Survival and immune response of drones of a Nosemosis tolerant honey bee strain towards N. ceranae infections

Honey bee colonies (Apis mellifera) have been selected for low level of Nosema in Denmark over decades and Nosema is now rarely found in bee colonies from these breeding lines. We compared the immune response of a selected and an unselected honey bee lineage, taking advantage of the haploid males to study its potential impact on the tolerance toward Nosema ceranae, a novel introduced microsporidian pathogen. After artificial infections of the N. ceranae spores, the lineage selected for Nosema tolerance showed a higher N. ceranae spore load, a lower mortality and an up-regulated immune response. The differences in the response of the innate immune system between the selected and unselected lineage were strongest at day six post infection. In particular genes of the Toll pathway were up-regulated in the selected strain, probably is the main immune pathway involved in N. ceranae infection response. After decades of selective breeding for Nosema tolerance in the Danish strain, it appears these bees are tolerant to N. ceranae infections.     

A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.     

The alkenyl migration mechanism catalyzed by extradiol dioxygenases: a hybrid DFT study

6-Hydroxymethyl-6-methylcyclohexa-2,4-dienone is a mechanistic probe which when incubated with an extradiol dioxygenase yields a 2-tropolone product. This observation was originally interpreted as evidence supporting a direct heterolytic 1,2-alkenyl migration mechanism for a ring expansion reaction catalyzed by this class of Fe(II)-dependent nonheme enzymes (Xin and Bugg in J Am Chem Soc 130:10422-10430, 2008). In the work reported in this contribution we used quantum chemical methods to test whether such a mechanism is energetically possible and we found that it is not, neither for the mechanistic probe nor for the native catalytic cycle intermediate. Models of increasing complexity were used to calculate energy barriers to the heterolytic 1,2-alkenyl migration and alternative radical mechanisms. It was found that the former involves substantially higher barriers than the latter. A tentative radical mechanism that accounts for the transformation of the probe substrate to 2-tropolone was also proposed, and it involves acceptable barriers.     

N–O bond cleavage mechanism(s) in nitrous oxide reductase

Quantum chemical calculations of active-site models of nitrous oxide reductase (N(2)OR) have been undertaken to elucidate the mechanism of N-O bond cleavage mediated by the supported tetranuclear Cu(4)S core (Cu(Z)) found in the enzymatic active site. Using either a minimal model previously employed by Gorelsky et al. (J. Am. Chem. Soc. 128:278-290, 2006) or a more extended model including key residue side chains in the active-site second shell, we found two distinct mechanisms. In the first model, N(2)O binds to the fully reduced Cu(Z) in a bent μ-(1,3)-O,N bridging fashion between the Cu(I) and Cu(IV) centers and subsequently extrudes N(2) while generating the corresponding bridged μ-oxo species. In the second model, substrate N(2)O binds loosely to one of the coppers of Cu(Z) in a terminal fashion, i.e., using only the oxygen atom; loss of N(2) generates the same μ-oxo copper core. The free energies of activation predicted for these two alternative pathways are sufficiently close to one another that theory does not provide decisive support for one over the other, posing an interesting problem with respect to experiments that might be designed to distinguish between the two. Effects of nearby residues and active-site water molecules are also explored.     

Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles

A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms.     

Changes in microbial activities and biomasses over a forest floor gradient in C-to-N ratio

Background and aims

Under chronically elevated N deposition, N retention mainly occur at high soil C-to-N ratio. This may be mediated through soil microbes, such as ectomycorrhizal (EM) fungi, saprotrophic fungi and bacteria, and the aim of this study was to evaluate the relationship between soil microbes and forest floor C-to-N ratios.

Methods

Soil samples from 33 Norway spruce (Picea abies (L.) H. Karst) forests in Denmark and southern Sweden in a forest floor C-to-N ratio gradient (ranging from 14 to 35) were analysed regarding the content of phospholipid fatty acids (PLFAs) to estimate their soil microbial community composition and the relative biomasses of different microbial groups. The relation of EM biomass to total fungal biomass was estimated as the loss of the fungal PLFA 18:2ω6,9 during incubation of soils and the production of EM mycelia was estimated using fungal in-growth mesh bags. The soil microbial variables were correlated to forest floor C-to-N ratio, NO ₃ ^- leaching, soil pH and stand age.

