The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:234-242
Inheritance of a Temperature-Modified Phenotype of the short antennae (sa) Mutation in a Moth, Ephestia kuehniella (Lepidoptera: Pyralidae)
From the Department of Genetics, Institute of Entomology, Czech Academy of Sciences, Branisovská 31, CZ-370 05 Ceské Budejovice, Czech Republic.
Address correspondence to Jaroslav Pavelka at the address aboveor e-mail: pavelka{at}entu.as.cz or pavelka{at}orange.lowtem.hokudai.ac.jp.
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Abstract |
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The autosomal recessive mutation short antennae (sa) causes considerable shortening of antennae in male and female Mediterranean flour moths (Ephestia kuehniella Zeller). However, the sa phenotype can be suppressed by several physical factors, making sa moths indistinguishable from wild-type moths (saWT). This can be done by subjecting larva and pupa to a higher temperature (25°C), to lithium ions, or to an alternate electric field. The first half of pupal development was found to be the sensitive period for the saWT phenotype. The saWT phenotype is stable and cannot be reverted to the original sa type by physical or chemical factors. The saWT phenotype is transmitted to future generations. When crossed with typical sa moths, the saWT phenotype is inherited either as a dominant character if carried by males or a semidominant character if carried by females. We compared proteins of the ejaculate, accessory gland secretions, and spermatophore in sa, saWT, and wild-type males and found considerable differences between sperm proteins of saWT, sa, and wild-type males. The saWT phenotype influences the mating success of males: saWT males mated successfully with any females, whereas typical sa males were less successful in mating and then mainly with females of the same phenotype.
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Introduction |
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One of the long-discussed questions of evolutionary biology is whether new heritable traits originate spontaneously and independently from the influence of external conditions. Current opinion accepts that better adapted genotypes have an advantage in the selective environment as opposed to the environment causing the adaptation. The latter option would mean that a new character could arise as a consequence of the environment during the course of life of an individual and be transmitted to the next generation. However, this phenotypic or "Lamarckian" mode of inheritance is at odds with basic rules of genetics. Such an observation indicates that the Weismann's barrier can be overcome when the offspring traits are inherited directly from the parental soma through the germ line (Bro
ek 1930; Johnston 1995; Kirk 1994). It is known that transitory treatment of certain single-cell systems or multicellular organisms with physical, chemical, or biological agents may induce en masse changes in particular characteristics that are then heritably transmitted. The molecular basis of this altered inheritance is quite different in different systems (Landman 1991). Suggested mechanisms include transmission of epigenetic information of gene expression by core histone acetylation/deacetylation (B
ek et al. 1998; Grewal et al. 1998), DNA methylation (Cubas et al. 1999), inherited epigenetic changes in the structure of chromatin (Jablonka and Lamb 1989), or reversion of a modified nonfunctional allele to the original functional state as a direct response to life conditions (Hall et al. 1983; Shapiro 1997; Storchová 1999). The evolutionary implication of mechanisms that allow acquired traits to be inherited are obvious: these processes will increase the fitness of a population in a selective environment. The phenomenon of phenotypic inheritance can be studied in the morphological mutation short antennae (sa) of the Mediterranean flour moth [Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)]. The sa mutation is inherited as a simple autosomal recessive and causes considerable shortening of antennae in moths of both sexes. When crossed with wild-type moths, the F1 moths are all long antennaed (Cotter 1960). However, the sa mutation exhibits unstable expression. Recently we found that it is temperature sensitive. At higher temperature the antennae of sa moths revert to the wild-type, which is then transmitted to subsequent generations. As far as we are aware, a similar phenomenon has not been described yet in sexually reproducing animals.
In Lepidoptera antennae develop from primordial structures know as imaginal antennal discs. For instance, in the silkmoth (Antheraea peryni), antennae develop from the leaf-shaped epidermal sac by means of segmental primary and secondary indentations that proceed from the periphery toward the centerline. The indentations are probably driven by long basal extensions of epidermal cells, the epidermal feet (Keil 1992). In Bombyx mori it was demonstrated that adult and larval antennae are produced by the same cells or their progeny (
vácha 1992). The ontogeny of body appendages from imaginal discs was most thoroughly studied in Drosophila melanogaster (Bate and Arias 1993).
