Spore-autonomous fluorescent protein expression identifies meiotic chromosome mis-segregation as the principal cause of hybrid sterility in yeast.

Genome-wide sequence divergence between populations can cause hybrid sterility through the action of the anti-recombination system, which rejects crossover repair of double strand breaks between nonidentical sequences. Because crossovers are necessary to ensure proper segregation of homologous chromosomes during meiosis, the reduced recombination rate in hybrids can result in high levels of nondisjunction and therefore low gamete viability. Hybrid sterility in interspecific crosses of Saccharomyces yeasts is known to be associated with such segregation errors, but estimates of the importance of nondisjunction to postzygotic reproductive isolation have been hampered by difficulties in accurately measuring nondisjunction frequencies. Here, we use spore-autonomous fluorescent protein expression to quantify nondisjunction in both interspecific and intraspecific yeast hybrids. We show that segregation is near random in interspecific hybrids. The observed rates of nondisjunction can explain most of the sterility observed in interspecific hybrids through the failure of gametes to inherit at least one copy of each chromosome. Partially impairing the anti-recombination system by preventing expression of the RecQ helicase SGS1 during meiosis cuts nondisjunction frequencies in half. We further show that chromosome loss through nondisjunction can explain nearly all of the sterility observed in hybrids formed between two populations of a single species. The rate of meiotic nondisjunction of each homologous pair was negatively correlated with chromosome size in these intraspecific hybrids. Our results demonstrate that sequence divergence is not only associated with the sterility of hybrids formed between distantly related species but may also be a direct cause of reproductive isolation in incipient species.

Ribosome Provisioning Activates a Bistable Switch Coupled to Fast Exit from Stationary Phase.

Observations of bacteria at the single-cell level have revealed many instances of phenotypic heterogeneity within otherwise clonal populations, but the selective causes, molecular bases, and broader ecological relevance remain poorly understood. In an earlier experiment in which the bacterium Pseudomonas fluorescens SBW25 was propagated under a selective regime that mimicked the host immune response, a genotype evolved that stochastically switched between capsulation states. The genetic cause was a mutation in carB that decreased the pyrimidine pool (and growth rate), lowering the activation threshold of a preexisting but hitherto unrecognized phenotypic switch. Genetic components surrounding bifurcation of UTP flux toward DNA/RNA or UDP-glucose (a precursor of colanic acid forming the capsules) were implicated as key components. Extending these molecular analyses-and based on a combination of genetics, transcriptomics, biochemistry, and mathematical modeling-we show that pyrimidine limitation triggers an increase in ribosome biosynthesis and that switching is caused by competition between ribosomes and CsrA/RsmA proteins for the mRNA transcript of a positively autoregulated activator of colanic acid biosynthesis. We additionally show that in the ancestral bacterium the switch is part of a program that determines stochastic entry into a semiquiescent capsulated state, ensures that such cells are provisioned with excess ribosomes, and enables provisioned cells to exit rapidly from stationary phase under permissive conditions.

Evolution of mating types in finite populations: The precarious advantage of being rare.

Sexually reproducing populations with self-incompatibility bear the cost of limiting potential mates to individuals of a different type. Rare mating types escape this cost since they are unlikely to encounter incompatible partners, leading to the deterministic prediction of continuous invasion by new mutants and an ever-increasing number of types. However, rare types are also at an increased risk of being lost by random drift. Calculating the number of mating types that a population can maintain requires consideration of both the deterministic advantages and the stochastic risks. By comparing the relative importance of selection and drift, we show that a population of size N can maintain a maximum of approximately N1/3 mating types for intermediate population sizes, whereas for large N, we derive a formal estimate. Although the number of mating types in a population is quite stable, the rare-type advantage promotes turnover of types. We derive explicit formulas for both the invasion and turnover probabilities in finite populations.

Diminishing Returns on Intragenic Repeat Number Expansion in the Production of Signaling Peptides.

