Morphological and Genetic Variation Among Populations of the Fiddler Crab Minuca burgersi (Holthuis, 1967) (Crustacea: Brachyura: Ocypodidae) from Shores of the Caribbean Basin and Western South Atlantic Ocean.

For this study, in addition to museum vouchers, 1437 specimens of Minuca burgersi (Holthuis, 1967) were collected from crab colonies at 105 locations in the western Atlantic Ocean to examine diversity in a species with a large geographic range. Both allometric and geometric morphometry were coupled with the molecular analysis of DNA to give a broader perspective of intraspecific variability in this species. A total of 1153 specimens from the Caribbean Sea and the Atlantic coast of South America demonstrated that M. burgersi from both regions are very similar in their pattern of growth. The average carapace width (CW) for Caribbean is larger than the average for South American males and females. However, size distribution based on CW is unimodal in Caribbean and bimodal in South American populations. The carapace length-width ratio is about 0.68 in females and 0.66 in males. South American males express asymmetric elongation of the cheliped in smaller CW intervals than Caribbean males. In a sample of 259 females, carapace shape is distinct between South American and Caribbean populations. Caribbean populations have less swelling in the branchial regions than South American populations. The swelling correlates primarily with geographic region and to a lesser degree with substrate and salinity. Molecular data from the 16S rDNA and cytochrome c oxidase subunit I (COI) reveal three clades within Minuca burgersi. Two clades are distributed in the Caribbean and the third in eastern South America. The timing of divergence between Caribbean and South American clades is coincident with an increased rate of water and sediment outflow from the Amazon as inferred from the geologic record. Current patterns and associated gene flow within the Caribbean were subsequently influenced by the closing of the Isthmus of Panama. We speculate that various populations may employ different larval dispersion mechanisms resulting in genetic heterogeneity. Consequently, there is considerable biological divergence among populations of M. burgersi in the Caribbean and South America.

Constraints on geographic variation in fiddler crabs (Ocypodidae: Uca) from the western Atlantic.

A key question in evolutionary biology is how intraspecific variation biases the evolution of a population and its divergence from other populations. Such constraints potentially limit the extent to which populations respond to selection, but may endure long enough to have macroevolutionary consequences. Previous studies have focused on the association between covariation patterns and divergence among isolated populations. Few have focused on geographic variation among semi-connected populations, however, even though this may be indicative of early selective pressures that could lead to long-term divergence and speciation. Here, we test whether covariation in the shape of the carapace of fiddler crabs (genus Uca Leach, 1814) is important for structuring geographic variation. We find that morphological divergence among populations is associated with evolvability in the direction of divergence in only a few species. The shape of the ancestral covariation matrix in these species differs from other species in having notably more variation concentrated along fewer directions (i.e. higher eccentricity). For most species, there is some evidence that covariation has constrained the range of directions into which populations have diverged but not the degree of divergence. These results suggest that even though fiddler crab populations have diverged morphologically in directions predicted by covariation, constraints on the extent to which divergence has occurred may only be manifested in species where variation patterns are eccentric enough to limit populations' ability to respond effectively in many directions.

Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage.

Increased nasopharyngeal colonization density has been associated with pneumonia. We used experimental human pneumococcal carriage to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. A total of 101 healthy subjects were screened for respiratory virus before pneumococcal intranasal challenge. Virus was associated with increased odds of colonization (75% virus positive became colonized vs. 46% virus-negative subjects; P=0.02). Nasal Factor H (FH) levels were increased in virus-positive subjects and were associated with increased colonization density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of coinfection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium. Binding was partially blocked by antibodies targeting the FH-binding protein Pneumococcal surface protein C (PspC). PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding to PspC in vivo masks this binding site, enabling FH to facilitate pneumococcal/epithelial attachment during viral infection despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce pneumococcal colonization, and may have enhanced protection in those with underlying viral infection.

CD56+ T cells are increased in kidney transplant patients following cytomegalovirus infection.

