons for mites grown under control conditions (25oC) are shown in Table 10. Descriptive statistics and means comparisons for mites grown in the treatment population (32°C) are also reported in Table 10. The mean sex ratio was significantly higher in the O generation at 32<>C. Fecundity and developmental rate did not vary between the P and O generations at 32<>C. No generational difference in sex ratio was observed within 98 Table 10. Number of families (N), means and standard errors (SE) of fecundity, development rate (DR) and sex ratio (SR) for mites that developed and oviposited at control (25°C) or treatment (32oC) temperatures for both parent (P) and offspring (O) generations. Different letters in same row designate that means are significantly different at the a=0.05 level. P Generation Q Generation Temperature/ T ra it N_ Mean 1SE1 U MeacUSE) Q.polr-pJ, Fecundity 236 48.92(1.29)a 970 43.38(0.55)b DR 236 9.68(Q.05)a 969 10.00(0.03)b SR 236 0.622(0.009)a 970 0.626(0.005)a Heat Treatment Fecundity 241 37.64(1.06)a 996 37.23(0.47)a DR 240 6.12(0.03)a 995 6.06(0.02)a SR 241 0.488(0.013)a 996 0.543(0.006)b 99 the control experiment. The mean fecundity was significantly higher in the parent generation. Mites in the parent generation also had progeny which developed faster. An ANOVA was calculated in order to see if replicate or trial had any effect on fecundity, developmental rate or sex ratio in either temperature or generation. The results are shown in Table 11. Trial had a significant effect on fecundity and developmental rate for mites grown in the hot environment for both parent and offspring generations, but no significant effect was found for sex ratio. Replicate had a significant effect only on developmental rate of the offspring generation in the control population. Heritability estimates for the three fitness traits are shown in Table 12. The heritability estimate for fecundity was significantly higher in the treatment population (0.15) compared to the control (0.10). Heritability estimates for developmental rate could not be compared since only one estimate could be calculated. The weighted parent-mean offspring regression was not calculated for developmental rate in the treatment experiment since the intraciass correlation was required for the weights and could not be obtained through the full sib correlations, since the p-values from Table 11. Summaries of a one-way analyses of variance for fecundity, development rate (DR) and sex ratio (SR) among replications within control (25°C) and treatment (32°C) temperatures. Complete analyses found in Appendices B and C. P arent O ffsprino Trait a E o-value E o-valu Control Temperature Fecundity 244 4.632 0.032 970 0.978 0.323 DR 244 0.519 0.472 969 337.187 0.001 SR 244 2.003 0.158 970 0.260 0.610 High Temperature Fecundity 272 6.193 0.002 996 23.236 0.001 DR 270 16.225 0.001 995 10.634 0.001 SR 272 1.602 0.203 996 1.047 0.351 100 101 Table 12. Heritability estimates in both treatment (32<>C) and control (25°C) environments. Methods of calculating the estimates are: full sib correlation (Full Sib), parent-mean offspring regression (Par-MnOff), and a weighted parent-mean offspring regression (Par- WtMnOff). N = The sample size, h2 = The heritability estimate, SE = The standard error of the heritability estimate. Trait/ Treatment G.ontml Method h! H2 {SB U t 2 { s a Fecunditv Full Sib 996 0.15 (0.06) 970 0.10 (0.06) Par-MnOff 241 NS* 236 NS* Par-WtMnOff 241 NS* 236 NS* Develoomental Rate Full Sib 995 NS* 969 0.50 (0.09) Par-MnOff 240 NS* 236 NS* Par-WtMnOff NA (see text) 236 NS* Sex Ratio Full Sib 996 0.43 (0.07) 970 0.29 (0.08) Par-MnOff 241 0.22 (0.01) 236 0.32 (0.01) Par-WtMnOff 241 0.26 (0.01) 236 0.35 (0.01) * If the ANOVA or slope of the regression was not significantly different from zero, NS (not significant) is denoted instead of a heritability estimate. 102 ANOVA s were not significantly different from 0.00. The slopes of all parent-offspring regressions were not significantly different from 0.00 and thus heritabilities could not be estimated by any of the regression methods for fecundity and development rate. The heritability estimates for sex ratio range between 0.22- 0.43 for the different methods of calculation and temperature regimes. The heritability estimate was higher for the treatment population by using the full sib correlation method, but lower when compared with either of the regression methods. D iscussion Not all of the traits and calculation methods yielded heritability estimates. Most of the fecundity and developmental rate calculations did not provide significant p-values for the ANOVA's or regressions so the heritability estimates could not be calculated. Only the full sib correlation could be calculated for estimating the heritability estimate for fecundity. This is not especially surprising, because heritabilities for fecundity tend to be very low and highly susceptible to any variation in the environment (Falconer, 1989). The full sib correlation method tends to bias 103 estimates upward because full sisters developed on the same leaf disk also, sample size was larger in the full sib correlation