Responses in pigmentation and anti-oxidant expression in Arctic Daphnia along gradients of DOC and UV exposure

Responses in carapace melanization and expression of the majoranti-oxidant catalase (CAT) and glutathione transferase (GST)in Arctic Daphnia were assessed in enclosures along a gradientof dissolved organic carbon (DOC). This gradient was createdby adding freeze-dried humic matter to 2 m³ UV-transparent enclosures,yielding final nominal concentrations of 1, 2.5, 5 and 10 mgC l^-1. The UV attenuation was strongly affected by additionsof DOC, and attenuation coefficients at 320 nm increased from3.0 in the control to approximately 3.5 and 11.0 m^-1 in the1 and 10 mg DOC treatments respectively. Most Daphnia showedpronounced carapace melanization, and the absorbance of short-waveradiation through the carapace was strongly related to the degreeof melanization. Nevertheless, the different UV climate in theenclosures did not cause any short-term adaptation in Daphniapigmentation over a 3 week period. The levels of CAT and GSTwere assessed over time in the control and at 10 mg DOC. Theseenzymes displayed opposite patterns, with somewhat lower activitiesof CAT at low DOC (control) relative to 10 mg DOC, while theopposite was found for GST. There was also a significant negativecorrelation between CAT and solar irradiation for GST in bothbags, while no effects were found for GST.     

Tyrant flycatchers coming out in the open: phylogeny and ecological radiation of Tyrannidae (Aves, Passeriformes)

Tyrant flycatchers constitute a substantial component of the land bird fauna in all South American habitats. Past interpretations of the morphological and ecological evolution in the group have been hampered by the lack of a well‐resolved hypothesis of their phylogenetic interrelationships. Here, we present a well‐resolved phylogeny based on DNA sequences from three nuclear introns for 128 taxa. Our results confirm much of the overall picture of Tyrannidae relationships, and also identify several novel relationships. The genera Onychorhynchus, Myiobius and Terenotriccus are placed outside Tyrannidae and may be more closely related to Tityridae. Tyrannidae consists of two main lineages. An expanded pipromorphine clade includes flatbills, tody‐tyrants and antpipits, and also Phylloscartes and Pogonotriccus. The spadebills, Neopipo and Tachuris are their closest relatives. The remainder of the tyrant flycatchers forms a well‐supported clade, subdivided in two large subclades, which differ consistently in foraging behaviour, the perch‐gleaning elaeniines and the sallying myiarchines, tyrannines and fluvicolines. A third clade is formed by the genera Myiotriccus, Pyrrhomyias, Hirundinea and three species currently placed in Myiophobus. Ancestral habitat reconstruction and divergence date estimation suggest that early divergence events in Tyrannida took place in a humid forest environment during the Oligocene. Large‐scale diversification in open habitats is confined to the clade consisting of the elaeniines, myiarchines, tyrannines and fluvicolines. This radiation correlates in time to the expansion of semi‐open and open habitats from the mid‐Miocene (c. 15 Mya) onwards. The pipromorphine, elaeniine and myiarchine–tyrannine–fluvicoline clades each employ distinct foraging strategies (upward striking, perch‐gleaning and sallying, respectively), but the degree of diversity in morphology and microhabitat exploitation is markedly different between these clades. While the pipromorphines and elaeniines each are remarkably homogenous in morphology and exploit a restricted range of microhabitats, the myiarchine–tyrannine–fluvicoline clade is more diverse in these respects. This greater ecological diversity, especially as manifested in their success in colonizing a wider spectrum of open habitats, appears to be connected to a greater adaptive flexibility of the search‐and‐sally foraging behaviour.     

A Systematic Approach towards Optimizing a Cohabitation Challenge Model for Infectious Pancreatic Necrosis Virus in Atlantic Salmon (Salmo salar L.)

A cohabitation challenge model was developed for use in evaluating the efficacy of vaccines developed against infectious pancreatic necrosis virus (IPNV) in Atlantic salmon (Salmo salar L) using a stepwise approach. The study involved identifying a set of input variables that were optimized before inclusion in the model. Input variables identified included the highly virulent Norwegian Sp strain NVI015-TA encoding the T₂₁₇A₂₂₁ motif having the ability to cause >90% mortality and a hazard risk ratio of 490.18 (p<0.000) for use as challenge virus. The challenge dose was estimated at 1x10⁷ TCID₅₀/mL per fish while the proportion of virus shedders was estimated at 12.5% of the total number of fish per tank. The model was designed based on a three parallel tank system in which the Cox hazard proportional regression model was used to estimate the minimum number of fish required to show significant differences between the vaccinated and control fish in each tank. All input variables were optimized to generate mortality >75% in the unvaccinated fish in order to attain a high discriminatory capacity (DC) between the vaccinated and control fish as a measure of vaccine efficacy. The model shows the importance of using highly susceptible fish to IPNV in the optimization of challenge models by showing that highly susceptible fish had a better DC of differentiating vaccine protected fish from the unvaccinated control fish than the less susceptible fish. Once all input variables were optimized, the model was tested for its reproducibility by generating similar results from three independent cohabitation challenge trials using the same input variables. Overall, data presented here show that the cohabitation challenge model developed in this study is reproducible and that it can reliably be used to evaluate the efficacy of vaccines developed against IPNV in Atlantic salmon. We envision that the approach used here will open new avenues for developing optimal challenge models for use in evaluating the efficacy of different vaccines used in aquaculture.     

Selection for High Oridonin Yield in the Chinese Medicinal Plant Isodon (Lamiaceae) Using a Combined Phylogenetics and Population Genetics Approach

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.     

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Ethylene production by loblolly pine seedlings associated with water stress

Effects of water stress on production of ethylene and its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), by loblolly pine ( Pinus taeda L.) seedlings from a Texas drought-hardy and a Virginia Coastal Plain source were investigated. Ethylene production rates in needles from the Virgnia source increased slightly with initial stress (-1.3 MPa), declined until water potential reached -1.6 MPa and then increased sharply at -2.5 MPa. The ethylene production rates in needles from the Texas also increased slightly with initial stress, then decreased with decreasing water potential. Ethylene production by root tissue was two to three times higher than needle tissue and decreased with decreasing water potential. ACC concentrations in needles of both seed sources decreased as water potential began decreasing. Below -1.4 MPa, ACC levels started increasing (Texas source) or remained constant until -2.8 MPa (Virginia source) at which time its level increased three-fold. Mean ACC levels in root tissue [122 nmol (g dry weight)⁻¹] were slightly higher than the mean levels in the needle tissue [92 nmol (g dry weight) ⁻¹]; roots apparently were more efficient in converting it to ethylene since ethylene production was two to three times higher than needle tissue. The modulation of ethylene synthesis by ACC synthase and ethyleneforming enzyme appeared to be influenced by stress level, organ and seed source.