BACKGROUND: CD56+ T cells previously have been identified as potentially cytotoxic lymphocytes, and relative numbers are increased in some infectious diseases. PATIENTS AND METHODS: Relative proportions of CD56+ T cells were measured by flow cytometry in groups of renal transplant patients differing in cytomegalovirus (CMV) status of donor (D) and recipient (R). These measurements were related to episodes of CMV viremia. RESULTS: Patient groups in which recipients (R+) or donors (D+/R-) were CMV+ had significantly higher proportions of CD56+ T cells (5.11 ± 0.69% and 5.42 ± 1.01%, respectively) than the D-/R- group (1.9 ± 0.35%; P = 0.0018 and 0.017, respectively). In the high-risk D+/R- group, it was found that patients who had post-transplant CMV viremia had higher levels than those who remained CMV negative (9.09 ± 2.34% vs. 3.16 ± 1.22%; P = 0.01). CD56+ T cells from R+ and D+/R- groups had higher proportions of both CD4+ and CD8+ cells than the D-/R- group. When activation markers were examined, some CD56+ T cells from both CMV+ groups had a TEM phenotype, with significantly more expressing CD45RO and NKG2C, and less expressing CD28, CD62L, CD127, and CD161 compared to the D-/R- group. Some CD56+ T cells showed specificity for CMV antigens and similar proportions of CD8+ cells were positive for class I HLA-CMV tetramers containing immunodominant CMV peptides compared to the majority CD56- T cells. CONCLUSION: The results show significant increases in proportions of CD56+ T cells in relation to CMV infection in renal transplant patients and suggest that these cells have a cytotoxic function against CMV-infected cells.

Decoupling of taxonomic diversity and morphological disparity during decline of the Cambrian trilobite family Pterocephaliidae.

Although discordance between taxonomic diversity and morphological disparity is common, little is known about the underlying dynamics that drive this decoupling. Early in the history of the Cambrian trilobite family Pterocephaliidae, there was an increase in taxonomic diversity and morphological diversity. As taxonomic diversity declined in the later history of the clade, range of variation stayed high and disparity continued to increase. However, per-branch rates of morphological evolution estimated from a recent phylogeny decreased with time. Neither within-trait nor within-species variation increased or decreased, suggesting that the declining rates of morphological evolution were more likely related to ecological opportunity or niche partitioning, rather than increasing intrinsic constraints. This is further supported by evidence for increased biofacies associations throughout the time period. Thus, the high disparity seen at low taxonomic diversity late in the history of this clade was due to extinction - either random or targeting mean forms - rather than increased rates of morphological evolution. This pattern also provides a scenario that could account for instances of low taxonomic diversity but high morphological disparity in modern groups.

Validation of a laboratory-developed real-time PCR protocol for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

OBJECTIVE: To evaluate a sensitive and specific, real-time PCR assay with internal control for Chlamydia trachomatis and Neisseria gonorrhoeae DNA detection in urine specimens. METHODS: The diagnostic performance of a laboratory-developed quadruplex assay (LDQA) targeting the cryptic plasmid and MOMP genes of C trachomatis, the porA pseudogene of N gonorrhoeae and a synthetic internal control was assessed using 1028 urine specimens. The LDQA was compared with the Roche COBAS Taqman CT test and the COBAS Amplicor NG assay with supplemental confirmation tests. The subsequent performance of the LDQA in detecting N gonorrhoeae was monitored in comparison with bacterial culture from swabs. RESULTS: 88 (8.6%) urines were determined as C trachomatis positive in the diagnostic evaluation. LDQA sensitivity and specificity were calculated to be 100% and 99.9%, respectively, for C trachomatis. The LDQA showed high specificity with isolates of other Neisseria species and gave complete concordance with resolved data for N gonorrhoeae detection. However, the incidence of N gonorrhoeae infection was low, with 17 (1.7%) positive patients. A post-implementation audit of 14 316 patients gave the LDQA N gonorrhoeae urine PCR protocol (porA, OPA, 16s rDNA) a sensitivity of 96.9% and specificity of 99.8% in comparison with bacterial culture from swabs. CONCLUSIONS: The LDQA was found to be an effective method for the detection of C trachomatis and N gonorrhoeae DNA in urine samples, and the PCR protocol has replaced bacterial culture for the screening of N gonorrhoeae in asymptomatic men and women in the laboratory.

