The Transformer Genes of the Fern Ceratopteris Simultaneously Promote Meristem and Archegonia Development and Repress Antheridia Development in the Developing Gametophyte

The sex of the haploid gametophyte of the fern Ceratopteris is determined by the presence or absence of the pheromone antheridiogen, which, when present, promotes male development and represses female development of the gametophyte. Several genes involved in sex determination in Ceratopteris have been identified by mutation. In this study, the epistatic interactions among new and previously described sex-determining mutants have been characterized. These results show that sex expression is regulated by two sets of genes defined by the FEM1 and TRA loci. Each promotes the expression of either male or female traits and simultaneously represses the expression of the other. A model describing how antheridiogen regulates the expression of these genes and the sex of the gametophyte is described. The observation that some gametophytic sex-determining mutants have phenotypic effects on the sporophyte plant indicates that sex determination in the Ceratopteris gametophyte is regulated by a mechanism that also regulates sporophyte development.     

Morphological functions of gibberellins in leptosporangiate fern gametophytes: insights into the evolution of form and gender expression

In addition to genetic load, the induction of maleness in leptosporangiate gametophytes by the pheromone antheridiogen may facilitate rates of outcrossing similar to those found in angiosperms. The antheridiogens that have been chemically identified are similar to gibberellins and probably evolved from this common plant hormone. The purposes of this study were to determine the functions of endogenous gibberellins in morphological development and gender expression in leptosporangiate fern gametophytes and to elucidate how antheridiogens may have evolved from gibberellin precursors. We grew gametophytes of Osmunda regalis and Athyrium filix‐femina on nutrient agar enriched with APOGEE, which blocks gibberellin synthesis. Osmunda regalis is a member of Osmundaceae, the only family in Osmundales, the sister group of all remaining extant leptosporangiate ferns. This family possesses a male‐first gender in isolation and lacks any known antheridiogen systems. In contrast, A. filix‐femina is a member of a derived family, Woodsiaceae, which possesses antheridiogen systems. Disruption of gibberellin synthesis retarded notch development and expression of both maleness and femaleness (i.e. gender status) in both species. On the basis of these results, we offer a simple model of gametophyte evolution driven by gender‐based fitness gain curves and the influence of exogenous gibberellins. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 599–615.     

狭眼凤尾蕨配子体发育及其成精子囊素对水蕨配子体发育的影响

对狭眼凤尾蕨（Pteris biaurita）配子体发育特征及其外源成精子囊素对模式植物水蕨（Ceratopteris thalictroides）在黑暗和光照条件下孢子萌发和配子体发育的影响进行了研究。结果表明：（1）狭眼凤尾蕨孢子深褐色,三裂缝,孢子萌发为书带蕨型,原叶体发育为水蕨型,无毛状体产生;培养发现,其配子体能产生精子器,但不产生颈卵器,当接种密度适中时,可进行无配子生殖。（2）在光照和黑暗条件下狭眼凤尾蕨成精子囊素有促进和抑制水蕨孢子萌发的作用,但效果均不显著。（3）在光照条件下,狭眼凤尾蕨成精子囊素可以延迟水蕨心脏形配子体分生组织缺刻的形成,但对其配子体形态和性别分化无明显影响;而在黑暗条件下狭眼凤尾蕨成精囊素对水蕨长条形配子体的形态发育具有一定影响,与对照组相比其顶端分生组织发达,整体呈长楔形,对性别分化影响不显著。可见,狭眼凤尾蕨和水蕨不具有同种成精子囊素系统。     

同形鳞毛蕨成精子囊素系统的初步研究

研究了同形鳞毛蕨成精子囊素对该种和水蕨孢子萌发和配子体发育的影响,结果表明：同形鳞毛蕨配子体能产生成精子囊素,该成精子囊素能抑制同种孢子的萌发,抑制作用随配子体成熟度的增加而增强;同形鳞毛蕨成精子囊素还可促进同种孢子发育为雄配子体;光照条件下,同形鳞毛蕨成精子囊素对水蕨孢子萌发和配子体发育影响不大,黑暗条件下,同形鳞毛蕨成精子囊素能显著的促使水蕨孢子提早萌发,但都不影响其孢子最终萌发率和配子体的性别分化,表明同形鳞毛蕨和水蕨的成精子囊素不属于同一系统。     

Characterization of mutations that feminize gametophytes of the fern Ceratopteris.

