Integrated genetic and morphological data support eco-evolutionary divergence of Angolan and South African populations of Diplodus hottentotus.

The genus Diplodus presents multiple cases of taxonomic conjecture. Among these the D. cervinus complex was previously described as comprising three subspecies that are now regarded as separate species: Diplodus cervinus, Diplodus hottentotus and Diplodus omanensis. Diplodus hottentotus exhibits a clear break in its distribution around the Benguela Current system, prompting speculation that Angolan and South African populations flanking this area may be isolated and warrant formal taxonomic distinction. This study reports the first integrated genetic [mitochondrial (mt)DNA and nuclear microsatellite] and morphological (morphometric, meristic and colouration) study to assess patterns of divergence between populations in the two regions. High levels of cytonuclear divergence between the populations support a prolonged period of genetic isolation, with the sharing of only one mtDNA haplotype (12 haplotypes were fully sorted between regions) attributed to retention of ancestral polymorphism. Fish from the two regions were significantly differentiated at a number of morphometric (69·5%) and meristic (46%) characters. In addition, Angolan and South African fish exhibited reciprocally diagnostic colouration patterns that were more similar to Mediterranean and Indian Ocean congeners, respectively. Based on the congruent genetic and phenotypic diversity we suggest that the use of hottentotus, whether for full species or subspecies status, should be restricted to South African D. cervinus to reflect their status as a distinct species-like unit, while the relationship between Angolan and Atlantic-Mediterranean D. cervinus will require further demo-genetic analysis. This study highlights the utility of integrated genetic and morphological approaches to assess taxonomic diversity within the biogeographically dynamic Benguela Current region.

Experimental manipulation of population-level MHC diversity controls pathogen virulence evolution in Mus musculus.

The virulence levels attained by serial passage of pathogens through similar host genotypes are much higher than observed in natural systems; however, it is unknown what keeps natural virulence levels below these empirically demonstrated maximum levels. One hypothesis suggests that host diversity impedes pathogen virulence, because adaptation to one host genotype carries trade-offs in the ability to replicate and cause disease in other host genotypes. To test this hypothesis, with the simplest level of population diversity within the loci of the major histocompatibility complex (MHC), we serially passaged Friend virus complex (FVC) through two rounds, in hosts with either the same MHC genotypes (pure passage) or hosts with different MHC genotypes (alternated passage). Alternated passages showed a significant overall reduction in viral titre (31%) and virulence (54%) when compared to pure passages. Furthermore, a resistant host genotype initially dominated any effects due to MHC diversity; however, when FVC was allowed to adapt to the resistant host genotype, predicted MHC effects emerged; that is, alternated lines show reduced virulence. These data indicate serial exposure to diverse MHC genotypes is an impediment to pathogen adaptation, suggesting genetic variation at MHC loci is important for limiting virulence in a rapidly evolving pathogen and supports negative frequency-dependent selection as a force maintaining MHC diversity in host populations.

Secondary contact and asymmetrical gene flow in a cosmopolitan marine fish across the Benguela upwelling zone.

The combination of oceanographic barriers and habitat heterogeneity are known to reduce connectivity and leave specific genetic signatures in the demographic history of marine species. However, barriers to gene flow in the marine environment are almost never impermeable which inevitably allows secondary contact to occur. In this study, eight sampling sites (five along the South African coastline, one each in Angola, Senegal and Portugal) were chosen to examine the population genetic structure and phylogeographic history of the cosmopolitan bluefish (Pomatomus saltatrix), distributed across a large South-east Atlantic upwelling zone. Molecular analyses were applied to mtDNA cytochrome b, intron AM2B1 and 15 microsatellite loci. We detected uncharacteristically high genetic differentiation (FST 0.15-0.20; P<0.001) between the fish sampled from South Africa and the other sites, strongly influenced by five outlier microsatellite loci located in conserved intergenic regions. In addition, differentiation among the remaining East Atlantic sites was detected, although mtDNA indicated past isolation with subsequent secondary contact between these East Atlantic populations. We further identified secondary contact, with unidirectional gene flow from South Africa to Angola. The directional contact is likely explained by a combination of the northward flowing offshore current and endogenous incompatibilities restricting integration of certain regions of the genome and limiting gene flow to the south. The results confirm that the dynamic system associated with the Benguela current upwelling zone influences species distributions and population processes in the South-east Atlantic.