Our study shows that the change of phenotype induced by external factors is inherited. We directed our research to better understand the dependence of phenotypic changes on temperature and other physical (light, electromagnetic field) or physiological (lithium ions) factors. To define mechanisms underlying heritable changes in the phenotype, we analyzed the possibility of extranuclear inheritance by transferring some factors via the male. Spermatophore, seminal fluid, and accessory gland secretions represent a considerable investment by the male in reproduction (Davey 1985; Kaitala and Wiklund 1995). The spermatophore and some components of seminal fluid contain materials destined to be digested by the female or incorporated directly into eggs (Boggs and Gilbert 1979; Gillot and Friedel 1977). To determine the effect of the inheritance of the acquired phenotype in a population, we studied the consequence of the phenotype on the mating success of the males.
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Materials and Methods |
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Flour Moth Strains, Rearing, and Handling
We used two strains of the Mediterranean flour moth [Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)]. The mutant strain Qy, homozygous for the autosomal recessive mutation short antennae (sa), was obtained from the stock cultures of W. B. Cotter, Jr. (Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY) and has been kept in single-pair cultures at the Institute of Entomology (Ceské Budejovice, Czech Republic) since 1991. The wild-type strain (WT-C) has been kept in a mass laboratory colony since 1984 (see Marec 1990). Stock cultures of both strains were reared in constant temperature rooms (20°C ± 1°C) at a 12-h:12-h light:dark regime, and at a humidity level of about 40%. Experimental and control cultures were kept at either 20°C ± 1°C or 25°C ± 1°C at the same conditions. At 20°C, development from egg laying to the emergence of the first imagoes required about 70–77 days, at 25°C about 40–42 days. In experiments with a lower (15°C) or a higher (30°C) temperature, cultures were kept in a temperature-controlled incubator. Larvae were fed milled wheat grains supplemented with a small amount of dried yeast. All experiments were done with single-pair cultures. Pairs were collected during copulation and placed individually into empty Petri dishes (6 cm in diameter). Females laid eggs for 3–4 days, then imagoes were removed and Petri dishes with eggs were put into plastic boxes with food. Hatching larvae migrated from the dish to the food where they completed their development. Details of rearing and handling methods are given in Marec (1990).
Factors Affecting Expression of the sa Character
In addition to the temperature treatments, two other factors were used for the suppression of sa character in the flour moth: electric field and lithium ions, because these two proved to have an effect (suppress wing development) in D. melanogaster mutants where expression pattern is sensitive to temperature (Pavelka and Jindrák, unpublished data; Pavelka et al. 1996). Homogeneous direct or alternating electric fields were generated by the electric field of an OMNI-BIO generator (Krivánek, Brno, Czech Republic) between two iron plates 2–3 cm apart. The alternating electric field had a sinusoidal character in all experiments.
Lithium chloride was used as a source of Li+ ions. The compound was dissolved in distilled water and mixed with an adequate amount of the wheat grout. This mixture was allowed to air dry and was then ground and added into the larval food. Based on the results of a preliminary test, concentrations ranging from 1.0 to 1.6 mg of LiCl per 1.0 g of food were found to be optimal for the treatment; the lower concentrations had no effect, whereas the higher concentrations were too toxic for the larvae.
The subsequent series of experiments was done to define the temperature-sensitive developmental period in sa mutants. The experimental schedule reflected the known Ephestia ontogeny at the above-mentioned conditions. Experimental sa cultures were reared at 25°C during different stages of the larval period and at 20°C for the remainder of their development. We used five different time windows at 25°C: (1) from the 1st to 3rd instar, (2) from the 1st to 4th instar, (3) from the 3rd instar to the adult stage, (4) only the 5th instar, and (5) only the pupal stage. Control sa cultures were kept at 20°C during their entire development.
