Current distribution and ecology of the critically endangered <Emphasis Type="Italic">Valencia letourneuxi</Emphasis> in Greece

The current population status of the critically endangered Greek valencia Valencia letourneuxi (Sauvage, 1880), is presented, based on a thorough survey of low-altitude aquatic systems of Western Greece. The species’ historical geographical range appears to have been reduced, with the westernmost and southernmost populations being currently extinct or near extinction respectively, and the remaining ones in a vulnerable state. According to univariate and multivariate analysis, V. letourneuxi exhibits habitat specificity, with a strong affinity towards spring-fed wetlands with clear waters and rich surface vegetation, being thus vulnerable to human-induced habitat modifications and hydrological changes. The impact of the above threats, as well as of the introduction of alien species such as the mosquitofish Gambusia holbrooki is discussed.     

Hurricane Disturbance Alters Secondary Forest Recovery in Puerto Rico

Land-use history and large-scale disturbances interact to shape secondary forest structure and composition. How introduced species respond to disturbances such as hurricanes in post-agriculture forest recovery is of particular interest. To examine the effects of hurricane disturbance and previous land use on forest dynamics and composition, we revisited 37 secondary forest stands in former cattle pastures across Puerto Rico representing a range of exposure to the winds of Hurricane Georges in 1998. Stands ranged from 21 to>80 yr since agricultural abandonment and were measured 9 yr posthurricane. Stem density decreased as stands aged, while basal area and species richness tended to increase. Hurricane disturbance exerted contrasting effects on stand structure, contingent on stand age. In older stands, the basal area of large trees fell, shifting to a stand structure characteristic of younger stands, while the basal area of large trees tended to rise in younger stands with increasing hurricane disturbance. These results demonstrate that large-scale natural disturbances can alter the successional trajectory of secondary forest stands recovering from human land use, but stand age, precipitation and soil series were better predictors of changes in stand structure across all study sites. Species composition changed substantially between census intervals, but neither age nor hurricane disturbance consistently predicted species composition change. However, exposure to hurricane winds tended to decrease the abundance of the introduced tree Spathodea campanulata, particularly in smaller size classes. In all sites the abundance of the introduced tree Syzygium jambos showed a declining trend, again most strongly in smaller size classes, suggesting natural thinning through succession.     

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.Download video file.^{(95M, mp4)}     

Unbiased estimation of gene diversity in samples containing related individuals: exact variance and arbitrary ploidy

Gene diversity, a commonly used measure of genetic variation, evaluates the proportion of heterozygous individuals expected at a locus in a population, under the assumption of Hardy-Weinberg equilibrium. When using the standard estimator of gene diversity, the inclusion of related or inbred individuals in a sample produces a downward bias. Here, we extend a recently developed estimator shown to be unbiased in a diploid autosomal sample that includes known related or inbred individuals to the general case of arbitrary ploidy. We derive an exact formula for the variance of the new estimator, H, and present an approximation to facilitate evaluation of the variance when each individual is related to at most one other individual in a sample. When examining samples from the human X chromosome, which represent a mixture of haploid and diploid individuals, we find that H performs favorably compared to the standard estimator, both in theoretical computations of mean squared error and in data analysis. We thus propose that H is a useful tool in characterizing gene diversity in samples of arbitrary ploidy that contain related or inbred individuals.     

Introgression at differentially aged hybrid zones in red-tailed chipmunks

Hybrid zones allow us to investigate the maintenance and the break down of reproductive isolation; they are a window into the speciation process. Tamias ruficaudus (red-tailed chipmunk) has a roughly ring-like distribution in the Inland Northwest and includes two morphologically well-differentiated subspecies, T. r. ruficaudus (in the eastern portion of its range) and T. r. simulans (in the western portion). These taxa meet at two contact zones: the Lochsa River in Idaho and 200 km to the north, near Whitefish, Montana. The Lochsa Zone is encompassed within the Clearwater River Drainage, which has been proposed as a glacial refugium for many taxa throughout the Pleistocene, whereas the Whitefish Zone was under the Cordilleran ice sheet during the most recent glacial maxima approximately 10,000 years ago. Mitochondrial DNA introgression has been documented at both contact zones, yet the subspecies remain significantly distinct with respect to bacular morphology and no intermediate morphologies have ever been reported. Here, we elucidate differentiation and introgression of the nuclear genome using ten microsatellite loci and compare findings to previously described mitochondrial DNA haplotype distribution and introgression. We found significant substructure in the nuclear data; each subspecies is divided into at least two genetically distinct demes. At the Lochsa contact zone, individuals restricted to the mtDNA zone of introgression form a distinct deme at microsatellite loci whereas in the younger, Whitefish contact zone, there is no hybrid-zone specific group. The genetic distances of the demes within these two subspecies indicate recent northward expansion.     

Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico

Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.Vibrio spp. occur naturally in estuarine and marine environments, and two species of this genus, V. vulnificus and V. parahaemolyticus, are responsible for the majority of reported vibrio illnesses in the United States (2). V. vulnificus infections are most commonly associated with the Gulf of Mexico, either via consumption of raw oysters harvested from these waters or wound infections following exposure to seawater. On average, about 50 cases of V. vulnificus septicemia are reported in the United States each year, with a case fatality rate of approximately 50% (31), the highest of any food-borne pathogen. In contrast, V. parahaemolyticus is the most common cause of seafood-associated bacterial gastroenteritis in the United States, with an estimated annual rate of 4,500 cases per year according to the Centers for Disease Control and Prevention. V. parahaemolyticus also causes wound infections, though these are less frequent and less severe compared to those caused by V. vulnificus (5). Primary septicemia can occur following V. parahaemolyticus infection, but it is relatively rare for this pathogen. In the United States, V. parahaemolyticus illness most often results from consumption of raw or undercooked seafood, particularly oysters.It is well established that vibrio densities correlate strongly with sea surface temperature (SST), with densities increasing as temperatures increase; however, with the exception of salinity, little is definitively known about the influence of other environmental parameters, such as turbidity and chlorophyll a (22, 33). Consequently, while SST has been estimated to explain approximately 50% of the annual variation of V. parahaemolyticus abundance in oysters harvested from the northern Gulf of Mexico (40), a considerable amount of variation remains unexplained. It is of interest to delineate the effects of other environmental parameters independent of SST, as these parameters may be associated with spatial and temporal variation of vibrio densities within seasonal periods when SST is relatively constant and risk of human exposure and illness is high. Moreover, the majority of what is known about V. parahaemolyticus in the environment is based on total populations; little information is available on the pathogenic subpopulations. Isolates containing genetic markers for pathogenicity factors, including the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) typically constitute <1% of the population in marine or postharvest oyster samples, but they account for >90% of clinical isolates (12). The basis for V. vulnificus pathogenicity remains unclear, as few pathogenicity factors have been described definitively (31). To address these data gaps, we monitored densities of culturable V. vulnificus containing vvh (the V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (the thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh, and trh in water, oysters, and sediment collected from coastal waters of Mississippi and Alabama. Associations between bacterial densities and environmental parameters were analyzed by regressing observations against sea surface temperature, chlorophyll a, turbidity, and salinity.     

Amino acid abundance and proteolytic potential in North American soils

Studies of nitrogen (N) cycling have traditionally focused on N mineralization as the primary process limiting plant assimilation of N. Recent evidence has shown that plants may assimilate amino acids (AAs) directly, circumventing the mineralization pathway. However, the general abundance of soil AAs and their relative importance in plant N uptake remains unclear in most ecosystems. We compared the concentrations and potential production rates of AAs and NH₄ ⁺, as well as the edaphic factors that influence AA dynamics, in 84 soils across the United States. Across all sites, NH₄ ⁺ and AA-N comprised similar proportions of the total bioavailable N pool (~20%), with NO₃ ⁻ being the dominant form of extractable N everywhere but in tundra and boreal forest soils. Potential rates of AA production were at least comparable to those of NH₄ ⁺ production in all ecosystems, particularly in semi-arid grasslands, where AA production rates were six times greater than for NH₄ ⁺ (P < 0.01). Potential rates of proteolytic enzyme activity were greatest in bacteria-dominated soils with low NH₄ ⁺ concentrations, including many grassland soils. Based on research performed under standardized laboratory conditions, our continental-scale analyses suggest that soil AA and NH₄ ⁺ concentrations are similar in most soils and that AAs may contribute to plant and microbial N demand in most ecosystems, particularly in ecosystems with N-poor soils.     

Proposal for a hydrogen bond network in the active site of the prototypic gamma-class carbonic anhydrase

The crystal structure of Cam, the prototypic gamma-class carbonic anhydrase, reveals active site residues Gln75, Asn73, and Asn 202 previously hypothesized to participate in catalysis. These potential roles were investigated for the first time by kinetic analyses of site-specific replacement variants of the zinc and cobalt forms of Cam. Gln75 replacement variants showed large decreases in k(cat)/K(m) relative to wild-type. Further, the Gln75 variants showed a loss of the pK(a) in pH versus k(cat)/K(m) profiles previously attributed to ionization of the metal-bound water yielding the hydroxyl group attacking CO(2). These results support the previously proposed role for Gln75 in hydrogen bonding with the catalytic hydroxyl orienting it for attack on CO(2). Kinetic analyses of Asn73 variants were consistent with a role in hydrogen bonding with Gln75 to position it for optimal interaction with the catalytic hydroxyl. Kinetic analyses of Asn202 variants showed substantial decreases in k(cat)/K(m) relative to the wild-type enzyme supporting the previously hypothesized role in polarizing CO(2) and facilitating attack from the metal-bound hydroxyl. On the basis of results presented here, and previously reported structural analyses, we present a catalytic mechanism involving Gln75, Asn73, and Asn202 that also suggests a role for Glu62 not previously recognized. Finally, the results suggest that the gamma-, beta-, and alpha-class carbonic anhydrases each independently evolved variations of a fundamental hydrogen bond network essential for catalysis.