Prebiotic carbohydrates modify the mucosa associated microflora of the human large bowel.

BACKGROUND AND AIMS: The mucosa associated flora of the large intestine is important in determining mucosal function although what controls its composition is unknown. This study has determined the effect of the prebiotic carbohydrates oligofructose and inulin on the mucosal flora. METHODS: An in vitro chemostat model of both planktonic and surface associated bacteria was used followed by an intervention study in 29 subjects undergoing colonoscopy. SUBJECTS: Fourteen subjects, recruited from colonoscopy waiting lists, supplemented their diet for two weeks with a mix of 7.5 g of oligofructose and 7.5 g inulin. Fifteen subjects were recruited at the time of colonoscopy and given no supplement. Multiple endoscopic biopsies were taken from the caecum, transverse and descending colon, and rectum. The mucosal flora was characterised by culture and to species level by cellular fatty acid profiles. Cell proliferation was assessed by immunohistochemical staining for minichromosome maintenance protein 2, Ki67, and proliferating cell nuclear antigen. RESULTS: In vitro prebiotics increased surface counts of bifidobacteria from 6.6 to 7.3 log(10) colony forming units (CFU) per slide (p<0.0006) with no significant changes in planktonic bacteria. In the feeding study, prebiotics increased mucosal bifidobacteria (log CFU/g mucosa (SEM)) in both the proximal (control 5.3 (0.4) v prebiotic 6.3 (0.3)) (p = 0.059) and distal (control 5.2 (0.3) v prebiotic 6.4 (0.3)) colon (p = 0.01). Lactobacilli were also increased (3.0 (0.1) v 3.7 (0.2) (p = 0.02) in the proximal and 3.1 (0.1) v 3.6 (0.2) (p = 0.04) in the distal colon, respectively). There were significantly more eubacteria in fed subjects but no changes in total anaerobes clostridia, bacteroides, or coliforms, nor in proliferation indices. CONCLUSION: Prebiotic carbohydrates can change the composition of the mucosa associated flora significantly.

Identification and quantitation of mucosal and faecal desulfovibrios using real time polymerase chain reaction.

BACKGROUND: Desulfovibrios produce sulphide, which is toxic to colonic epithelial cells. These bacteria have previously been linked to ulcerative colitis. Traditional methods of culturing these organisms are slow, and often unreliable, while molecular approaches are either non-quantitative or lack sensitivity. AIMS: To develop a sensitive method for quantitating desulfovibrios in stools and biopsy tissue, and to investigate the effects of age and disease on these bacteria. METHODS: Rectal biopsies were taken from 10 colitis patients and 10 healthy controls. Stool samples were obtained from 10 healthy infants (mean age 1.01 (0.18) years), 10 healthy young adults (26.7 (1.2) years), and 10 healthy elderly people (71.7 (1.2) years). Primers were designed and developed for analysing Desulfovibrio populations in the bowel using real time polymerase chain reaction (PCR). RESULTS: The PCR primers were highly specific for desulfovibrios. Large numbers (approximately 10(6)-10(7)/g) occurred in biopsies in colitis patients and healthy subjects, and no disease related differences were observed. Measurements of mucosal desulfovibrios over 12 months showed marked changes in some patients. Infants (10(6)-10(7)/g) and elderly people (10(7)-10(8)/g) had significantly higher numbers of desulfovibrios in stools compared with young adults (10(5)/g). CONCLUSIONS: Real time PCR analysis of desulfovibrios was an efficient and accurate method for studying these potentially harmful microorganisms. Desulfovibrios were ubiquitous in the bowel, irrespective of age. As rectal mucosae were heavily colonised in health and disease, if these bacteria play a role in colitis, some host defect, possibly in sulphide detoxication pathways or in bacterial antigen handling, is required for manifestations of pathogenicity.