Phenotyping
The effects of the temperature treatments, lithium, and electric field on the phenotypic expression of the sa gene were evaluated by classifying the moths into four categories based on the morphology of their antennae (Figure 1): (1) sa short (sa), with a typical expression of the sa allele, meaning moths with very short antennae; (2) sa wild type (saWT), with long antennae similar to the wild population; (3) sa nearly wild (saNW), with nearly wild length antennae; and (4) sa mixed (saM), with one short antenna and one medium or long antenna. The antennae were also observed for fine morphology of antennal segments with a scanning electron microscope using routine technology for preparing the samples. Samples were covered with gold by means of the sputtercoater POLARON E5100.
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Inheritance of Phenotype
The persistence of the acquired phenotype through subsequent generations was studied by intercrossing phenotypes (saWT x saWT, sa x sa) that resulted from the temperature treatments for up to six generations, while rearing the offspring at normal conditions (20°C). For retesting that the sa phenotype was caused by one gene (Cotter 1960), standard crosses of WT-C moths with sa moths were done in the P, F1, and F2 generations.
Protein Analyses
Ejaculates transferred by males to females via the spermatophore were analyzed electrophoretically. A male of a particular phenotype and known genotype was mated to a randomly chosen virgin female of the same strain. Immediately after the copulation ended, the female was dissected. The following components were removed for analysis: (1) the spermatophore sac from the bursa copulatrix, (2) the seminal fluid from the spermatophore containing both the eupyrene and apyrene sperm, and (3) the secretion of male accessory glands from the bursa copulatrix. Ten samples of each category were stored at -20°C, and later homogenized and submitted to SDS-PAGE electrophoresis. Polyacrylamide-gel electrophoresis was done using the BIO-RAD MINI-PROTEAN slab gel apparatus according to the manufacturer's procedures. Unless otherwise stated, all chemicals were supplied by SIGMA-ALDRICH.
Mating Success
For testing the mating success of males of different sa phenotypes (sa, saWT) and WT-C males, 100 L glass aquaria with net covers were used. Before starting the experiments, the space was divided into three parts by glass barriers to separate sexes and phenotypes. Virgin females were released into the middle section. Males of different phenotypes were released into the right and left parts. To allow identification, insects of different types were marked with different colors on the thorax. In the early morning, that is, at the beginning of the photophase period when flour moths exhibit a high level of sexual activity, the glass barriers were removed to allow mating.
Statistical Analyses
The effect of each factor (temperature, lithium chloride concentration, and electric field) on the expression of mutation sa in both sexes was tested with a three-way analysis of variance (ANOVA). A log transformation was performed on the data using log(x + 1) to handle zero values in the dataset and achieve normality and homogeneity of variance. Single statistical comparisons with control data were performed using the Mann–Whitney two-tailed test at the P < .01 significance level. The results from the mating success experiments were analyzed by one-way ANOVA.
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Results |
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Morphology of the Antennae in sa Moths
The structure of antennae in sa and WT-C moths was studied by using both the optical microscope and scanning electron microscope. The number of antennal segments in sa moths was reduced to 15–19 segments, whereas 51–55 and 53–56 segments were found in saWT and WT-C moths, respectively (Table 1). The antennae of saWT and WT-C moths did not differ morphologically from each other. In sa moths (Figure 2), various malformations of the last antennal segments were observed. These malformations are likely to affect important olfactory sensillae (Figure 3A,B versus C,D). The apical antennal segment was often narrowed and elongated or shortened and extended. In many cases, an aperture in the apex was seen.
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Higher Temperature Affecting Expression of the sa Character
Most control sa moths kept at 20°C showed standard expression of the sa mutant phenotype (Tables 2 and 3). Only a small proportion of moths showed saWT or saM phenotypes. When sa cultures were exposed to a temperature of 25°C during their entire development, almost all imagoes lost the sa phenotype (F1 generation) (Tables 2 and 3) and were classified as saWT moths.