Protein pheromone expression levels predict and respond to the formation of social dominance networks.

Communication signals are key regulators of social networks and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odours of dominants and nondominants differentially influence behaviour, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analysing expression levels before, during and after competition for reproductive resources over 3 days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species.

Experimental viral evolution reveals major histocompatibility complex polymorphisms as the primary host factors controlling pathogen adaptation and virulence.

Using an experimental evolution approach, we recently demonstrated that the mouse-specific pathogen Friend virus (FV) complex adapted to specific major histocompatibility complex (MHC) genotypes, which resulted in fitness tradeoffs when viruses were exposed to hosts possessing novel MHC polymorphisms. Here we report the analysis of patterns of pathogen adaptation and virulence evolution from viruses adapting to one of three hosts that differ across the entire genome (A/WySn, DBA/2J and BALB/c). We found that serial passage of FV complex through these mouse genotypes resulted in significant increases in pathogen fitness (156-fold) and virulence (11-fold). Adaptive responses by post-passage viruses also resulted in host-genotype-specific patterns of adaptation. To evaluate the relative importance of MHC versus non-MHC polymorphisms as factors influencing pathogen adaptation and virulence, we compared the magnitude of fitness tradeoffs incurred by post-passage viruses when infecting hosts possessing either novel MHC polymorphisms alone or hosts possessing novel MHC and non-MHC polymorphisms. MHC polymorphisms alone accounted for 71% and 83% of the total observed reductions in viral fitness and virulence in unfamiliar host genotypes, respectively. Strikingly, these data suggest that genetic polymorphisms within the MHC, a gene region representing only -0.1% of the genome, are major host factors influencing pathogen adaptation and virulence evolution.

Sensory quality of meat from lambs fed garlic.

The objective of this study was to determine whether the inclusion of garlic in a ration would have a negative impact on the flavour of lamb. The study used meat from 31 Merino wether lambs fed diets with varying levels of garlic (0%, 1.8% and 3.6%) for 10 weeks. Cooked samples of meat from the lambs were assessed for flavour and acceptability as lamb by an untrained consumer panel. There was no difference (P>0.05) between the treatments in flavour score, but the 3.6% garlic treatment group scored significantly higher in terms of acceptability as lamb (P<0.05) and was commented on positively by the panellists more frequently than the meat from any other treatment (P<0.05). These results suggest that the inclusion of garlic into the animals' feed did not have a negative impact on the flavour of the lamb and, at the high rate (3.6%), made the meat more acceptable to the panellists.

Experimental infection magnifies inbreeding depression in house mice.

It is often assumed that inbreeding reduces resistance to pathogens, yet there are few experimental tests of this idea in vertebrates, and no tests for the effects of moderate levels of inbreeding more commonly found in nature. We mated wild-derived mice with siblings or first cousins and compared the resistance of their offspring to Salmonella infection with outbred controls under laboratory and seminatural conditions. In the laboratory, full-sib inbreeding reduced resistance to Salmonella and survivorship, whereas first-cousin inbreeding had no detectable effects. In competitive population enclosures, we found that first-cousin inbreeding reduced male fitness by 57% in infected vs. only 34% in noninfected control populations. Our study provides experimental evidence that inbreeding reduces resistance and ability to survive pathogenic infection, and moreover, it shows that even moderate inbreeding can cause significant fitness declines under naturalistic conditions of social stress, and especially with exposure to infectious agents.

The physiological function of the coelom in starfish larvae and its evolutionary implications.

