Airway submucosal glands from cystic fibrosis swine suffer from abnormal ion transport across the serous acini, collecting duct, and ciliated duct.

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.

Evolution of gamete attraction molecules: evidence for purifying selection in speract and its receptor, in the pantropical sea urchin Diadema.

Many free-spawning marine invertebrates, such as sea urchins, lack any courtship or assortative mating behavior. Mate recognition in such cases occur at the gametic level, and molecules present on the sperm and egg are major determinants of species-specific fertilization. These molecules must also coevolve in relation to each other in order to preserve functional integrity. When sea urchins release their gametes in seawater, diffusible molecules from the egg, termed sperm-activating peptides, activate and attract the sperm to swim toward the egg, initiating a series of interactions between the gametes. Although the compositions and diversity of such sperm-activating peptides have been characterized in a variety of sea urchins, little is known about the evolution of their genes. Here we characterize the genes encoding the sperm-activating peptide of the egg (speract) and its receptor on the sperm, and examine their evolutionary dynamics in the sea urchin genus Diadema, in the interest of determining whether they are involved in reproductive isolation between the species. We found evidence of purifying selection on several codon sites in both molecules and of selectively neutral evolution in others. The diffusible speract peptide that activates sperm is invariant across species, indicating that Diadema egg peptides do not discriminate between con- and hetero-specific sperm at this stage of the process. Speract and its receptor do not contribute to reproductive isolation in Diadema.

Intracellular Ca2+ oscillation frequency and amplitude modulation mediate epithelial apical and basolateral membranes crosstalk.

Since the early seminal studies on epithelial solute transport, it has been understood that there must be crosstalk among different members of the transport machinery to coordinate their activity and, thus, generate localized electrochemical gradients that force solute flow in the required direction that would otherwise be thermodynamically unfavorable. However, mechanisms underlying intracellular crosstalk remain unclear. We present evidence that crosstalk between apical and basolateral membrane transporters is mediated by intracellular Ca2+ signaling in insect renal epithelia. Ion flux across the basolateral membrane is encoded in the intracellular Ca2+ oscillation frequency and amplitude modulation and that information is used by the apical membrane to adjust ion flux accordingly. Moreover, imposing experimentally generated intracellular Ca2+ oscillation modulation causes cells to predictably adjust their ion transport properties. Our results suggest that intracellular Ca2+ oscillation frequency and amplitude modulation encode information on transmembrane ion flux that is required for crosstalk.

Lipopolysaccharides induce a RAGE-mediated sensitization of sensory neurons and fluid hypersecretion in the upper airways.

Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

cAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

Mate Choice and the Persistence of Maternal Mortality.

Maternal mortality remains one of the leading causes of death in women of reproductive age in developing countries, and a major concern in some developed countries. It is puzzling why such a condition has not been reduced in frequency, if not eliminated, in the course of evolution. Maternal mortality is a complex phenomenon caused by several physiological and physical factors. Among the physical factors, maternal mortality due to fetopelvic disproportion remains controversial. Several explanations including evolution of bipedal locomotion, rapid brain growth, and nutritional changes and life style changes in settler communities have been proposed. The influences of human reproductive biology and sexual selection have rarely been considered to explain why maternal mortality persisted through human evolution. We entertain the hypothesis that irrespective of the causes, the risks of all factors causing maternal mortality would be aggravated by disassortative mating, specifically male preference for younger females who are generally small statured and at higher risk of obstetric complications. Maternal mortality arising due to sexual selection and mate choice would have the long-term effect of driving widowers toward younger women, often resulting in "child marriage," which still remains a significant cause of maternal mortality globally. Evolutionarily, such a male driven mating system in polygamous human populations would have prolonged the persistence of maternal mortality despite selection acting against it. The effects may extend beyond maternal mortality because male-mate choice driven maternal mortality would reduce average reproductive life spans of women, thus influencing the evolution of menopause.

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea.

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Evolution in the fast lane: rapidly evolving sex-related genes in Drosophila.

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.

Young species of cupuladriid bryozoans occupied new Caribbean habitats faster than old species.