Signaling peptides enable communication between cells, both within and between individuals, and are therefore key to the control of complex physiological and behavioral responses. Since their small sizes prevent direct transmission to secretory pathways, these peptides are often produced as part of a larger polyprotein comprising precursors for multiple related or identical peptides; the physiological and behavioral consequences of this unusual gene structure are not understood. Here, we show that the number of mature-pheromone-encoding repeats in the yeast α-mating-factor gene MFα1 varies considerably between closely related isolates of both Saccharomyces cerevisiae and its sister species Saccharomyces paradoxus. Variation in repeat number has important phenotypic consequences: Increasing repeat number caused higher pheromone production and greater competitive mating success. However, the magnitude of the improvement decreased with increasing repeat number such that repeat amplification beyond that observed in natural isolates failed to generate more pheromone, and could actually reduce sexual fitness. We investigate multiple explanations for this pattern of diminishing returns and find that our results are most consistent with a translational trade-off: Increasing the number of encoded repeats results in more mature pheromone per translation event, but also generates longer transcripts thereby reducing the rate of translation-a phenomenon known as length-dependent translation. Length-dependent translation may be a powerful constraint on the evolution of genes encoding repetitive or modular proteins, with important physiological and behavioral consequences across eukaryotes.

Ribosome reinitiation can explain length-dependent translation of messenger RNA.

Models of mRNA translation usually presume that transcripts are linear; upon reaching the end of a transcript each terminating ribosome returns to the cytoplasmic pool before initiating anew on a different transcript. A consequence of linear models is that faster translation of a given mRNA is unlikely to generate more of the encoded protein, particularly at low ribosome availability. Recent evidence indicates that eukaryotic mRNAs are circularized, potentially allowing terminating ribosomes to preferentially reinitiate on the same transcript. Here we model the effect of ribosome reinitiation on translation and show that, at high levels of reinitiation, protein synthesis rates are dominated by the time required to translate a given transcript. Our model provides a simple mechanistic explanation for many previously enigmatic features of eukaryotic translation, including the negative correlation of both ribosome densities and protein abundance on transcript length, the importance of codon usage in determining protein synthesis rates, and the negative correlation between transcript length and both codon adaptation and 5' mRNA folding energies. In contrast to linear models where translation is largely limited by initiation rates, our model reveals that all three stages of translation-initiation, elongation, and termination/reinitiation-determine protein synthesis rates even at low ribosome availability.

Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae.

Anopheles gambiae mosquitoes are major African vectors of malaria, a disease that kills more than 600,000 people every year. Given the spread of insecticide resistance in natural mosquito populations, alternative vector control strategies aimed at reducing the reproductive success of mosquitoes are being promoted. Unlike many other insects, An. gambiae females mate a single time in their lives and must use sperm stored in the sperm storage organ, the spermatheca, to fertilize a lifetime's supply of eggs. Maintenance of sperm viability during storage is therefore crucial to the reproductive capacity of these mosquitoes. However, to date, no information is available on the factors and mechanisms ensuring sperm functionality in the spermatheca. Here we identify cellular components and molecular mechanisms used by An. gambiae females to maximize their fertility. Pathways of energy metabolism, cellular transport, and oxidative stress are strongly regulated by mating in the spermatheca. We identify the mating-induced heme peroxidase (HPX) 15 as an important factor in long-term fertility, and demonstrate that its function is required during multiple gonotrophic cycles. We find that HPX15 induction is regulated by sexually transferred 20-hydroxy-ecdysone (20E), a steroid hormone that is produced by the male accessory glands and transferred during copulation, and that expression of this peroxidase is mediated via the 20E nuclear receptor. To our knowledge, our findings provide the first evidence of the mechanisms regulating fertility in Anopheles, and identify HPX15 as a target for vector control.

Reflections on a life with Monte Carlo in Medical Physics.

The author reminisces about some of his experiences working with Monte Carlo techniques for Medical Physics applications.

Molecular quantification of Saccharomyces cerevisiae α-pheromone secretion.