Gametophytes of the fern Ceratopteris are either male or hermaphroditic. Their sex is epigenetically determined by the pheromone antheridiogen, which is secreted by the hermaphrodite and induces male and represses female development in other young, sexually undetermined gametophytes. To understand how antheridiogen represses the development of female traits at the genetic level, 16 new mutations that feminize the gametophyte in the presence of antheridiogen were identified and characterized. Seven are very tightly linked to the FEM1 locus previously described. Nine others define another locus named NOTCHLESS1 (NOT1), as several of the not1 mutants lack a meristem notch. Some not1 mutations also affect sporophyte development only when homozygous, indicating that the not1 mutations are recessive and that NOT1 is also required for normal sporophyte development. The epistatic interactions among FEM1, NOT1, and other sex-determining genes are described. This information was used to expand the genetic model of the sex-determining pathway in Ceratopteris. On the basis of this model, we can say that the presence of antheridiogen leads to the activation of the FEM1 gene, which not only promotes the differentiation of male traits, but also represses female development by activating the NOT1 gene. NOT1 represses the TRA genes necessary for the development of female traits in the gametophyte.     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

Gametophyte development of Woodwardia radicans (L.) Sm.: Effect of population density and antheridiogen on sex expression

Abstract

Gametophyte development of Woorwardia radicans was studied in controlled culture conditions, as well as the effect of population density and antheridiogen on sex expression. The pattern of gametophyte development is not affected by temperature, while sex expression of gametophytes is affected by sowing density, by the presence in the culture medium of «antheridiogen» and by the nutritional conditions of gametophytes. The probable effects of antheridiogen and population density on the observed developmental pattern are considered.     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

Growth, sex determination and reproduction of Dryopteris filix-mas (L.) Schott gametophytes under varying nutritional conditions

KORPELAINEN, H., 1994. Growth, sex determination and reproduction of Dryopteris filix-mas (L.) Schott gametophytes under varying nutritional conditions. Gametophyte isolates originating from two populations of Dryopteris filix-mas (L.) Schott were grown in culture media containing different amounts of nutrients. Both nutrition and source population significantly affected gametophyte growth, sex, reproduction and mortality. Taking into account the most optimal nutritional condition for the selfing of gametophytes originating from individual source sporophytes, the proportions of hermaphrodites reproducing by intragametophytic selfing in the two populations varied from 33 to 96% and from 54 to 100%, respectively. It is emphasized that when examining the amount of genetic load only hermaphrodites, not all individuals, should be included, and genetic load should be estimated from the growth experiments where the intensity of reproduction is at the maximum. It was detected that hermaphroditic gametophytes are considerably larger than males or asexuals. It follows that gametophyte size is decisive in sex determination. It is suggested that the effect of antheridiogen hormones, which is considered to be an important factor in gametophyte sex determination, is indirect. Antheridiogens would actually affect size, and size would influence sex expression. The ecology of fern mating systems, and the different genetic and nongenetic factors which promote intergametophytic matings are discussed.     

The tradeoff between reproductive and food resources in the crayfish Orconectes virilis

The conflict between mating and foraging is one of the many tradeoffs associated with reproduction. The relative attraction to sex pheromone and food stimuli by male virile crayfish (Orconectes virilis) were tested using two experiments. In the first experiment, the responses by males to pheromone and food stimuli alone were assessed. In the second experiment, males were exposed to both pheromone and food stimuli in a choice maze. To determine the effects of feeding motivation, the males in the choice experiment were fed either one day or seven days prior to observation. Male crayfish exhibited similar responses to the pheromone and food stimuli individually. Males exhibited a preference for the pheromone stimulus over the food stimulus in the choice trials, and that preference that was not influenced by starvation.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Sex-linked control of sex pheromone behavioral responses in European corn-borer moths (Ostrinia nubilalis) confirmed with TPI marker gene

In two races of European corn-borer moths (ECB), the E-race females emit and males respond to 99:1 sex pheromone blend of (E)/(Z)-11-tetradecenyl acetates, whereas the Z-race females and males produce and respond to the opposite 3:97 pheromone blend of (E)/(Z)-11-tetradecenyl acetates, respectively. We previously have shown that female production of the final blend ratio is under control of a major autosomal locus but that the sequence of male upwind flight responses to the blend is controlled by a sex-linked (Z-linked) locus. This sex-linked control of behavioral responses in crosses of E and Z ECB now is confirmed by use of sex-linked TPI (triose phosphate isomerase) allozyme phenotypes to determine the origin of the sex chromosomes in F₂ populations. F₁ males from reciprocal E × Z crosses generate similar behavioral-response profiles in wind-tunnel studies, with moderate numbers responding to the Z pheromone and intermediate blends (35%–65% Z), but very few responding to the E pheromone. The F₂ behavioral-response profiles indicate that they are composed of 1:1 mixtures of hybrids and paternal profiles. Analysis of TPI allozyme differences allowed us to separate male F₂ populations into individuals whose Z chromosomes both originated from their grandfathers, and individuals who had one Z chromosome originating from each grandparent. With these partitioned F₂s, the TPI homozygotes exhibited behavioral-response profiles very much like their grandfathers, whereas the TPI hybrids produced response profiles similar to their heterozygous F₁ fathers. These results demonstrate incontrovertibly that the response to sex pheromone in male ECB is controlled by a sex-linked gene that is tightly linked to the TPI locus and therefore is independent of the locus controlling pheromone blend production in females.     