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Data obtained to define the temperature-sensitive developmental period in sa mutants are presented in Figure 4. When sa cultures were reared at 25°C from the 1st to 3rd instar, and then at 20°C until adulthood (Figure 4B), the number of emerged moths that lost expression of the sa mutation was comparable with that in the control sa cultures kept for the whole development at 20°C (Figure 4A). In contrast, strong suppression of the sa phenotype was achieved when larvae were kept at 20°C until the 4th instar, and then at 25°C starting with the 5th instar until adulthood (Figure 4C). Similarly, shortening the exposure to 25°C at the 5th larval instar was sufficient for sa suppression (Figure 4D–F). However, when only the pupal stage was exposed to 25°C, the number of imagoes with the suppressed phenotype decreased considerably (Figure 4G,H). Our results suggest that the period of sensitivity to higher temperature is very short and involves the end of the 5th larval instar and the beginning of the pupal stage.
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Other Factors Affecting Expression of the sa Character
When lithium ions were added to the standard food at concentrations of 1.0–1.6 mg of LiCl per 1.0 g of food, a dose-dependent suppressive effect was achieved (Figure 5, Table 4). Besides the sa normal and sa wild-type categories, we also found a "nearly wild type" phenotype (saNW). At the concentration of 1.6 mg, almost all moths showed the saWT phenotype, similar to treatment with high temperature. However, this concentration prolonged the development of sa cultures considerably: from about 70 days to 110–150 days. A direct current electric field had no visible effect on sa expression (Figure 6A,B). On the contrary, an alternating current electric field with an intensity of 50 Hz increased the occurrence of saWT (Figure 6C) (for control, see Figure 7; for the statistical test, Table 5).
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Inheritance of Phenotype
When the saWT moths (F1 generation) were crossed with each other, the suppressive effect of higher temperature persisted to the next generation, even when the cultures were kept at 20°C (F2 generation) (Figure 8, Table 6) or at a lower temperature (15°C, data not shown). The same results were obtained at 20°C in the F3 and F4 (see Figure 8) and in the following generations (until F5 and F6, data not shown). However, when a rare sa moth emerged from these cultures and was mated with sa individuals, most progeny showed the sa phenotype (statistical data correspond to those in Table 2 and Figure 7).
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Phenotypes of individuals belonging to the offspring of single females (mated with single males) were evaluated. Although there was some phenotypic variability in the offspring of these pairs, it usually did not deviate greatly from the average (see Table 2). Moreover, the offspring sometimes (about 1–3 per 15 pairs) reversed phenotype from their parents (respective of parental phenotype). For example, in the control experiment (Figure 7), this situation occurred twice. These circumstances strengthen our belief that there probably had not been a reversion at the DNA level to the wild type (by mutation), but that the trait is carried to the next generation by an epigenetic mechanism.
It is evident that the saWT phenotype is dominant or semidominant over the sa phenotype, depending on the cross type (Figure 9, Table 7; for control, see Figure 7) Surprisingly, almost complete dominance of the saWT phenotype was observed in crosses between saWT males and sa females (Figure 9B), whereas reciprocal crosses produced sa, saNW, and saWT moths in similar proportions (Figure 9A). In the former crosses, the portion of saWT moths in the progeny was similar to that in the intercrosses of saWT F1 moths (see Figure 8; F2 and F3+4 in the upper half of the scheme).
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In the crosses between sa and WT-C moths, the phenotypic ratio in F2 moths was 3:1, as we expected (data not shown); in the F1, only wild-type individuals appeared. For this type of analysis, those imagoes were scored as the sa representatives in which the sa mutation was expressed, that is, all imagoes with shorter antennae (sa, saNW, saM), while in other experiments, individual types (sa, saNW, saM) were always distinguished. Thus the Mendelian segregation of the sa gene was confirmed. In addition, we concluded that the WT-C strain did not change the phenotype of sa moths to the saWT phenotype in the next generation, as the saWT strain did.
We also found that in progeny of saWT moths, the original sa phenotype cannot be recovered by treatment of the progeny with various physical factors. No change was noticed when different sources of light were used during development (daylight, electric bulb, fluorescent tube, infrared light) or when the cultures were kept under a constant light regime. Similarly, various combinations of low and high temperatures or treatment with an electric field (direct at 85 kV/m or alternating at 55 kV/m) had no effect on the saWT phenotype (data not shown).