analysis. Developmental rate is also a trait expected to have low genetic variation and is also highly sensitive to the environment. The estimate for development rate could only be obtained from the control population. The parent-weighted mean offspring regressions could not be calculated because weights could not be obtained when the ANOVA of full sibs did not have a significant (a 0.05) p-value. Heritability of sex ratio was significant, however. Thus, all three methods of estimating heritability were calculated. Estimates for sex ratio could be obtained, in part, because the numerical value for sex ratio is accurately measured compared to those fecundity and developmental rate. Each family consists of several males and females from which the proportion female was obtained. Heritability estimates could not be calculated for development rate perhaps, in part, due to it being recorded at no finer resolution than by day (integer). These records are very coarse, especially given that only one value was recorded for each day and mites emerged throughout each day. Therefore, measurement insensitivity caused minor differences in development rate to be missed. Fecundity was 104 also recorded in integers, but each female laid several eggs so the records were sufficiently accurate for heritability to be estimated. Phenotypic variation is often discussed in terms of plasticity in expression of the trait. The norm of reaction for a trait is the range of phenotypes that are specified by a particular genotype under different environments (Hartl and Clark, 1989). When reaction norms are not parallel, they can be measured by the genotype x environment interaction (Vqe) terms from the factorial analysis of variance. These interaction terms are a measure of phenotypic plasticity, although there should be considerable care taken in interpreting their analyses (Gregorius and Namkoong, 1986; Yamada et al., 1988). Norms of reaction and adaptive phenotypic plasticity are most widely associated with plants (Bradshaw, 1965; Schlichting and Levin, 1986; Schlichting, 1986) but have been empirically studied inDrosophila (Gupta and Lewontin, 1982; Scheiner and Lyman, 1989; Scharloo, 1989), carabid beetles (Desender, 1989), milkweed bugs (Groeters and Dingle, 1988), cockroaches (Parker, 1984) and avians (van Noordwijk, 1989). The evolution of adaptive plasticity is also of great interest to biologists (Charnov, 1982; Via and Lande, 1985; Via, 1988; Schlichting, 1989; West-Eberhard, 1989). Waddington's (1959) description of canalization is synonymous with what Falconer (1989) called environmental sensitivity and are closely aligned conceptually to the revived form of phenotypic plasticity (.eg . Bradshaw, 1965). To establish that plasticity is adaptive, and has thus evolved, requires the minimum stipulation that there is additive genetic variation of this trait. The unit upon which this selection operates, the limit to the variation in responses and the importance of these responses within the population structure are still highly conjectural. Empirical tests of plasticity theories are important to further understanding (Schlichting, 1989). Trade-offs among life history traits leading to negative correlations were assumed to be necessary for maintaining genetic variation, but recent data challenged this stipulation (Scheiner et al., 1989). Contrary to Bradshaw's (1965) and others prediction that selection for phenotypic flexibility would operate antagonistically to genetic variation, Scheiner and Goodnight (1984) showed, with simultaneous measure of plastic and genetic variation in a grass, that no correlation existed between the two types of variances. 106 Sex ratio in spider mites offers a unique extension of these empirical studies: to a hapiodiploid species that, as argued for plants, exist in a spaciotemporally unstable, unpredictable environment. The consistent observation that sex ratio is highly variable has not been evaluated within the context of adaptive phenotypic plasticity, nor has the genetics of plasticity been evaluated for any life history trait in a nonsocial hapiodiploid species except for within this chapter. Sex ratio regulation is believed to be adaptive, as evidenced by different phenotypic values in different environments (Wrensch and Young, 1978; Young and Wrensch, 1981). Relatively more males are produced away from optimal conditions and this is believed to lead to a faster rate of evolution in these less favorable environments (Wrensch, 1993). Sex ratio is expected to be adjusted downward, and its variance increased, above or below the optimum temperatures for each strain of mites (Wrensch, 1993). Since the local laboratory population should be adapted to 25°C, mites grown and ovipositing at 32<>C should exhibit an adjustment in sex ratio. The results of the current experiment do agree with these 107 predictions of a decrease in sex ratio and an increase in its variance, with sex ratio being adjusted in both parent and offspring generations. Higher heritabilities are also predicted in deviant environments, suggesting more additive genetic variation exposed to selection (Wrensch, 1993). This experiment provides mixed results when comparing heritabilities between deviant (32°C) and normal (25°C) environments. Full sib correlations give estimates which concur with the predictions of sex ratio theory. The heritability estimate for mites grown at high temperature was nearly twice that of those grown in control conditions. Even the estimate for fecundity was slightly higher in the treatment population. This result was surprising since heritabilities are generally expected to decrease in 'deviant' environments (Falconer, 1989) and might suggest an interesting possible side effect of the hapiodiploid mating system. 