Growth and gender in the gametophyte of Blechnum spicant L

The growth and gender in the gametophyte of Blechnum spicant L. were strongly affected by its origin, spore or mature homogenized gametophytes, and also by the addition of plant growth regulators to the culture medium. Spore-derived gametophytes cultured in Murashige and Skoog liquid medium were females and heart-shaped, whereas those derived from mature homogenized gametophytes were shorter and male or asexual (1:3) because of the release of antheridiogen to the culture medium. In the latter, maleness was especially increased by the addition of BA. This cytokinin is a strong inductor of maleness in homogenized cultures; however, even though BA influenced sexual organ formation in spore-derived gametophytes, it does not change the␣female sexual pattern that these gametophytes have. Separate male and female populations of gametophytes were obtained in this work.     

Do antheridiogens act via gametophyte size? A study of Woodwardia radicans (Blechnaceae)

For many plants, sex is not fixed by genotype but determined by environmental conditions during development. In homosporous pteridophytes, sex is environmentally determined by the presence or absence of antheridiogens, maleness-inducing pheromones. It has been proposed that antheridiogens primarily reduce growth rate, with small gametophyte size responsible for maleness. To test this hypothesis, the effects of antheridiogen and intergametophytic competition on gender expression and gametophyte size were studied in a culture experiment with Woodwardia radicans. We found that (1) antheridiogen inhibited growth of gametophytes; and (2) slow growth favored maleness, whereas fast growth favored femaleness, irrespective of the presence or absence of antheridiogen. Both conclusions are consistent with the hypothesis that, in W. radicans, antheridiogen effect is mediated by size. They also agree with the "size-advantage" hypothesis in which energetic limitations associated with relatively small individual size impose a less severe limitation for male reproductive success than for female reproductive success. The results are also discussed with regard to a genetic sex-determining pathway that has recently been identified.     

Searching for pheromone strains in the pecan nut casebearer

The pecan nut casebearer, Acrobasis nuxvorella Nuenzig (Lepidoptera: Pyralidae), is the most damaging insect pest of pecan, Carya illinoinensis (Wang) K. Koch (Juglandaceae), in the USA and Mexico. A pheromone monitoring program for A. nuxvorella has been developed to assist pecan growers in the timing of insecticide applications. The discovery that there are two pheromone types produced by A. nuxvorella has led to complications in the implementation of pheromone monitoring programs. One pheromone (referred to as standard) is attractive to moths in the southern USA, but not in Mexico. The other pheromone (referred to as Mexican) is attractive to moths in the southern USA and in Mexico. Because most male lepidopterans respond only to a specific pheromone, it was suspected that there were two pheromone strains of A. nuxvorella, one exclusively present in the northern distribution of A. nuxvorella (USA strain) and the other widely distributed from Sonora, Chihuahua, and Durango in northern Mexico to Texas, Georgia, and Oklahoma in the USA (Mexican strain). The goal of this research was to determine whether differences in pheromone response are sufficient to genetically isolate A. nuxvorella into pheromone strains. To confirm the existence of the two alleged pheromone strains, amplified fragment length polymorphism (AFLP) markers were obtained and analyzed. Four primer combinations were used to obtain a total of 483 polymorphic AFLP markers. Our results indicated that the standard and Mexican pheromones did not group A. nuxvorella into pheromone strains. However, three genetically distinct populations of A. nuxvorella were identified. Two of those three populations are sympatric throughout the southern USA and one is allopatric relative to the other two and occurs exclusively in pecan growing regions of Mexico.     

Inter- and Intraspecific Effects of Volatile and Nonvolatile Sex Pheromones on Males, Mating Behavior, and Hybridization in Eretmocerus mundus and E. eremicus (Hymenoptera: Aphelinidae)

Eretmocerus species (Hym. Aphelinidae) are solitary parasitoids of Bemisia tabaci (Gennadius). Mate finding and mating behavior of two species, E. mundus and E. eremicus, were studied under laboratory conditions. We used three populations of Eretmocerus: typical arrhenotokous populations of E. eremicus (from USA) and E. mundus (from Spain), and an atypical thelytokous population of E. mundus (from Australia). We studied the intra- and interspecific responses of males to volatile and nonvolatile components of the female sex pheromones, mating behavior, and hybridization between populations and species. In both arrhenotokous populations, males reacted to volatile pheromones by walking toward conspecific virgin females. Males also reacted to nonvolatile pheromones by spending more time on and around patches on leaves of poinsettia plants that had been exposed to virgin females. Males of E. eremicus showed the same reaction to the nonvolatile sex pheromone of E. mundus females, but E. mundus males did not show any reaction to the nonvolatile sex pheromone of E. eremicus. There was no response of males of both species to thelytokous females of E. mundus. In both species three phases were distinguished in the mating behavior: premating, mating, and postmating. The duration of the phases differed between the three populations. Successful copulation between the two Eretmocerus species did not occur. In contrast, we recorded some successful copulations between Australian males and Spanish females of E. mundus, but they did not produce any hybrid females.     