Protein Analyses
Because the transmission of the saWT phenotype into the following generation must be realized via sperm and eggs, we tried to find some changes if possible. As shown above, the saWT phenotype is incompletely inherited from the mother but almost fully inherited from the father. Therefore we analyzed ejaculate proteins transferred by males of a particular phenotype to females during copulation. The proteins of the eggs were not analyzed because of their great number. We suppose that comparing different ejaculates is more informative than, for example, comparing antennae proteins, because in antennae there is a "factor" causing changes of the phenotype in following generations (DNA, RNA, maybe proteins). Analyzed proteins originated from sperm, spermatophore, and accessory gland secretions. Figure 10A shows that the pattern of bands from the sperm sample of WT-C and sa males differs considerably, in particular in the region of 30–40 kDa. In the sperm sample of saWT males, several protein bands are absent compared with samples of sa males. In the ejaculate without sperm (Figure 10B), there was no difference between saWT and sa males, but a large difference was found between WT-C males and both sa phenotypes. Similar results were obtained when the spermatophore proteins were examined (Figure 10C), but the saWT and sa differ from each other in proteins greater than 45 kDa in size. In the accessory gland secretion, a very small amount of proteins was detected and thus it was impossible to interpret the pattern obtained (not shown). We can conclude that different protein bands appeared between WT-C and sa (sa and saWT) strains in the fluid, and different bands were observed between the sperm and spermatophore in sa phenotypes.
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Mating Success
The last set of experiments revealed that the sa moths did not mate randomly (Table 8): sa males mated with sa females twice as frequently as with saWT females. That is, sa males all had less success in mating. The sa males were most successful with the sa females. In contrast, saWT and WT-C males succeeded in mating with all female phenotypes. WT-C males showed much higher mating success in competition with sa males. Finally, WT-C males were not significantly more successful than those of the saWT phenotype.
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Discussion |
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In the present study, we found that higher temperature, lithium ions, and alternating current electric fields suppress (the electric field only partially) the sa mutation: that is, sa moths with wild-type antennae (saWT) emerge instead of mutant ones, and this changed phenotype is transmitted to subsequent generations. The sensitive stage for the suppression phenotype occurs during the late 5th instar larval and pupal phase of the life cycle. Morphologies of antennae of different types were studied using scanning electron microscopy. The sa mutant phenotype was suppressed better through the paternal rather than the maternal lineage and the saWT phenotype is remarkably stable through the subsequent generations. In addition, saWT does not respond to various physical factors after the initial suppression event. Spontaneous reversal to the sa phenotype did not occur after treatment with diverse physical factors, although from time to time the progeny of one pair of saWT moths reverted back to the sa phenotype. It was demonstrated that the sa phenotype results from a single mutation. Great differences were found in the profiles of ejaculatory proteins between saWT and sa males. The saWT males were more successful (almost as successful as the wild males) in mating as the sa males.
One of the possible mechanisms by which physical factors such as temperature, lithium ions, or electric field can possibly change gene expression was suggested by experiments describe by Voet and Voet (1990) with lithium ions. Li+ ions were used to damage the phosphoinositoid cascade which altered the Ca2+ distribution in the cell (Voet and Voet 1990). We suggest that the effect of Li+ ions is somehow connected with the metabolism of Ca2+ ions, because Li+ ions interfere with the function of calcium channels. We presume the effect of an electric field is exerted on an organic structure that involves electric potentials, for example, cell membranes. Ion metabolism could be also influenced by temperature (Chopra and Singh 1994). This hypothesis could explain the change in gene expression (by temperature, lithium ions, electric field) in the first generation, but it does not explain the transmission of the effect onto subsequent generations. Temperature has been shown to influence gene expression in many organisms. For example, expression of the genes Curly and curled in D. melanogaster (see Lindsley and Zimm 1992) are known to be temperature dependent. However, in these cases, the reduced or lost expression is not transferred to the progeny when they are not exposed to the environmental factor (Pavelka et al. 1996).