The coelom in the bipinnaria larva of Asterias acts as a buoyancy tank. The concentrations of magnesium and sulphate in the coelomic fluid are lower than in seawater, reducing the density. The coelomic epithelium is a secretory epithelium, probably secreting sodium or chloride ions that then draw in the counter ion and water. The rate of urine production is very high for an isotonic marine animal, compensating for the large surface/volume ratio of the coelom. This function would account for the precocious development of the coelom and its association with an excretory duct. It is proposed that the coeloms of other pelagic larvae such as the actinotroch of Phoronis and of echiuran larvae have a similar function and that this may have been an original function of the coelom, although in many phyla this function has been modified or lost.

Accumulated background variation among H2 mutant congenic strains: elimination through PCR-based genotyping of F2 segregants.

Many commercially and privately available congenic strains of laboratory animals were founded decades ago and are likely to differ from one another by dozens of fixed mutational differences at background loci. This problem is often ignored despite growing evidence that such background variation exists. Eliminating this confounding variation can be largely accomplished by crossing congenic strains to produce F2 segregants that are homozygous (or heterozygous) for relevant genes. Discriminating F2 homozygotes can be difficult when strain differences are minor, as are mutant mouse strains differing at single major histocompatibility loci (H2 mutant congenics). Here, we describe a two-step polymerase chain reaction (PCR) method utilizing heteroduplex analysis and sequence specific primers (SSP-PCR) that efficiently discriminates the F2 progeny of two such H2 mutant congenic mice crosses (bm1xB6 and bm1xbm3). A third H2 mutant cross cannot be resolved by heteroduplexing, but is discriminated (albeit less efficiently) with SSP-PCR alone. This sensitive application can be extended to any congenic mutant strains.

Discovery of novel, orally active dual NK1/NK2 antagonists.

Exploration of the SAR around selective NK2 antagonists, SR48968 and ZD7944, led to the discovery that naphth-1-amide analogues provide potent dual NK1 and NK2 antagonists. ZD6021 inhibited binding of [3H]-NKA or [3H]-SP to human NK1 and NK2 receptors, with high-affinity (K(i)=0.12 and 0.62nM, respectively). In functional assays ZD6021 had, at 10(-7)M, in human pulmonary artery pK(B)=8.9 and in human bronchus pK(B)=7.3, for NK1 and NK2, respectively. Oral administration of ZD6021 to guinea pigs dose-dependently attenuated ASMSP induced extravasation of plasma proteins, ED(50)=0.5mg/kg, and NK2 mediated bronchoconstriction, ED(50)=13mg/kg.

Size and fluctuating asymmetry of morphometric characters in mice: their associations with inbreeding and t-haplotype.

Fluctuating asymmetry (FA), a ubiquitous type of asymmetry of bilateral characters, often has been used as a measure of developmental instability in populations. FA is expected to increase in populations subjected to genetic stressors such as inbreeding or environmental stressors such as toxins or parasites, although results have not always been consistent. We tested whether FA in four skeletal size characters and mandible shape was greater in a population of wild-derived mice reared in the laboratory and subjected to one generation of inbreeding (F = 0.25) versus that in an outbred group (F = 0.00). FA did not significantly differ between the inbred and outbred groups, despite the fact that these two groups differed dramatically in fitness under seminatural population conditions. As far as we know, this is the first study to evaluate the relationship between FA and inbreeding in wild house mice, and our general conclusion is opposite that of earlier work on laboratory inbred strains of mice and their hybrids. Size for two of the characters was significantly less in inbreds than in outbreds, however, and there was a significant difference between inbreds and outbreds in the signed differences of right and left sides in one character (humerus length). Some of the mice in both groups also were heterozygous or homozygous carriers of the t-complex. Because mice carrying this chromosome 17 variant are known to have reduced fitness, we also tested whether they had greater FA than mice carrying non-t-haplotypes. The overall level of a composite FA index calculated from all four characters was in fact significantly higher in the t-bearing mice. These combined results suggest that FA is not a generally sensitive proxy measure for fitness, but can be associated with fitness reductions for certain genetic stressors.

Paternity in horseshoe crabs when spawning in multiple-male groups.