RELEVANCE OF PLANT VOLATILES TO SEX PHERO‐MONE IN LURING APHIDS IN THE FIELD1

Abstract Outdoor trials were made to test the responses of male aphids to pheromone lures in fields with different host‐plants, in order to know whether host‐plants would affect catches of males to pheromone lures. In peach orchard, males of three aphid species, i.e. Myzus persicae, Hyalopterus amygdali and Rhopalosiphum nymphaeae, were found in traps baited with aphid sex pheromone nepetalactone and nepetalactol. The most numerous numbers was M, persicae and it was also attracted by plant‐derived nepetalactone alone in peach orchard and cabbage garden. Schizaphis graminum males were caught in water traps in wheat field. In apple orchard, males of two aphid species, Aphis cifricola and Sappaphis sinipiricola were caught in significantly larger numbers in the pheromone traps than in the control traps. Field study indicated that the sex pheromone of H. amygduli comprises either one or both of nepetalactone and nepetalactol, and nepetalactone may be one component of sex pheromones of A cifricola and S sinipiricola Another field trial had shown that catches were increased if volatiles from a steam‐distilled extract of peach leaves were released simultaneously with nepetalactone. Elec‐troantennograms (EAGs) were recorded from males of M. persicae to volatiles of peach leaves and buds.     

DIFFERENTIAL ANTHERIDIOGEN RESPONSE AND EVOLUTIONARY MECHANISMS IN CYSTOPTERIS

It has been demonstrated that antheridiogens determine gametangial initiation in cultures of some homosporous fern gametophytes. Studies of natural populations have implicated these compounds as breeding system controllers. In Cystopteris, response to antheridiogen varied significantly among species. While Cystopteris protrusa responded to exogenous antheridiogen, C. bulbifera did not. The allopolyploid derivative of these two species, C. tennesseensis, had an intermediate level of response. This marked physiological difference among morphologically similar taxa is valuable in delineating species and suggests a mechanism that may be associated with interspecific hybridization.     

Electrophysiological and Behavioral Responses of Female Beet Armyworm Spodoptera exigua (Hübner) to the Conspecific Female Sex Pheromone

Electroantennogram (EAG) recordings showed that female Spodoptera exigua can detect their own sex pheromones (two single components and their mixture), displaying a similar dose–response pattern to that of males, although intensities of female responses were much less at all doses compared with males. Furthermore, the female calling behavior was inhibited and late-shifted by the presence of the female sex pheromone. When the pheromone components were presented, the calling female proportion in the peak calling period was significantly reduced and the calling peak time and calling termination time postponed, compared with controls. Although the calling behavior was inhibited, the pheromone titer of treated females was not different to the control, implying a reduced pheromone biosynthesis in the pheromone glands of treated moths. However, observations during the olfactometer experiments revealed that there were no obvious behavioral responses of females exposed to sex pheromone stimuli including whole gland extracts, 0.1, 1 or 10 μg binary pheromone mixtures.     

Homology of olfactory receptor neuron response characteristics inferred from hybrids between Asian and European corn borer moths (Lepidoptera: Crambidae)

First generation hybrid males from crosses between the Asian corn borer (ACB), Ostrinia furnacalis, and the “univoltine Z-strain” European corn borer (ECB), Ostrinia nubilalis, were examined with respect to behavioral and physiological responses to ACB and ECB pheromones. The hybrid males often flew to the pheromone of ECB Z-strain, but very rarely to the ACB pheromone. We mapped the tuning profiles of each ORN of the F₁ hybrids with respect to the relevant pheromone components and a common behavioral antagonist by employing differential cross-adaptation and varying doses of the ligands. In the trichoid sensilla of F₁ hybrid males, the three co-compartmentalized ORNs produced spikes that were very difficult to distinguish by size, unlike the parental populations. Comparing the responses to ACB and ECB components at different doses reveals overlapping profiles similar to males of both parental types, but more responsiveness to the ECB pheromone components. We were unable to detect any differences in the ORN tuning profiles when comparing males with different behavioral phenotypes. While the two ECB pheromone races have similar ORN tuning properties that are different from those in ACB, the spike-amplitude patterns of ECB E-strain and ACB have greater homology when compared to ECB Z-strain.