Saccharomyces cerevisiae yeast cells court each other by producing an attractive sex pheromone specific to their mating type. Cells detect the sex pheromone from potential mates using a well-defined intracellular signalling cascade that has become a model for studying signal transduction. In contrast, the factors contributing to the production of pheromone itself are poorly characterized, despite the widespread use of the S. cerevisiae α-pheromone secretion pathway in industrial fungal protein expression systems. Progress in understanding pheromone secretion has been hindered by a lack of a precise and quantitative pheromone production assay. Here, we present an ELISA-based method for the quantification of α-pheromone secretion. In the absence of pheromone from the opposite mating type, we found that each cell secretes over 550 mature α-pheromone peptides per second; 90% of this total was produced from MF α1. The addition of a-pheromone more than doubled total α-pheromone secretion. This technique offers several improvements on current methods for measuring α-pheromone production and will allow detailed investigation of the factors regulating pheromone production in yeast.

Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito.

Insect seminal fluid proteins are powerful modulators of many aspects of female physiology and behaviour including longevity, egg production, sperm storage, and remating. The crucial role of these proteins in reproduction makes them promising targets for developing tools aimed at reducing the population sizes of vectors of disease. In the malaria mosquito Anopheles gambiae, seminal secretions produced by the male accessory glands (MAGs) are transferred to females in the form of a coagulated mass called the mating plug. The potential of seminal fluid proteins as tools for mosquito control demands that we improve our limited understanding of the composition and function of the plug. Here, we show that the plug is a key determinant of An. gambiae reproductive success. We uncover the composition of the plug and demonstrate it is formed through the cross-linking of seminal proteins mediated by a MAG-specific transglutaminase (TGase), a mechanism remarkably similar to mammalian semen coagulation. Interfering with TGase expression in males inhibits plug formation and transfer, and prevents females from storing sperm with obvious consequences for fertility. Moreover, we show that the MAG-specific TGase is restricted to the anopheline lineage, where it functions to promote sperm storage rather than as a mechanical barrier to re-insemination. Taken together, these data represent a major advance in our understanding of the factors shaping Anopheles reproductive biology.

Molecular characterization and evolution of a gene family encoding male-specific reproductive proteins in the African malaria vector Anopheles gambiae.

BACKGROUND: During copulation, the major Afro-tropical malaria vector Anopheles gambiae s.s. transfers male accessory gland (MAG) proteins to females as a solid mass (i.e. the "mating plug"). These proteins are postulated to function as important modulators of female post-mating responses. To understand the role of selective forces underlying the evolution of these proteins in the A. gambiae complex, we carried out an evolutionary analysis of gene sequence and expression divergence on a pair of paralog genes called AgAcp34A-1 and AgAcp34A-2. These encode MAG-specific proteins which, based on homology with Drosophila, have been hypothesized to play a role in sperm viability and function. RESULTS: Genetic analysis of 6 species of the A. gambiae complex revealed the existence of a third paralog (68-78% of identity), that we named AgAcp34A-3. FISH assays showed that this gene maps in the same division (34A) of chromosome-3R as the other two paralogs. In particular, immuno-fluorescence assays targeting the C-terminals of AgAcp34A-2 and AgAcp34A-3 revealed that these two proteins are localized in the posterior part of the MAG and concentrated at the apical portion of the mating plug. When transferred to females, this part of the plug lies in proximity to the duct connecting the spermatheca to the uterus, suggesting a potential role for these proteins in regulating sperm motility. AgAcp34A-3 is more polymorphic than the other two paralogs, possibly because of relaxation of purifying selection. Since both unequal crossing-over and gene conversion likely homogenized the members of this gene family, the interpretation of the evolutionary patterns is not straightforward. Although several haplotypes of the three paralogs are shared by most A. gambiae s.l. species, some fixed species-specific replacements (mainly placed in the N- and C-terminal portions of the secreted peptides) were also observed, suggesting some lineage-specific adaptation. CONCLUSIONS: Progress in understanding the signaling cascade in the A. gambiae reproductive pathway will elucidate the interaction of this MAG-specific protein family with their female counterparts. This knowledge will allow a better evaluation of the relative importance of genes involved in the reproductive isolation and fertility of A. gambiae species and could help the interpretation of the observed evolutionary patterns.