Transposable elements could explain the sa suppression effect. Such an explanation would be supported by non-Mendelian transmission of the trait to subsequent generations. When mated with the wild type, however, the phenotype of the offspring of the sa strain maintained at low temperature (20°C) was transmitted as a single Mendelian trait. Thus it is not likely that the sa mutation is located on a transposable element.
It could be argued that the loss of sa expression was caused by reversion of the original mutant allele to a wild-type allele. However, this conventional hypothesis can be excluded for several reasons. First, a reverted mutation could never occur in the whole population, but would be expected only in some individuals. Second, the progeny of reverted mutants would follow Mendelian segregation and their phenotype would be more or less constant, whereas saWT moths showed irregular deviations from the wild type. Finally, in the progeny of reverted adults, the change to the original state, as was sometimes seen in our experiments, never occurred.
Another hypothesis for the mechanism of sa suppression is that the effect is controlled by a regulatory protein that possesses prion-like properties that is transferred via eggs or sperm. The relationship between prions and heat shock proteins has been discussed by Rother et al. (1997). They reported that scrapie prions modified the expression or localization of heat shock proteins, and one can see a certain analogy with the sa suppression induced by temperature. This hypothesis could be supported by the fact that the phenomenon arises under the influence of various physical factors without disturbing the DNA sequence. However, prions are transferred clonally from cell to cell, whereas we trace the transfer of the saWT trait only through germ cells.
Another alternative hypothesis could involve epigenetic causes of sa suppression. These may include acetylation of histones or modification of chromatin structure (Bek et al. 1998; Mielnicki et al. 1999; Turner 1998). This hypothesis seems to be supported by the results of experiments with Li+ ions, which do not necessarily need to damage the phosphoinositoid cascade, but can probably affect the chromatin directly. For example, in the sea urchin, Strongylocentrotus purpuratus, a delay in development, accompanied by a delay in the expression of a late class histone H2B gene, was observed in lithium-treated embryos (Nocente-McGrath et al. 1991). Transformations in the identity of imaginal structures in D. melanogaster can also result from epigenetic causes. These transformations can arise in a variety of ways aside from mutations in genes characterized as selector genes. Some observations raise the possibility that mutations which cause cell death in imaginal tissue could perhaps lead to a process akin to transdetermination in situ (Bate and Arias 1993). Although the phenomenon of sa suppression corresponds with the definition of epigenetic effects in several respects, it differs with respect to transmission to later generations.
The observed phenomenon can be also interpreted as Lamarckian inheritance. Although it is usually assumed that Lamarckian inheritance does not and cannot occur, molecular mechanisms by which nonmutational changes acquired in one generation can be transmitted to the next are now known. These mechanisms involve changes in chromatin structure rather than changes in DNA base sequences (Jablonka and Lamb 1990). It is becoming increasingly clear that chromatin modification plays a fundamental part in transcriptional control (Ashraf and Ip 1998). Lamarckian inheritance is supported by the occurrence of the change of expression in the whole population at the same time and it's transmission into subsequent generations. On the other hand, most examples of such a mechanism have been demonstrated in clonal cells (Grimes et al. 1980; Landman 1991) and do not compare to the phenomenon presented in this study because the factor changing the phenotype in sa mutants is transferred via the reproductive process.
Considering only the phenomenon of phenotype inheritance, it appears to be analogous to genomic imprinting, because the saWT phenotype was transmitted almost completely from the father and incompletely from the mother. Extranuclear (cytoplasmic) inheritance is unlikely because the trait would be expected to be transmitted maternally. The mechanism and causes of sa suppression have not been satisfactorily explained, and thus a relationship with imprinting and extranuclear inheritance remains unclear. Similar conclusions were reported by Jirtle (1999), who studied genomic imprinting in tumor cells. He noted that the parental level of nutrition, stress, and exposure to chemical and physical agents could function as imprint-altering factors and result in Lamarckian-like inheritance. This might appear like the observed phenomenon. But the stability of the phenotype in subsequent generations and its inheritance through both paternal and maternal lines (better through paternal line) does not correspond to the genetic imprinting.