Unpaired or satellite male horseshoe crabs, Limulus polyphemus, are attracted to and often form a group around a pair (a female with an attached male) that is nesting in the high intertidal zone. These males are engaged in sperm competition. We observed nesting pairs and their associated satellites in the wild, collected and reared their eggs and used genetic markers to examine paternity. We found that the unpaired, satellite males are highly successful at fertilizing eggs; two satellites can leave the attached male with few fertilizations. Two satellites together are each as successful as one spawning with a pair. A satellite's location around the female greatly affects his success, and males compete for access to a position over the dorsal canal between the prosoma and opisthosoma of the female and under the front margin of the paired male where they are most likely to fertilize eggs. Although eggs and sperm retain their viability for some time after spawning, nearly all eggs are fertilized by the satellites that are around the nesting pair at the time of egg laying and by the attached male. A number of factors including beach current, female size and male behaviour affect the outcome of sperm competition in this externally fertilizing species. Copyright 2000 The Association for the Study of Animal Behaviour.

Nonpeptide tachykinin receptor antagonists. II. Pharmacological and pharmacokinetic profile of SB-222200, a central nervous system penetrant, potent and selective NK-3 receptor antagonist.

The pharmacological and pharmacokinetic profile of SB-222200 [(S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car boxami de], a human NK-3 receptor (hNK-3R) antagonist, was determined. SB-222200 inhibited (125)I-[MePhe(7)]neurokinin B (NKB) binding to Chinese hamster ovary (CHO) cell membranes stably expressing the hNK-3 receptor (CHO-hNK-3R) with a K(i) = 4.4 nM and antagonized NKB-induced Ca(2+) mobilization in HEK 293 cells stably expressing the hNK-3 receptor (HEK 293-hNK-3R) with an IC(50) = 18.4 nM. SB-222200 was selective for hNK-3 receptors compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 250 nM). In HEK 293 cells transiently expressing murine NK-3 receptors (HEK 293-mNK-3R), SB-222200 inhibited binding of (125)I-[MePhe(7)]NKB (K(i) = 174 nM) and antagonized NKB (1 nM)-induced calcium mobilization (IC(50) = 265 nM). In mice oral administration of SB-222200 produced dose-dependent inhibition of behavioral responses induced by i.p. or intracerebral ventricular administration of the NK-3 receptor-selective agonist, senktide, with ED(50) values of approximately 5 mg/kg. SB-222200 effectively crossed the blood-brain barrier in the mouse and rat. The inhibitory effect of SB-222200 against senktide-induced behavioral responses in the mouse correlated significantly with brain, but not plasma, concentrations of the compound. Pharmacokinetic evaluation of SB-222200 in rat after oral administration (8 mg/kg) indicated sustained plasma concentrations (C(max) = about 400 ng/ml) and bioavailability of 46%. The preclinical profile of SB-222200, demonstrating high affinity, selectivity, reversibility, oral activity, and central nervous system penetration, suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors, in particular in the central nervous system.

Chicken beta-globin 5'HS4 insulators function to reduce variability in transgenic founder mice.

Transgenic mice and rats play a vital role in the study of human diseases and the advancement of drug development within the pharmaceutical industry. In this report we describe a method which improves the yield of phenotypically desirable transgenic founder mice resulting from each microinjection session and consequently reduces animal requirements for transgenic production. We have shown that by flanking a tyrosinase minigene with tandem copies of the chicken beta-globin 5'HS4 insulator there is a significant reduction in variability between transgenic lines, with resulting mice exhibiting similar levels of coat pigmentation. Furthermore, the presence of insulators leads to visually identical tissue distribution of pigmentation including skin, fur, eyes, points, and foot pads. The overall impact of insulators is to reduce the number of transgenic founders required in order to identity animals with an appropriate expression level and tissue distribution.

Male-male competition magnifies inbreeding depression in wild house mice.