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DISCUSSION The experiments described within the body of this document provide valuable insight to the genetics of sex ratio regulation in an agriculturally important pest species. Results show that spontaneous thelytoky is probably extremely rare in T. urticae. Males were found to have no effect on their progeny sex ratio, and probably little or no effect on their mate's fecundity. These findings are essential to interpreting the experimental results of sex ratio genetic analyses. They prove that sire effects may be disregarded when designing heritability experiments, and that the a particular sire would not influence analyses because sex ratio is solely under the control of the female. The mean sex ratio of all experiments reported within this document is approximately 0.63, much lower than the 0.75 proportion female reported by other authors. Although the sex ratio reported here is differs from that reported by others, it is not a 113 114 concern. The sex ratios reported in this document are from means of the first 5-7 days of oviposition, but 85-95% of the eggs are laid by day 11 (Young et al., 1986). Since the first day of oviposition is heavily biased towards males (Helle, 1967), and these present calculations include the first week of oviposition, a slightly lower estimate for sex ratio is not surprising. Sex ratio is an interesting life history trait which has received long-standing attention for its ecological and evolutionary importance (Charnov, 1982; Wrensch, 1993). Essentially all arthropods with haplodiploid mating systems are characterized by female biased sex ratios (Wrensch and Ebbert, 1993). The degree and changes in the proportion females result in important consequences within agricultural systems and for evolutionary interpretations. Sex ratio ultimately affects the number of dispersers, the probability of their success and thus the chance of colonization onto new hosts (Kennedy and Smitley, 1985; Mitchell, 1970), the rate of evolution to host defenses (Crozier, 1985) and pesticide resistance (Havron et al., 1991; Havron et al., 1991) and changes in the environment and microclimate (Wrensch, 1993). 115 Historically, sex ratio was recognized as an important factor in biological control programs using parasitic wasps (Kfir and Luck, 1979; Waage, 1982a) but was generally underrated as an important life history trait and fitness component affecting the intrinsic rate of increase (rm) and the evolution of beneficial species (Sm) (Wrensch, 1993; Waage 1982a; Waage, 1982b; Waage and Hassell, 1982) and their prey. Sex ratio regulation in haplodiploids results in a change in both the mean and variance, with a decreased proportion of females and increased variance as a result of environmental stress (Young et al., 1986), thus indicating that this trait is highly adaptive. It is argued that increasing the proportion of males in the progeny provides an efficient way to reconstitute the original genotype or to increase the efficiency of selection to new conditions (See Introduction section of this document). Researchers working with haplodiploid arthropods are generally convinced that sex ratio regulation has a genetic basis, but most of the experimental work is restricted to hymenopteran parasitoids (King, 1987). However, a large body of work on spider mites is available (Helle and Sabelis, 1985; Wrensch, 1979; Wrensch, 1985) and some research relevant to understanding sex ratio genetics is starting to accumulate (Mitchell, 1972; Overmeer and Harrison, 1969; Takafugi and Ishi, 1989; Wrensch, 1993; Young et al., 1985; Young et al., 1986). This document provides one of the first clear studies that shows sex ratio regulation to be highly heritable in mites and is the first to indicate that its phenotypic plasticity is probably also heritable. Heritability was significantly higher for mites grown in elevated temperatures (32oC) compared to those grown under control (25oC) conditions. Such findings indicate a large amount of additive genetic variance is present, which is a prerequisite for selection. However, the ability for directional selection to operate on progeny sex ratio is not symmetrical. It is the proportion of males that are increased as a response to environmental stimuli. Thus far, no selection experiments have increased the proportion female. Although comparing heritability estimates in different environments gives some information as to the inheritance of the phenotypic plasticity of this trait, further studies