Variation in human intestinal microbiota with age.

The large intestinal microbiota plays an important role in normal bowel function and the maintenance of host health, through the formation of short chain fatty acids, modulation of immune system reactivity and development of colonisation resistance. However, the effects of ageing on bacterial community structure in the colon are not well documented. Aim of this study is to assess bacterial species diversity in the human faecal microbiota with respect to age and Clostridium difficile infection. Bacterial populations were quantified from stool samples obtained from children (16 months to seven years), young adults (21-34 years), healthy elderly people (67-88 years) and patients diagnosed with Clostridium difficile-associated diarrhoea (68-73 years). Microbial diversity was assessed to species level for samples from the latter three subject groups. Marked interindividual variations occurred in microbial composition at genus and species levels. The faecal microbiota of children was found to be bacteriologically less complex whilst advancing age was associated with decreased bifidobacteria and increased bacteroides species diversity. Changes in microbial composition with age or disease will alter the metabolic capacity of the gut microbiota and has important implications for therapies aimed at modulating the large intestinal microbiota.

Changes in predominant bacterial populations in human faeces with age and with Clostridium difficile infection.

The bacterial composition of human faeces can vary greatly with factors such as age and disease, although relatively few studies have monitored these events, particularly at species level. In this investigation, bacteria were isolated from faecal samples from healthy young adults and elderly subjects, and elderly patients with Clostridium difficile-associated diarrhoea (CDAD). The organisms were identified to species level on the basis of their cellular fatty acid profiles with the MIDI system. In some groups of bacteria, species diversity was found to change with age despite the overall numbers of organisms being similar at genus level. Bacteroides thetaiotaomicron, B. ovatus and Prevotella tannerae were common gram-negative anaerobes isolated from young adults. Bacteroides species diversity increased in the faeces of healthy elderly people. Bifidobacterial species diversity decreased with age, with Bifidobacterium adolescentis and Bif. angulatum being the most common isolates. CDAD patients were characterised by greater diversity of facultative species, lactobacilli and clostridia, but greatly reduced numbers of bacteroides, prevotella and bifidobacteria. Such bacterial population changes in the normal microbiota could result in metabolic conditions favourable for the establishment of pathogenic micro-organisms, such as clostridia, and would have considerable effects on the biochemical capacity of the large intestine as a whole. Alterations in the community structure of bifidobacteria and lactobacilli have relevance for dietary and therapeutic interventions such as the use of pre- or probiotics that aim to modify the composition or metabolic activities of the intestinal microflora in a beneficial way, particularly in elderly people or individuals at risk of CDAD.

Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum.

Clostridium septicum is responsible for several diseases in humans and animals. The bacterium is capable of a simple kind of multicellular behavior known as swarming. In this investigation, environmental and physiologic factors affecting growth and swarm cell formation in C. septicum were studied over a range of dilution rates (D = 0.02 to 0.65 h(-1)) in glucose-limited, glucose-excess, and mucin-limited chemostats. Cellular differentiation was observed at low specific growth rates, irrespective of the carbon and energy source, showing that swarming occurred in response to nutrient depletion. Differential expression of virulence determinants was detected in swarm cells. Hemolysin was secreted by short motile rods but not swarm cells, whereas in cultures grown with glucose, only swarm cells formed DNase, hyaluronidase, and neuraminidase. However, neuraminidase and, to a lesser degree, hyaluronidase were induced in short motile rods in mucin-limited cultures. Both swarm cells and short rods were cytotoxic to Vero cells. Mucin was chemotaxic to C. septicum, and large amounts of mucin-degrading enzymes (beta-galactosidase, N-acetyl beta-glucosaminidase, glycosulfatase, and neuraminidase) were produced. Synthesis of these enzymes was catabolite regulated. In chemostat experiments, glycosulfatase secretion occurred only in swarm cells at low dilution rates in mucin-limited cultures. Determinations of oligosaccharide utilization demonstrated that N-acetylglucosamine, galactose, and N-acetylgalactosamine were the main carbon sources for C. septicum in mucin. Neuraminic acid was not assimilated, showing that neuraminidase does not have a direct nutritional function in this pathogen.

Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles.

BACKGROUND: The normal intestinal microflora plays an important role in host metabolism and provides a natural defence mechanism against invading pathogens. Although the microbiota in adults has been extensively studied, little is known of the changes that occur in the microflora with aging. These may have important consequences in elderly people, many of whom are receiving antibiotic therapy and who are most susceptible to intestinal dysbiosis. AIMS: To characterise the major groups of faecal bacteria in subjects of different ages using a combination of cultural, molecular, and chemotaxonomic approaches. METHODS: Comparative microbiological studies were made on four different subject groups: children (16 months to seven years, n=10), adults (21-34 years, n=7), healthy elderly subjects (67-88 years, n=5), and geriatric patients (68-73 years, n=4) diagnosed with Clostridium difficile diarrhoea. Selected faecal bacteria were investigated using viable counting procedures, 16S ribosomal RNA (rRNA) abundance measurements, and the occurrence of specific signature fatty acids in whole community fatty acid methyl ester profiles. RESULTS: The principal microbiological difference between adults and children was the occurrence of higher numbers of enterobacteria in the latter group, as determined by viable counts (p<0.05) and 16S rRNA (p<0.01) measurements. Moreover, a greater proportion of children's faecal rRNA was hybridised by the three probes (bifidobacteria, enterobacteria, bacteroides-porphyromonas-prevotella) used in the study, indicating a less developed gut microbiota. Species diversity was also markedly lower in the Clostridium difficile associated diarrhoea group, which was characterised by high numbers of facultative anaerobes and low levels of bifidobacteria and bacteroides. Although it was a considerably less sensitive diagnostic tool, cellular fatty acid analysis correlated with viable bacterial counts and 16S rRNA measurements in a number of bacteria, including bacteroides. CONCLUSIONS: Polyphasic analysis of faecal bacteria showed that significant structural changes occur in the microbiota with aging, and this was especially evident with respect to putatively protective bifidobacteria. Reductions in these organisms in the large bowel may be related to increased disease risk in elderly people.

Evaluation of 16s rRNA and cellular fatty acid profiles as markers of human intestinal bacterial growth in the chemostat.

Chemostats were used to study the effects of carbon and nitrogen limitation and specific growth rate on 16S rRNA synthesis and cellular fatty acid (CFA) profiles in four human intestinal bacteria (Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Clostridium bifermentans and Cl. difficile). Cellular fatty acid synthesis varied with dilution rate and nutrient availability in different species, but these cellular constituents were relatively stable phenotypic characteristics in Bact. thetaiotaomicron, where branched chain and hydroxy CFA were good taxonomic markers. Conversely, CFA in the Gram-positive bacteria varied markedly with changes in growth environment. For example, in chemostats, cyclopropane CFA were only synthesized in Cl. bifermentans and Cl. difficile under N-limited conditions. Similarly, Dimethyl acetal (DMA) fatty acids in Bif. adolescentis were primarily produced during N-limited growth, and this was inversely related to dilution rate. At low growth rates, 16S rRNA concentrations (microg rRNA per ml culture) correlated with viable bacterial counts, but were more closely related to specific growth rate when expressed as a function of cell mass (microg rRNA per mg dry weight bacteria). However, this did not reveal differences in bacterial population size and rRNA concentration in C-limited cultures. Thus, at low dilution rates, C limitation strongly reduced rRNA synthesis in Cl. bifermentans, despite viable cell counts being similar to those in N-limited cultures. These results indicate that, while 16S rRNA is a useful indicator of microbial activity, cell growth rate does not necessarily relate to rRNA concentration under all nutritional conditions. Consequently, bowel habit and diet will affect both CFA and rRNA content in bacteria isolated from intestinal samples, and this should be taken into consideration when interpreting such data measurements.