The breadth of habitat occupied by a species, and the rate at which a species can expand into new habitats has important ecological and evolutionary consequences. Here we explore when extant species of free-living cupuladriid bryozoans expanded into new benthic Caribbean habitats that emerged during the final stages of formation of the Isthmus of Panama. Habitat breadth was estimated using the abundances of over 90,000 colonies in ten cupuladriid species, along with the ecological and sedimentary characteristics of the samples in which they occurred. Data reveal that all species expanded their habitat breadths during the last 6 Myr, but did so at a different tempo. 'Young' species - those that originated after 5 Ma - expanded relatively quickly, whereas 'old' species - those that originated before 9 Ma - took a further 2 Myr to achieve a comparable level of expansion. We propose that, like invasive species, young species are less restrained when expanding their habitat breadths compared to older well-established species. Understanding the mechanism causing this restraint requires further research.

RAGE-dependent potentiation of TRPV1 currents in sensory neurons exposed to high glucose.

Diabetes mellitus is associated with sensory abnormalities, including exacerbated responses to painful (hyperalgesia) or non-painful (allodynia) stimuli. These abnormalities are symptoms of diabetic peripheral neuropathy (DPN), which is the most common complication that affects approximately 50% of diabetic patients. Yet, the underlying mechanisms linking hyperglycemia and symptoms of DPN remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) channel plays a central role in such sensory abnormalities and shows elevated expression levels in animal models of diabetes. Here, we investigated the function of TRPV1 channels in sensory neurons cultured from the dorsal root ganglion (DRG) of neonatal mice, under control (5mM) and high glucose (25mM) conditions. After maintaining DRG neurons in high glucose for 1 week, we observed a significant increase in capsaicin (CAP)-evoked currents and CAP-evoked depolarizations, independent of TRPV1 channel expression. These functional changes were largely dependent on the expression of the receptor for Advanced Glycation End-products (RAGE), calcium influx, cytoplasmic ROS accumulation, PKC, and Src kinase activity. Like cultured neurons from neonates, mature neurons from adult mice also displayed a similar potentiation of CAP-evoked currents in the high glucose condition. Taken together, our data demonstrate that under the diabetic condition, DRG neurons are directly affected by elevated levels of glucose, independent of vascular or glial signals, and dependent on RAGE expression. These early cellular and molecular changes to sensory neurons in vitro are potential mechanisms that might contribute to sensory abnormalities that can occur in the very early stages of diabetes.

Cystic fibrosis swine fail to secrete airway surface liquid in response to inhalation of pathogens.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR-/- swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.

Female Choice or Male Sex Drive? The Advantages of Male Body Size during Mating in Drosophila Melanogaster.

The mating success of larger male Drosophila melanogaster in the laboratory and the wild has been traditionally been explained by female choice, even though the reasons are generally hard to reconcile. Female choice can explain this success by virtue of females taking less time to mate with preferred males, but so can the more aggressive or persistent courtships efforts of large males. Since mating is a negotiation between the two sexes, the behaviors of both are likely to interact and influence mating outcomes. Using a series of assays, we explored these negotiations by testing for the relative influence of male behaviors and its effect on influencing female courtship arousal threshold, which is the time taken for females to accept copulation. Our results show that large males indeed have higher copulation success compared to smaller males. Competition between two males or an increasing number of males had no influence on female sexual arousal threshold;-females therefore may have a relatively fixed 'arousal threshold' that must be reached before they are ready to mate, and larger males appear to be able to manipulate this threshold sooner. On the other hand, the females' physiological and behavioral state drastically influences mating; once females have crossed the courtship arousal threshold they take less time to mate and mate indiscriminately with large and small males. Mating quicker with larger males may be misconstrued to be due to female choice; our results suggest that the mating advantage of larger males may be more a result of heightened male activity and relatively less of female choice. Body size per se may not be a trait under selection by female choice, but size likely amplifies male activity and signal outputs in courtship, allowing them to influence female arousal threshold faster.

Evolutionary Consequences of Male Driven Sexual Selection and Sex-Biased Fitness Modifications in Drosophila melanogaster and Members of the simulans Clade.