DNA methylation may be a potential cause of the observed suppression effect. The Lcyc mutant of Antirrhinum is analogous to the sa mutant of E. kuehniella. A substantial analogy to our observations was described in a naturally occurring mutant of Linaria vulgaris. Cubas et al. (1999) showed that the mutant carries a defect in Lcyc, a homologue of the cycloidea gene that controls dorsoventral asymmetry in Antirrhinum. The Lcyc gene is extensively methylated and transcriptionally silent in the mutant. This modification is heritable and cosegregates with the mutant phenotype. Occasionally the mutant reverts phenotypically during somatic development, which is correlated with the demethylation of Lcyc and restoration of gene expression (Cubas et al. 1999).
Maybe the observed phenomenon is similar to Waddington's "genetic assimilation," the inheritance of some apparently "acquired" characters (Waddington 1953, 1956). Waddington described so-called phenocopies that developed after being exposed to external factors and were transferred into the following generations even without further influence of these factors. The phenomenon was called genetic assimilation (Waddington 1953, 1956). Rutherford and Lindquist (1998) have recently provided a molecular framework for these hypotheses. They discovered in Drosophila that heat shock protein Hsp90 seems to buffer the effects of cryptic variants by aiding the formation of appropriate secondary and tertiary structures. When Hsp90 buffering is compromised, for example, by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored (Rutherford and Lindquist 1998). Nevertheless, our results are not identical to the experiments of Waddington, where the selection was necessary to increase the frequency of phenocopies. In saWT the percentage of "phenocopies" predominated immediately in the next generation and was maintained at the same level, but our observations show similarity to "phenocopies" in many ways.
Our finding that electrophoretic bands of sperm proteins in sa moths differ from those in saWT and WT-C moths implies that the transfer of the saWT phenotype to following generations was mediated by changes in the content or composition of sperm proteins. In proteins transferred during copulation, we found differences between sa moths and WT-C control moths but not between sa and saWT moths. This implies that the sperm proteins are mainly responsible for the inheritance of the saWT phenotype. Such changes in the protein content of sperm are a very promising line of evidence for understanding this phenomenon. The suppression of the sa mutant phenotype was correlated more with changes in the ejaculatory proteins than with the treatments that generated the phenotypic alternation (temperature or lithium). We conclude that a structural change in a regulatory protein or a transcription factor was modified in sa moths. The altered protein is then transferred via germ line, particularly via sperm, to subsequent generations.
To evaluate the situation under natural conditions (under lower temperatures), we compared the mating success of four categories of moths. Low mating success in sa males most probably reflects the lack of proper reproductive behavior where aberrant antennae prevent appropriate pheromonal stimulation of the olfactory sensillae (contrary to Cotter 1960). The importance of olfactory sensillae in the mating of moths and butterflies has been demonstrated in many species (Kanaujia and Kaissling 1985; Schneider 1969). Our competition experiments between sa and saWT or WT-C imagoes indicate that the absence of olfactory sensillae in sa moths is the main reason for the higher mating success of saWT or WT-C males compared to sa ones.
An essential item of sa suppression is the fact that information is transmitted to subsequent generations through mechanisms other than DNA. We are inclined to speculate that various kinds of inheritance of acquired characters can be transmitted at the protein level via mechanisms similar to the Hsp90 system (Rutherford and Lindquist 1998). The fact that information can be transmitted to subsequent generations by a mechanism other than DNA is of great interest. We have shown that the induced phenotype has an advantage in male reproduction, which can lead to the spread of the induced phenotype in the population. Because of the inherited saWT phenotype, the sa allele can be preserved, which might otherwise have been selected against and eliminated.
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Acknowledgments |
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The authors wish to thank Katja Hora, Franti
ek Marec and Jirí Král for helpful suggestions and Mrs. Ivana Kollárová for technical assistance. |
Footnotes |
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Corresponding Editor: Stephen Schaeffer
Received December 31, 1999
Accepted January 15, 2001
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References |
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