Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening

The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound α-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1_REC) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1_REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1_REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through α-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1_REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.     

A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its alpha-glucosidase receptor in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the approximately 60-kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino-acid sequence confirmed its identity as an alpha-glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19-nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full-length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N-terminal 45-kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense-mediated decay pathway of mRNA degradation. In-gel assays confirmed that a single alpha-glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N-terminus of the receptor.     

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.     

Spatiotemporal Variation of Bacterial Assemblages in a Shallow Subtropical Coastal Lagoon in Southern Brazil

A study on the bacterioplankton of Conceição Lagoon (27°34′ S–48°27′ W), Southern Brazil, was carried out in July 2005 (austral winter) and January 2006 (austral summer) to characterize the bacterial spatiotemporal distribution and to determine the heterotrophic and photoautotrophic bacterial dominance in hypoxic/oxic stratified waters. Bacterial abundance increased significantly (p?5 (winter) to 3.21?×?10⁶ cells mL^?1 (summer), heterotrophic coccus/rod-shaped (HCR) cells from 7.00?×?10⁴ to 3.60?×?10⁶ cells mL^?1, and heterotrophic filamentous (HF) bacteria from 2.90?×?10³ to 2.74?×?10⁵ cells mL^?1. Bacterial biovolumes also increased in summer with mean biovolumes of CCY ranging from 0.38 to 1.37 μm³, HCR cells from 0.31 to 1.12 μm³, and HF from 3.32 to 11.34 μm³. Principal component analysis showed that salinity, temperature, and light were the abiotic factors that better explained the temporal variability of bacterial assemblages. Bacterial heterotrophy dominated in the lagoon, excepted by the southern and part of central sector in January 2006, when autotrophic-dominated microbial community occurred. Spatially, bacterial assemblages were influenced by nutrient gradient, oxygen, and salinity with a positive relationship between biovolumes and nutrients and a negative relationship between abundance of coccus cyanobacteria and nutrients. area revealed a singular temporal pattern with hypoxic bottom waters in winter and oxygen-rich waters appearing in summer related with the availability of light and predominant microbes. Thus, oxygen consumption/production is likely to be regulated by the amount of light reaching the bottom, stimulating the production of oxygen by oxygenic phototrophs.     

Time‐shifted reproductive behaviours among fall armyworm (Noctuidae: Spodoptera frugiperda) host strains: evidence for differing modes of inheritance

The noctuid moth Spodoptera frugiperda consists of two strains associated with different larval host plants (most notably corn and rice). These strains exhibit differential temporal patterns of female calling and copulation during scotophase, with the corn strain more active earlier in the night. We investigated strain‐specific constraints in reproductive timing, mating interactions between the two strains, and the mode of inheritance of timing of female calling, male calling, copulation and oviposition. We observed an allochronic shift of all reproductive behaviours by approximately 3 h and a parallel shift of nonreproductive locomotor activity, suggesting involvement of the circadian clock. The corn strain was more variable in the timing of calling and copulation than the rice strain. Rice strain females were more restricted in the timing of copulation than rice strain males, while such differences between the sexes were not apparent in the corn strain. There were significant interactions between the strains affecting onset times of copulation and male calling. The four investigated reproductive traits differed in their modes of inheritance: timing of female and male calling exhibited strong maternal effects, timing of copulation was controlled by a combination of maternal effects and corn strain dominant autosomal factors, and timing of oviposition was inherited in a corn strain dominant fashion. We conclude that the allochronic separation of reproduction between fall armyworm strains is asymmetric, less pronounced than previously thought, and under complex genetic control.     

Physical and chemical factors influencing transport of microorganisms through porous media.

Resting-cell suspensions of bacteria isolated from groundwater were added as a pulse to the tops of columns of clean quartz sand. An artificial groundwater solution (AGW) was pumped through the columns, and bacterial breakthrough curves were established and compared to test the effects of ionic strength of the AGW, cell size (by using strains of similar cell surface hydrophobicity but different size), mineral grain size, and presence of heterogeneities within the porous media on transport of the bacteria. The proportion of cells recovered in the effluent ranged from nearly 90% for AGW of a higher ionic strength (I = 0.0089 versus 0.00089 m), small cells (0.75-micron-diameter spheres versus 0.75 by 1.8-micron rods), and coarse-grained sand (1.0 versus 0.33 mm) to less than 1% for AGW of lower ionic strength, large cells, and fine-grained sand. Differences in the widths of peaks (an indicator of dispersion) were significant only for the cell size treatment. For treatments containing heterogeneities (a vein of coarse sand in the center of a bed of fine sand), doubly peaked breakthrough curves were obtained. The first peak represents movement of bacteria through the transmissive coarse-grained vein. The second peak is thought to be dominated by cells which have moved (due to dispersion) from the fine-grained matrix to the coarse-grained vein near the top of the column and thus had been retarded, but not retained, by the column. Strength of effects tests indicated that grain size was the most important factor controlling transport of bacteria over the range of values tested for all of the factors examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteome analysis of resting human neutrophils

Neutrophils constitute the first line of host defense against pathogens. In the present study 2-D gel electrophoresis-mass spectrometry technology was employed to analyze the human resting neutrophils proteome. One hundred and two conserved spots were subjected to peptide mass fingerprinting, yielding 22 identifications. Among the identified proteins, nine are related to the inflammatory process, two polypeptides are assigned to metabolic functions and five are classified as structural.     

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.     

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.     

Ectoparasites are more vulnerable to host extinction than co-occurring endoparasites: evidence from metazoan parasites of freshwater and marine fishes

Hydrobiologia - The extinction of fish species can direct and indirectly affect many groups of associated species, among which parasite communities can be the most susceptible. However, the...