Molecular and cellular components of the mating machinery in Anopheles gambiae females.

Anopheles gambiae mosquitoes are the principal vectors of malaria. A major determinant of the capacity of these mosquitoes as disease vectors is their high reproductive rate. Reproduction depends on a single insemination, which profoundly changes the behavior and physiology of females. To identify factors and mechanisms relevant to the fertility of A. gambiae, we performed a comprehensive analysis of the molecular and cellular machinery associated with copulation in females. Initial whole-body microarray experiments comparing virgins with females at 2 h, 6 h, and 24 h after mating detected large transcriptional changes. Analysis of tissue localization identified a subset of genes whose expression was strikingly regulated by mating in the lower reproductive tract and, surprisingly, the gut. In the atrium of virgin females, where the male seminal fluid is received, our studies revealed a "mating machinery" consisting of molecular and structural components that are turned off or collapse after copulation, suggesting that this tissue loses its competence for further insemination. In the sperm storage organ, we detected a number of mating-responsive genes likely to have a role in the maintenance and function of stored sperm. These results identify genes and mechanisms regulating the reproductive biology of A. gambiae females, highlighting considerable differences with Drosophila melanogaster. Our data inform vector control strategies and reveal promising targets for the manipulation of fertility in field populations of these important disease vectors.

Replacement correction factors for plane-parallel ion chambers in electron beams.

PURPOSE: Plane-parallel chambers are recommended by dosimetry protocols for measurements in (especially low-energy) electron beams. In dosimetry protocols, the replacement correction factor P(repl) is assumed unity for "well-guarded" plane-parallel chambers in electron beams when the front face of the cavity is the effective point of measurement. There is experimental evidence that ion chambers which are not well-guarded (e.g., Markus) have nonunity P(repl) values. Monte Carlo simulations are employed in this study to investigate the replacement correction factors for plane-parallel chambers in electron beams. METHODS: Using previously established Monte Carlo calculation methods, the values of P(repl) are calculated with high statistical precision for the cavities of a variety of plane-parallel chambers in a water phantom irradiated by various electron beams. The dependences of the values of P(repl) on the beam quality, phantom depth, as well as the guard ring width are studied. RESULTS: In the dose fall-off region for low-energy beams, the P(repl) values are very sensitive to depth. It is found that this is mainly due to the gradient effect, which originates from the fact that the effective point of measurement for many plane-parallel chambers should not be at the front face of the cavity but rather shifted toward the center of the cavity by a fraction of a millimeter. Using the front face of the cavity as the effective point of measurement, the calculated values of P(repl) at d(ref) are not unity for some well-guarded plane-parallel chambers. The calculated P(repl) values for the Roos chamber are close to 1 for all electron beams. The calculation results for the Markus chamber are in good agreement with the measured values. CONCLUSIONS: The appropriate selection of the effective point of measurement for plane-parallel chambers in electron beams is an important issue. If the effective point of measurement is correctly accounted for, the P(repl) values would be almost independent of depth. Both the guard ring width and the ratio of the collecting volume diameter to the cavity thickness can influence the values of P(repl) For a diameter to thickness ratio of 5 (e.g., NACP02 chamber), the guard width has to be 6 mm for the chamber to be considered as well-guarded, i.e., have a P(repl) value of 1.00.

Experimental evolution of a sexually selected display in yeast.