are needed. Comparisons of inbred lines of mites, under several temperature regimes, will allow proper partitioning of the variance into its components (Falconer, 1989) and thus provide an estimate of the genotype-environment interaction(V q e )- An accurate estimate of the genetic correlation would be able to provide information on whether sex ratio is controlled by similar or different genes in the different environments. APPENDICES APPENDIX A. Analyses of Variance of Fecundity and Sex Ratio by Sire. Trial A: ANOVA of Fecundity by Sire Analysis of Variance Table Source: DF: Sum Sguares: Mean Sguare: F-test: Between groups 91 33529.225 368.453 1.208 Within groups 365 111296.617 304.922 o = .116 Total 456 144825.842 Model II estimate of between component variance = 12.819 118 119 ANOVA of Sex Ratio by Sire Analysis of Variance Table Source: DF:______Sum Squares: Mean Square: F-test: Between groups 91 2.124 1.023 .9 Within groups 365 9.469 1.026 p = .7244 Total 456 11.594 I Model II estimate of between component variance = -.001 Trial..,g: ANOVA of Fecundity by Sire Analysis of Variance Table Source:______DR______Sum Squares: Mean Square: F-test: Between groups 61 24003.994 393.508 1.589 Within groups 276 68361.662 247.687 p = .0069 Total 337 92365.657 Model II estimate of between component variance = 26.875 120 ANOVA of Sex Ratio by Sire Analysis of Variance Table Source: DF: Sum Squares: Mean Square: F-test: Between groups 61 1.414 .023 .86 Within groups 276 7.439 .027 p = .757 Total 337 8.853 Model II estimate of between component variance = -.001 APPENDIX B. Analyses of Variance of Development Rate, Fecundity and Sex Ratio by Replicate for the Control Population (25<>C). Parent Control: ANOVA of Fecundity by Replicate Analysis of Variance Table Source: DF: Sum Squares: Mean Square: F -test: Between groups 1 1921.916 1921.916 4.632 Within groups 242 100414.232 414.935 D = .0324 Total 243 102336.148 Model II estimate of between component variance = 13.147 121 122 ANOVA of Developmental Rate by Replicate Analysis of Variance Table Source: DF: Sum Sguares: Mean Square: F-test: Between groups 1 .284 .284 .519 Within groups 242 132.417 .547 0 = .4721 Total 243 132.701 Model II estimate of between component variance = -.002 ANOVA of Sex Ratio by Replicate Analysis of Variance Table Source: DR______Sum Squares: Mean Square: F-test: Between groups 1 .039 .039 2.003 Within groups 242 4.665 .019 p = .1583 Total 243 4.703 Model II estimate of between component variance = 1.686E-4 123 Offspring Control ANOVA of Fecundity by Replicate Analysis of Variance Table Source: DF: Sum Squares: Mean Square: F-test: Between groups 1 290.358 290.358 1.978 Within orouos 968 287305.543 296.803 Id = .3229 Total 969 287595.901 Model II estimate of between component variance = -.014 124 ANOVA of Development Rate by Replicate Analysis of Variance Table Source:______DR______Sum Squares: Mean Square: F-test: Between groups 1 192.825 192.825 337.187 Within groups 967 552.991 .572 p = .0001 Total 968 745.816 Model II estimate of between component variance = .408 ANOVA of Sex Ratio by Replicate Analysis of Variance Table Source: ______DR______Sum Squares: Mean Square: F-test: Between groups 1 .006 .006 .26 Within groups 966 23.578 .024 p = .6105 Total 969 23.584 Model II estimate of between component variance = -3.820E-5 APPENDIX C. Analyses of Variance of Development Rate, Fecundity and Sex Ratio by Replicate for the Hot Population (32<>C). ANOVA of Fecundity by Replicate Analysis of Variance Table Source: DF: Sum Squares: Mean Square: F -test: Between aroups 2 3608.662 1804.331 6.193 Within aroups 269 78374.54 291.355 D = .0023 Total 271 81983.202 Model II estimate of between component variance ■= 17.236 125 126 ANOVA of Development Rate by Replicate Analysis of Variance Table Source:______DR______Sum Squares: Mean Square: F-test: Between groups 2 7.81 3.905 16.225 Within groups 268 64.5 .241 p = .0001 Total 270 72.31 Model II estimate of between component variance <= .042 ANOVA of Sex Ratio by Replicate Analysis of Variance Table Source:______DR______Sum Squares: Mean Square: F-test: Between groups2 .165 .083 1.602 Within groups 269 13.878 .052 p = .2034 Total 271 14.043 Model II estimate of between component variance = 3.539E-4 127 Offspring Hot: ANOVA of Fecundity by Replicate Analysis of Variance Table Source: DF: Sum Sguares: Mean Sguare: F -test: Between groups 2 9606.433 4803.217 23.236 Within groups 993 205270.831 206.718 p = .0001 Total 995 214877.264 Model II estimate of between component variance = 13.847 ANOVA of Development Rate by Replicate Analysis of Variance Table Source: DF: Sum Squares: Mean Square: F-test: Between groups 2 6.397 3.198 10.634 Within groups 992 298.357 .301 p = .0001 Total 994 304.754 Model II estimate of between component variance = .009 128 ANOVA of Sex Ratio by Replicate Analysis of Variance Table Source:______DR______Sum Squares: Mean Square: F-test: Between groups 2 .083 .042 1.047 Within groups 993 39.452 .04 P = .3512 Total 995 39.535 Model II estimate of between component variance = 5.677E-6 BIBLIOGRAPHY Alberti, G. and A. 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