A palliative care education model for the hospital setting.

This paper describes an integrated, needs-led palliative care education model for qualified nurses, developed by collaboration between a hospital-based palliative care team of a large NHS Trust and a specialist palliative care unit, in order to serve its common healthcare users. The aim of this model was to develop the knowledge and skills gained through clinical practice and ongoing education in order to achieve a basic level of expertise, and to enhance communication and collaboration between the hospital and community-based nursing services throughout the geographical area for for patients requiring palliative care.

A test of pollinator specificity and morphological convergence between nectarivorous birds and rainforest tree flowers in New Guinea.

Interactions between flowering trees in a representative sample of vegetation, and the birds that fed at their flowers, were studied for 2 years in lowland tropical hill forest in New Guinea. All 2,200 trees in a 3-ha plot were tagged, identified, mapped, and monitored monthly. Approximately 60% of all individual trees flowered during the study; all species that these flowering individuals belonged to were evaluated for bird visitation. Approximately 13% of the 164 resident species of New Guinea avifauna at the study site, especially honeyeaters and parrots, visited flowers. In the forest inventory plot, approximately 15-22% of all 86 tree species that flowered during the study were visited by birds; most of these tree species were canopy species. Results showed that there was no statistically significant correlation between bird species grouped by bill morphology and flower species grouped as morphotypes and ranked by nectar accessibility, although strong but unexpected bird/plant associations were evident. These associations may be related to variables such as body mass or perch size. These results are discussed in comparison with results from the Neotropics and Australia, and in terms of morphological convergence and pollinator specificity in pollination systems.

Evaluating a basic-needs strategy and population policies: the BACHUE approach.

PIP: The BACHUE model, a dynamic simulation technique developed within the International Labour Organization's World Employment Program, has been applied to the Philippines. The model simulates behavior and consequences in a number of key areas: fertility, marriage, migration, savings and expenditure, and labor force participation for households and a macro-model for demand, ouput, employment, and income. The design and development of the model are discussed in detail. The model was run for a series of 13 experiments ranging from nationlization of modern sectors, increasing self-employment, movement toward labor-intensive techniques, changes in growth rates of various sectors, and a reduction in fertility by 2% over 1976-1985, an increase over the 1% assumed in the base run. Runs R-2 to R-11 all showed that a change in basic needs is associated with significant declines in fertility, largely because of increasing education and decreasing mortality. Better economic conditions in rural areas also reduced migration. R-13 which examined the effects of a family planning program of moderate size on ultimate fertility, showed that even by year 2000 the effects were small. The population is reduced 5% over the run which assumes negative income tax and government subsidies to poor families but the gain in income per adult is less than 4%. Any real improvement in income as the result of family planning will take 40-50 years to achieve. Economic incentives, on the other hand, have much faster demographic results. The models also show that rural-urban migration is responsive to policy changes. Planners are cautioned that the model is not a picture of the entire range of human behavior but is an adjunct for use in analyzing interaction between policies.^ieng

On the evaluation of population and employment policy.

PIP: The Bachue series of planning models, which contain economic, demographic, and where possible social variables integrated into a theoretical schema purporting to represent interrelationships between these variables in the real world, are described as a background for discussion of planning for employment and income distribution. The basic structure of models in the Bachue series is described and illustrated using examples from the model for the Philippines. Bachue-type models are useful for evaluating policies, measuring the cost-effectiveness of policy instruments and the trade-offs between them, and identifying redundancies and complementarities between different policies rather than for generating projections. The models analyze the effects of different types of demographic and economic change in terms of such outputs as employment, income, income distribution and population size and structure. Examples of education, migration, and public works policies from the Philippines are used to show how these analyses are conducted and how their results are interpreted.^ieng