The fundamental principle underlying sexual selection theory is that an allele conferring an advantage in the competition for mates will spread through a population. Remarkably, this has never been demonstrated empirically. We have developed an experimental system using yeast for testing genetic models of sexual selection. Yeast signal to potential partners by producing an attractive pheromone; stronger signallers are preferred as mates. We tested the effect of high and low levels of sexual selection on the evolution of a gene determining the strength of this signal. Under high sexual selection, an allele encoding a stronger signal was able to invade a population of weak signallers, and we observed a corresponding increase in the amount of pheromone produced. By contrast, the strong signalling allele failed to invade under low sexual selection. Our results demonstrate, for the first time, the spread of a sexually selected allele through a population, confirming the central assumption of sexual selection theory. Our yeast system is a powerful tool for investigating the genetics of sexual selection.

Male sexual ornament size is positively associated with reproductive morphology and enhanced fertility in the stalk-eyed fly Teleopsis dalmanni.

BACKGROUND: Exaggerated male ornaments and displays often evolve in species where males only provide females with ejaculates during reproduction. Although "good genes" arguments are typically invoked to explain this phenomenon, a simpler alternative is possible if variation in male reproductive quality (e.g. sperm number, ejaculate content, mating rate) is an important determinant of female reproductive success. The "phenotype-linked fertility hypothesis" states that female preference for male ornaments or displays has been selected to ensure higher levels of fertility and has driven the evolution of exaggerated male traits. Females of the stalk-eyed fly Teleopsis dalmanni must mate frequently to maintain high levels of fertility and prefer to mate with males exhibiting large eyespan, a condition-dependent sexual ornament. If eyespan indicates male reproductive quality, females could directly increase their reproductive success by mating with males with large eyespan. Here we investigate whether male eyespan indicates accessory gland and testis length, and then ask whether mating with large eyespan males affects female fertility. RESULTS: Male eyespan was a better predictor of two key male reproductive traits--accessory gland and testis length--than was body size alone. This positive relationship held true over three levels of increasing environmental stress during the maturation of the adult accessory glands and testes. Furthermore, females housed with a large eyespan male exhibited higher levels of fertility than those with small eyespan males. CONCLUSION: Male eyespan in stalk-eyed flies is subject to strong directional mate preference and is a reliable indicator of male reproductive quality--both because males with larger eyespan have bigger accessory glands and testes, and also as they confer higher fertility on females. Fertility enhancement may have arisen because males with larger eyespan mated more often and/or because they transferred more sperm or other substances per ejaculate. The need to ensure high levels of fertility could thus have been an important selective force in the coevolution of female preference and male eyespan in stalk-eyed flies. Our results support the phenotype-linked fertility hypothesis and suggest that it might be of general importance in explaining the evolution of exaggerated male ornaments and displays in species where males only provide females with ejaculates during reproduction.

Sexual selection: the importance of long-term fitness measures.

New results from a 20-year study of free-living song sparrows confirm that attractive males contribute more offspring than less attractive males. They also reveal that the offspring of preferred males produce more descendents themselves. Females prefer males with a large song repertoire, which further work shows is a condition-dependent indicator of male quality.

Monte Carlo study of si diode response in electron beams.

Silicon semiconductor diodes measure almost the same depth-dose distributions in both photon and electron beams as those measured by ion chambers. A recent study in ion chamber dosimetry has suggested that the wall correction factor for a parallel-plate ion chamber in electron beams changes with depth by as much as 6%. To investigate diode detector response with respect to depth, a silicon diode model is constructed and the water/silicon dose ratio at various depths in electron beams is calculated using EGSnrc. The results indicate that, for this particular diode model, the diode response per unit water dose (or water/diode dose ratio) in both 6 and 18 MeV electron beams is flat within 2% versus depth, from near the phantom surface to the depth of R50 (with calculation uncertainty <0.3%). This suggests that there must be some other correction factors for ion chambers that counter-balance the large wall correction factor at depth in electron beams. In addition, the beam quality and field-size dependence of the diode model are also calculated. The results show that the water/diode dose ratio remains constant within 2% over the electron energy range from 6 to 18 MeV. The water/diode dose ratio does not depend on field size as long as the incident electron beam is broad and the electron energy is high. However, for a very small beam size (1 X 1 cm(2)) and low electron energy (6 MeV), the water/diode dose ratio may decrease by more than 2% compared to that of a broad beam.

