Molecular-based estimate of species number,phylogenetic relationships and divergence times for the genus Stenotaenia (Chilopoda,Geophilomorpha) in the Italian region

Stenotaenia is one of the largest and most widespread genera of geophilid centipedes in the Western Palearctic, with a very uniform morphology and about fifteen species provisionally recognized. For a better understanding of Stenotaenia species-level taxonomy, we have explored the possibility of using molecular data. As a preliminary assay, we sampled twelve populations, mainly from the Italian region, and analyzed partial sequences of the two genes COI and 28S. We employed a DNA-barcoding approach, complemented by a phylogenetic analysis coupled with divergence time estimation. Assuming a barcoding gap of 10–16% K2P pairwise distances, we found evidence for the presence of at least six Stenotaenia species in the Italian region, which started diverging about 50 million years ago, only partially matching with previously recognized species. We found that small-sized oligopodous species belong to a single clade that originated about 33 million years ago, and obtained some preliminary evidence of the related genus Tuoba being nested within Stenotaenia.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.     

Selenium phytoremediation potential of Stanleya pinnata

Disposal of saline irrigation wastewater in hydrologically closed sinks in the semi-arid western U.S. has concentrated selenium-rich salts to hazardous levels and phytoextraction, along with plant-enhanced volatilization of methyl-selenides, is an active area of research. Here, we provide an overview of our ongoing studies of Stanleya pinnata (Brassicaceae), a previously unstudied candidate that is a primary accumulator (hyperaccumulator) of Se that is widespread and broadly adapted in the western U.S. When grown in sand culture under uniform greenhouse conditions, 16 populations representing S. pinnata's broad biogeographical range differed in shoot Se concentration by 1.4- to 3.6-fold, and the shoot concentrations were positively correlated with the indigenous soil Se levels at the collection sites. Thus, S. pinnata exhibits significant ecotypic variation in Se accumulation. All populations accumulated SeO₄ ^2- preferentially over SO₄ ^2- consistent with S. pinnata's classification as a primary Se accumulator, while hydroponically-grown Brassica juncea consistently accumulated sulfate preferentially over selenate. The Se in S. pinnata shoots was predominately in the soluble amino-acid pool, which may serve as direct precursor to volatile forms such as dimethyldiselenide; inorganic forms (e.g. selenate) dominated in B. juncea. Preliminary results suggest that S. pinnata may volatilize unusually large quantities of Se when grown at high sulfate concentrations, an unexpected result not heretofore reported in any species. In a sand–culture experiment, S. pinnata exhibited excellent tolerance of excess boron, but only moderate tolerance of salinity, and superior genotypes will likely be needed for phytoremediation of highly salinized soils and sediments. Stanleya pinnata is a perennial that responded favorably to repeated cuffing in the greenhouse, a trait that could prove valuable in field-scale phytoremediation. Environmental concerns about Se are common in the western USA, and S. pinnata is a potentially useful species for phytoremediation due to its broad adaptation to western soils and environments, and its uptake, metabolism and volatilization of Se.     

Recruitment of Black Howler Fruit Trees in Fragmented Forests of Northern Belize

We examined recruitment of trees whose seeds are dispersed by black howler monkeys (Alouatta pigra) in forest fragments within the Community Baboon Sanctuary in north-central Belize. In the fragments, most other large frugivores are absent and howlers dominate the large frugivore community. Consequently, we expected to observe an increased representation of howler fruit trees among the sapling community. To test this prediction preliminarily we observed howler feeding behavior for one year and conducted adult tree and sapling transects in 6 locations where howlers were present. We sampled a seventh site where howlers were absent for vegetation only. We found that in 4 of 6 sites there were proportionately fewer howler fruit saplings when compared to adult tree samples. However, when recruitment of howler fruit trees in the 6 sites was compared to a site where howlers were absent, 11 of 12 species had relatively higher recruitment. The lack of recruitment among howler fruit trees in general likely reflects differences in responses of individual species, as well as disruptions from human activities to natural processes within the forests. The observed lower recruitment patterns of howler trees suggest that over time, the abundance of the trees will likely decline and thus affect the foraging behavior and possibly survival of howlers in the fragmented forests.     

Robustness of a meta-network to alternative habitat loss scenarios

Studying how habitat loss affects the tolerance of ecological networks to species extinction (i.e. their robustness) is key for our understanding of the influence of human activities on natural ecosystems. With networks typically occurring as local interaction networks interconnected in space (a meta-network), we may ask how the loss of specific habitat fragments affects the overall robustness of the meta-network. To address this question, for an empirical meta-network of plants, herbivores and natural enemies we simulated the removal of habitat fragments in increasing and decreasing order of area, age and connectivity for plant extinction and the secondary extinction of herbivores, natural enemies and their interactions. Meta-network robustness was characterized as the area under the curve of remnant species or interactions at the end of a fragment removal sequence. To pinpoint the effects of fragment area, age and connectivity, respectively, we compared the observed robustness for each removal scenario against that of a random sequence. The meta-network was more robust to the loss of old (i.e. long-fragmented), large, connected fragments than of young (i.e. recently fragmented), small, isolated fragments. Thus, young, small, isolated fragments may be particularly important to the conservation of species and interactions, while contrary to our expectations larger, more connected fragments contribute little to meta-network robustness. Our findings highlight the importance of young, small, isolated fragments as sources of species and interactions unique to the regional level. These effects may largely result from an unpaid extinction debt, whereby younger fragments are likely to lose species over time. Yet, there may also be more long-lasting effects from cultivated lands (e.g. water, fertilizers and restricted cattle grazing) and network complexity in small, isolated fragments. Such fragments may sustain important biological diversity in fragmented landscapes, but maintaining their conservation value may depend on adequate restoration strategies.     

Zebrafish Caudal Fin Angiogenesis Assay—Advanced Quantitative Assessment Including 3-Way Correlative Microscopy

Background

Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics.

Objective

To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way.

Approach & Results

Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level.

Conclusions

The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element