Results

Fungal proportions of microbial biomass, EM to total fungi and EM mycelial production were all positively related to C-to-N ratio, while NO ₃ ^- leaching was negatively related to C-to-N ratio.

Conclusions

Both EM and saprotrophic fungi change with forest floor C-to-N ratios and appear to play a central role in N retention in forest soil. A better understanding of the mechanisms behind this process may be revealed if the role of recalcitrant fungal metabolites for N retention (and soil C sequestration) can be identified. Research along this line deserves further studies.     

Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.     

Myxobolus groenlandicus n. sp. (Myxozoa) distorting skeletal structures and musculature of Greenland halibut Reinhardtius hippoglossoides (Teleostei: Pleuronectidae)

A specimen of Greenland halibut Reinhardtius hippoglossoides (Walbaum, 1792) caught on the west coast of Greenland (Qasigiannguit) was found to possess serious pathological changes in the body musculature. A series of cartilaginous cylindrical structures organized symmetrically at the position of the proximal pterygiophores had changed the musculature and produced irreversible distortions (cavities and holes) in the fillet of the processed fish, leaving it with no value for the industry. Histopathological investigation showed that these structures consisted of hypertrophic cartilage containing numerous myxospore-producing plasmodia. Morphometric and molecular analyses of the parasites showed that both spore morphology and rDNA sequences complied with characteristics of the genus Myxobolus, but no full affiliation with a known species could be found. The parasite is a previously undescribed species, and the name Myxobolus groenlandicus n. sp. is assigned to this new myxobolid.     

Mould and mycotoxin contamination of pig feed in northwest Croatia

The aim of this study was to investigate the contamination of pig feed with moulds and the occurrence of mycotoxins. A total of 30 feed samples were collected at different animal feed factories in the north-western part of Croatia. Mycological analysis showed that the total number of moulds ranged from 1?×?10³ to 1?×?10⁵?cfu/g with samples contaminated with Aspergillus spp. (63?%), Penicillium spp. (80?%), and Fusarium spp. (77?%). A determination of aflatoxin B1 (AFB₁), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T-2 toxin (T-2) and fumonisin (FUM) concentration was done using the validated ELISA method. The mean concentrations of AFB₁ (0.5?±?0.6???g/kg), OTA (1.53?±?0.42???g/kg) and FUM (405?±?298???g/kg) were below the maximum levels or recommended values in the EU in all the investigated samples. The observed results indicated an increased contamination of pig feed with Fusarium mycotoxins DON and ZEA with mean concentrations of 817?±?447 and 184?±?214???g/kg, higher than recommended in 40 and 17?% of the analysed samples, respectively.     

Identification of subpopulations from connectivity matrices

Dispersal on the landscape/seascape scale may lead to complex spatial population structure with non‐synchronous demography and genetic divergence. In this study we present a novel approach to identify subpopulations and dispersal barriers based on estimates of dispersal probabilities on the landscape scale. A theoretical framework is presented where the landscape connectivity matrix is analyzed for clusters as a signature of partially isolated subpopulations. Identification of subpopulations is formulated as a minimization problem with a tuneable penalty term that makes it possible to generate population subdivisions with varying degree of dispersal restrictions. We show that this approach produces superior results compared to alternative standard methods. We apply this theory to a dataset of modeled dispersal probabilities for a sessile marine invertebrate with free‐swimming larvae in the Baltic Sea. For a range of critical connectivities we produce a hierarchical partitioning into subpopulations spanning dispersal probabilities that are typical for both genetic divergence and demographic independence. The mapping of subpopulations suggests that the Baltic Sea includes a fine‐scale (100–600 km) mosaic of invisible dispersal barriers. An analysis of the present network of marine protected areas reveal that protection is very unevenly distributed among the suggested subpopulations. Our approach can be used to assess the location and strength of dispersal barriers in the landscape, and identify conservation units when extensive genotyping is prohibitively costly to cover necessary spatial and temporal scales, e.g. in spatial management of marine populations.