Males have evolved a variety of behavioral, morphological, and physiological traits to manipulate their mates in order to maximize their chances of success. These traits are bound to influence how females respond to male behaviors and influence the nature of sexual selection/conflict. A common consequence of aggressive male mating strategies in Drosophila melanogaster is the reduction of female lifespan. Our study shows that this is common across members of the simulans clade. Reduced life expectancy of females implies that female contribution to a population is less than that of males per generation. Fitness differences between the sexes in every generation will invariably affect overall population fitness. How natural selection responds to the female deaths and thereby the unequal fitness of the sexes has rarely been addressed. We shed light on this issue and provide evidence, which suggests that additional gains of fitness by males due to their longevity and continued mating may provide one explanation as to why the loss of female fitness may be "invisible" (effectively neutral) to natural selection. Male driven sexual selection and additional, transgenerational gains of male fitness can be an important force of evolutionary change and need to be tested with other organisms.

Sex and speciation: Drosophila reproductive tract proteins- twenty five years later.

The protein electrophoresis revolution, nearly fifty years ago, provided the first glimpse into the nature of molecular genetic variation within and between species and showed that the amount of genetic differences between newly arisen species was minimal. Twenty years later, 2D electrophoresis showed that, in contrast to general gene-enzyme variation, reproductive tract proteins were less polymorphic within species but highly diverged between species. The 2D results were interesting and revolutionary, but somewhat uninterpretable because, at the time, rapid evolution and selective sweeps were not yet part of the common vocabulary of evolutionary biologists. Since then, genomic studies of sex and reproduction-related (SRR) genes have grown rapidly into a large area of research in evolutionary biology and are shedding light on a number of phenomena. Here we review some of the major and current fields of research that have greatly contributed to our understanding of the evolutionary dynamics and importance of SRR genes and genetic systems in understanding reproductive biology and speciation.

Is speciation accompanied by rapid evolution? Insights from comparing reproductive and nonreproductive transcriptomes in Drosophila.

The tempo and mode of evolutionary change during speciation have remained contentious until recently. While much of the evidence claiming speciation is an abrupt and rapid process comes from fossil data, recent molecular phylogenetics show that the background of gradual evolution is often broken by accelerated rates of molecular evolution during speciation. However, what kinds of genes affect or are affected by speciation remains unexplored. Our analysis of 4843 protein-coding genes in five species of the Drosophila melanogaster subgroup shows that while ~70% of genes follow clock-like evolution, between 17-19.67% of loci show signatures of accelerated rates of evolution in recently formed species. These genes show 2-3-fold higher rates of substitution in recently diverged species compared to older species. This fraction of loci affects a diverse range of functions. Only a small proportion of reproductive genes experience speciation-related accelerated changes but many sex-and -reproduction related genes show an interesting pattern of persistent rapid evolution suggesting that sex-and-reproduction related genes are under constant selective pressures. The identification of loci associated with accelerated evolution allows us to address the mechanisms of rapid evolution and speciation, which in our study appears to be a combination of both selection and rapid demographical changes.

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits.

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.

Rapidly evolving genes of Drosophila: differing levels of selective pressure in testis, ovary, and head tissues between sibling species.

Investigations of rapidly evolving sex- and reproduction-related genes are expected to reveal important information about the process of speciation and species divergence. We screened testis, ovary, and head tissues to identify and characterize rapidly evolving genes (REGs) between closely related species. The results show differential patterns of evolution of genes expressed in reproductive and nonreproductive tissues. (1) There is a differential distribution of REGs in the Drosophila genome, with most REGs localized in the testis, followed by ovary, and then head. (2) Sequence analysis indicates that differential selective pressures are driving the rapid evolution of genes expressed in sex and nonsex tissues. Testis REGs from our data, on average, yielded higher rates of nonsynonymous substitutions relative to transcripts in ovary and head, indicating stronger selective pressures on the male reproductive system. (3) We identified REGs in the testis, ovary, as well as in head tissue that show evidence of evolving under positive selection. Identification of rapidly evolving sex genes is important for detailed investigations of cryptic female choice, sexual conflict, and faster male evolution and is pertinent to our understanding of the process of species divergence and speciation.