Assigning sex to pre-adult stalk-eyed flies using genital disc morphology and X chromosome zygosity.

BACKGROUND: In stalk-eyed flies (Diopsidae) the eyes and antennae are laterally displaced at the ends of elongated eyestalks. Eyespan and the degree of sexual dimorphism in eyespan vary considerably between species and several sexually dimorphic species show sexual selection through female mate preference for males with exaggerated eyespan. The genes on which selection acts to regulate eyespan remain to be identified. This could be achieved by comparing gene expression during eyestalk development in males and females if the sex of pre-adult flies could be reliably assigned. Here we describe two techniques, one morphological and one microsatellite-based, that identify the sex of stalk-eyed fly larvae and pupae. RESULTS: We showed that genital discs of the stalk-eyed fly Teleopsis dalmanni have two highly distinct morphologies, compact ("C") and lobed ("L"). Segment composition (revealed by Engrailed expression) was consistent with C morphology being typical of males and L morphology of females. We confirmed the proposed association between disc morphology and sex by evaluating the combined heterozygosity of four X-linked microsatellite markers. We demonstrated that individuals with C genital discs had hemizygous (male) genotypes while those with L discs were heterozygous (female) genotypes. Similar dimorphism in genital disc morphology was observed in eight other species spanning three representative Diopsid genera. In every case the segment composition supported C morphology being male and L morphology female. We assigned larval sex by C or L morphology and compared cell division frequencies in male and female eye-antennal discs in two species (T. dalmanni and Diasemopsis meigenii) sexually dimorphic for eyespan. The number of mitotic (anti-H3-labelled) cells did not differ between the sexes in either species. CONCLUSION: We have made novel use of two complementary techniques for identifying the sex of pre-adult stalk-eyed flies. These procedures will facilitate studies of the evolution of sexually dimorphic development in a variety of other species. Morphology and En expression in male and female genital discs are highly conserved within each genus of Diopsidae. Finally, sexual dimorphism for eyespan in two Diopsid species is unlikely to be due to an increased rate of cell division at the third larval instar in males.

Variation in preference for a male ornament is positively associated with female eyespan in the stalk-eyed fly Diasemopsis meigenii.

There is currently much interest in mate preferences for sexual ornaments. However, few studies have focused on individual variation in mate preference despite its importance for the rate and direction of sexual selection. Females of the sexually dimorphic stalk-eyed fly, Diasemopsis meigenii, exhibit an unambiguous rejection response towards unattractive males bearing small ornaments. We investigated individual mate preferences using repeated sequential sampling of female rejection or acceptance responses to a wide range of male ornament phenotypes. We found significant variation in the strength of individual preference. In addition, preference was positively associated with female eyespan, a condition-dependent trait putatively linked to visual acuity.

Mating-induced reduction in accessory reproductive organ size in the stalk-eyed fly Cyrtodiopsis dalmanni.

BACKGROUND: Internal reproductive organ size is an important determinant of male reproductive success. While the response of testis length to variation in the intensity of sperm competition is well documented across many taxa, few studies address the importance of testis size in determining other components of male reproductive success (such as mating frequency) or the significance of size variation in accessory reproductive organs. Accessory gland length, but not testis length, is both phenotypically and genetically correlated with male mating frequency in the stalk-eyed fly Cyrtodiopsis dalmanni. Here we directly manipulate male mating status to investigate the effect of copulation on the size of both the testes and the accessory glands of C. dalmanni. RESULTS: Accessory gland length was positively correlated with male mating frequency. Copulation induced a significant decrease in accessory gland size. The size of the accessory glands then recovered slowly over the next 8-48 hours. Neither testis length nor testis area was altered by copulation. CONCLUSION: These results reveal that the time course of accessory gland recovery corresponds to field observations of mating behaviour and suggest that accessory gland size may limit male mating frequency in C. dalmanni.