Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.     

Seed dormancy and germination of Ficus lundellii and tropical forest restoration

We investigated seed dormancy and germination in Ficus lundellii Standl. (Moraceae), a native species of Mexico's Los Tuxtlas tropical rain forest. In an 8-h photoperiod at an alternating diurnal (16/8 h) temperature of 20/30 degrees C, germination was essentially complete (96%) within 28 days, whereas in darkness, all seeds remained dormant. Neither potassium nitrate (0.05-0.2%) applied continuously nor gibberellic acid applied either continuously (10-200 ppm) or as a 24 hour pretreatment (2000 ppm) induced germination in the dark. Germination in the light was not reduced by a 24-h hydrochloric acid (0.1-1%) pretreatment, but it was reduced both by a 24-h pretreatment with either H(2)O(2) (0.1-5 M) or 5% HCl, or by more than 5 days of storage at 40 degrees C (4.5% seed water content). In a study with a 2-dimensional temperature gradient plate, seeds germinated fully and rapidly in the light at a constant temperature of 30 degrees C, and fully but less rapidly in the light at alternating temperatures with low amplitudes (< 12 degrees C) about the optimal constant temperature. The base, optimal and ceiling temperatures for rate of germination were estimated as 13.8, 30.1 and 41.1 degrees C, respectively. In all temperature regimes, light was essential for the germination of F. lundellii seeds.     

Cell death localization in situ in laboratory reared honey bee (Apis mellifera L.) larvae treated with pesticides

In this study, cell death detected by DNA fragmentation labeling and phosphatidylserine (PS) localization was investigated in the honey bee (Apis mellifera L.) midgut, salivary glands and ovaries after treating larvae with different pesticides offered via an artificial diet. To do this, honey bee larvae reared in an incubator were exposed to one of nine pesticides: chlorpyrifos, imidacloprid, amitraz, fluvalinate, coumaphos, myclobutanil, chlorothalonil, glyphosate and simazine. Following this, larvae were fixed and prepared for immunohistologically detected cellular death using two TUNEL techniques for DNA fragmentation labeling and Annexin V to detect the localization of exposed PS specific in situ binding to apoptotic cells. Untreated larvae experienced ∼10% midgut apoptotic cell death under controlled conditions. All applied pesticides triggered an increase in apoptosis in treated compared to untreated larvae. The level of cell death in the midgut of simazine-treated larvae was highest at 77% mortality and statistically similar to the level of cell death for chlorpyrifos (65%), imidacloprid (61%), myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any pesticide-treated larvae. Indications of elevated apoptotic cell death in salivary glands and ovaries after pesticide application were detected. Annexin V localization, indicative of apoptotic cell deletion, had an extensive distribution in the midgut, salivary glands and ovaries of pesticide-treated larvae. The data suggest that the tested pesticides induced apoptosis in tissues of honey bee larvae at the tested concentrations. Cell death localization as a tool for a monitoring the subclinical and sub-lethal effects of external influences on honey bee larval tissues is discussed.     

Macroalgal meadow habitats support fish and fisheries in diverse tropical seascapes

Canopy‐forming macroalgae can construct extensive meadow habitats in tropical seascapes occupied by fishes that span a diversity of taxa, life‐history stages and ecological roles. Our synthesis assessed whether these tropical macroalgal habitats have unique fish assemblages, provide fish nurseries and support local fisheries. We also applied a meta‐analysis of independent surveys across 23 tropical reef locations in 11 countries to examine how macroalgal canopy condition is related to the abundance of macroalgal‐associated fishes. Over 627 fish species were documented in tropical macroalgal meadows, with 218 of these taxa exhibiting higher local abundance within this habitat (cf. nearby coral reef) during at least one life‐history stage. Major overlap (40%–43%) in local fish species richness among macroalgal and seagrass or coral reef habitats suggest macroalgal meadows may provide an important habitat refuge. Moreover, the prominence of juvenile fishes suggests macroalgal meadows facilitate the triphasic life cycle of many fishes occupying diverse tropical seascapes. Correlations between macroalgal canopy structure and juvenile abundance suggests macroalgal habitat condition can influence levels of replenishment in tropical fish populations, including the majority of macroalgal‐associated fishes that are targeted by commercial, subsistence or recreational fisheries. While many macroalgal‐associated fishery species are of minor commercial value, their local importance for food and livelihood security can be substantial (e.g. up to 60% of landings in Kenyan reef fisheries). Given that macroalgal canopy condition can vary substantially with sea temperature, there is a high likelihood that climate change will impact macroalgal‐associated fish and fisheries.     

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.     

Comparative Genomics for Gene Isolation in Legumes

The similarity in gene order between closely related taxa suggests that genomic information from model systems should facilitate gene isolation and characterization in target crops. If this is the case, a great deal of effort and investment can be saved by focusing attention on a few model systems that have appropriate applicability. Pea （Pisura sativum） provides a good test case： its genome is large and the insertion sites for repetitive elements, which comprise the bulk of its genome, are highly polymorphic （Jing et al., 2005; Vershinin et al., 2003）, so we expect a great deal of structural polymorphism between the genomes of Pisum lines. Yet the genetic map of Pisum is essentially coUinear with Medicago （Choi et al., 2004; Kalo et al., 2004）.     

A stability analysis of time to flowering as a screen for responsiveness to temperature and photoperiod in cowpea (Vigna unguiculata)

Summary Twenty-one genotypes of cowpea (Vigna unguiculata), comprising landraces and varieties, were grown in 22 photothermal environments in Nigeria and Niger, West Africa, and a stability analysis of days from sowing to flowering (f) was carried out. Cowpeas are rarely insensitive to photoperiod; they are typically quantitative shortday plants wherein f is delayed when photoperiod (P) is longer than the critical photoperiod (P _c ). Therefore, in order to quantify genotypic variation in temperature sensitivity, genotype f was regressed against the mean trial f in circumstances where P c (i.e. approximately 13 hd^-1) and mean temperature (T) was between 19° and 28° C. Correspondingly, in order to assess genotypic variation in photoperiod sensitivity, trials where T was near optimal (25°–28° C) but where P ranged from 10–14.5 hd^-1 were used. These stability analyses detected no significant differences (P>0.05) between genotypes 9n temperature sensitivity but revealed significant differences (P<0.001) in photoperiod sensitivity. Regression coefficients from the stability analysis were strongly correlated (r=0.94, 19df) with a photoperiod sensitivity constant, c, determined from a photothermal flowering model. A stability analysis of f from field trials can therefore identify and quantify genotypic variation in response to temperature and photoperiod in cowpea.Abbreviations f days from sowing to flowering - P mean photoperiod - P _c critical photoperiod - P _ce ceiling photoperiod - T mean temperature - T _b base temperature - T _o optimum temperature - SDP short-day plant