The detrimental effects of inbreeding on vertebrates are well documented for early stages of the life cycle in the laboratory. However, the consequences of inbreeding on long-term survival and reproductive success (Darwinian fitness) are uncertain for vertebrates in the wild. Here, we report direct experimental evidence for vertebrates that competition increases the harmful effects of inbreeding on offspring survival and reproduction. We compared the fitness of inbred (from full-sib matings) and outbred wild house mice (Mus domesticus) in large, seminatural enclosures. Inbred males sired only one-fifth as many surviving offspring as outbred males because of their poor competitive ability and survivorship. In laboratory conditions, inbreeding had relatively minor effects on male reproductive success and no effect on survivorship. Seminatural conditions did not increase inbreeding depression for females, probably because females were not competing for any critical resources. The overall reduction in fitness from inbreeding was 57%, which is 4.5 times as great as previous estimates from the laboratory. These results have important implications for medicine, conservation, evolutionary biology, and functional genomics.

Untrained mice discriminate MHC-determined odors.

Immune recognition occurs when foreign antigens are presented to T-lymphocytes by molecules encoded by the highly polymorphic genes of the major histocompatibility complex (MHC). House mice (Mus musculus) prefer to mate with individuals that have dissimilar MHC genes. Numerous studies indicate that mice recognize MHC identity through chemosensory cues; however, it is unclear whether odor is determined by classical, antigen-presenting MHC loci or closely linked genes. Previous studies have relied on training laboratory mice and rats to distinguish MHC-associated odors, but there are several reasons why training experiments may be inappropriate assays for testing if MHC genes affect odor. The aim of this study was to determine whether classical MHC genes affect individual odors and whether wild-derived mice can detect MHC-associated odors without training. In the first experiment, we found that wild-derived mice can be trained in a Y-maze to detect the odors of mice that differ genetically only in the MHC region. In the second and third experiments, we used a naturalistic habituation assay and found that wild-derived mice can, without training, distinguish the odors of mice that differ genetically only at one classical MHC locus (dm2 mutants).

Gross appearance of the turkey blastoderm at oviposition.

The blastoderm (fertilized ovum) and unfertilized germinal disc (UGD) of fresh laid eggs and eggs stored prior to incubation exhibit subtle but definable morphological variations. Such variations may lead to difficulty when attempting to determine true flock fertility based on the appearance of the blastoderm/UGD. The objectives of this study were to define and categorize such morphological variations and to determine whether sperm influence the frequency distribution of the different categories. Eleven categories of blastoderms were defined based on the relative density and appearance of the area alba, area pellucida, area opaca, and the periblast. The majority of the blastoderms were included in the first four categories. Unfertilized germinal discs were divided into six categories and were best differentiated from the blastoderms by the presence of vacuoles around its central dense area. They were also discernible from blastoderms based on their overall denser appearance. Differences in the frequency distribution of some of the UGD categories between virgin and inseminated hens may be due to the effect that supernumary sperm may have on the organization of the UGD (no fertilization but supernumary sperm present) or blastoderm (fertilized but failed to develop). It is recommended that before starting true fertility determinations during fresh egg breakouts, one should study the appearance of the UGD from virgin hens and then the blastoderm from inseminated hens. One then will learn to appreciate the subtle differences in shape and density of the blastoderm/UGD structural components.

MHC-disassortative mating preferences reversed by cross-fostering.

House mice (Mus musculus domesticus) avoid mating with individuals that are genetically similar at the major histocompatibility complex (MHC). Mice are able recognize MHC-similar individuals through specific odour cues. However, to mate disassortatively for MHC genes, individuals must have a referent, either themselves (self-inspection) or close kin (familial imprinting), with which to compare the MHC identity of potential mates. Although studies on MHC-dependent mating preferences often assume that individuals use self-inspection, laboratory experiments with male mice indicate that they use familial imprinting, i.e. males learn the MHC identity of their family and then avoid mating with females carrying 'familial' MHC alleles. To determine if female mice use familial imprinting, we cross-fostered wild-derived female mouse pups into MHC-dissimilar families, and then tested if this procedure reversed their mating preferences compared with in-fostered controls. Our observations of the female's mating behaviour in seminatural social conditions and the genetic typing of their progeny both indicated that females avoided mating with males carrying MHC genes of their foster family, supporting the familial imprinting hypothesis. We show that MHC-dependent familial imprinting potentially provides a more effective mechanism for avoiding kin matings and reducing inbreeding than self-inspection.