Functional investigation of a QTL affecting resistance to Haemonchus contortus in sheep

This study reports a functional characterization of a limited segment (QTL) of sheep chromosome 12 associated with resistance to the abomasal nematode Haemonchus contortus. The first objective was to validate the identified QTL through the comparison of genetically susceptible (N) and resistant (R) sheep produced from Martinik × Romane back-cross sheep. The R and N genotype groups were then experimentally infected with 10 000 H. contortus larvae and measured for FEC (every three days from 18 to 30 days post-challenge), haematocrit, worm burden and fertility. Significant differences in FEC and haematocrit drop were found between R and N sheep. In addition, the female worms recovered from R sheep were less fecund. The second step of the characterization was to investigate functional mechanisms associated with the QTL, thanks to a gene expression analysis performed on the abomasal mucosa and the abomasal lymph node. The gene expression level of a candidate gene lying within the QTL region (PAPP-A2) was measured. In addition, putative interactions between the chromosome segment under study and the top ten differentially expressed genes between resistant MBB and susceptible RMN sheep highlighted in a previous microarray experiment were investigated. We found an induction of Th-2 related cytokine genes expression in the abomasal mucosa of R sheep. Down-regulation of the PAPP-A2 gene expression was observed between naïve and challenged sheep although no differential expression was recorded between challenged R and N sheep. The genotyping of this limited region should contribute to the ability to predict the intrinsic resistance level of sheep.     

Link between Domoic Acid Production and Cell Physiology after Exchange of Bacterial Communities between Toxic Pseudo-nitzschia multiseries and Non-Toxic Pseudo-nitzschia delicatissima

Bacteria are known to influence domoic acid (DA) production by Pseudo-nitzschia spp., but the link between DA production and physiology of diatoms requires more investigation. We compared a toxic P. multiseries to a non-toxic P. delicatissima, investigating links between DA production, physiological parameters, and co-occurring bacteria. Bacterial communities in cultures of both species were reduced by antibiotic treatment, and each of the diatoms was inoculated with the bacterial community of the other species. The physiology of P. delicatissima was minimally affected by the absence of bacteria or the presence of alien bacteria, and no DA was detected. P. multiseries grew faster without bacteria, did not produce a significant amount of DA, and exhibited physiological characteristics of healthy cells. When grown with alien bacteria, P. multiseries did not grow and produced more DA; the physiology of these cells was affected, with decreases in chlorophyll content and photosynthetic efficiency, an increase in esterase activity, and almost 50% mortality of the cells. The alien bacterial community had morphological and cellular characteristics very different from the original bacteria, and the number of free-living bacteria per algal cell was much higher, suggesting the involvement of bacteria in DA production.     

Effect of cis/trans isomerism of beta-carotene on the ratios of volatile compounds produced during oxidative degradation

beta-Carotene is, when cleaved, an important source of flavor and aroma compounds in fruits and flowers. Among these aroma compounds, the main degradation products are beta-ionone, 5,6-epoxy-beta-ionone, and dihydroactinidiolide (DHA), which are associated by flavorists and perfumers with fruity, floral, and woody notes. These three species can be produced by degradation of beta-carotene through an attack by enzyme-generated free radicals and a cleavage at the C9-C10 bond. This study investigated the influence of cis/trans isomerism at the C9-C10 bond on the production of beta-carotene degradation compounds, first with a predictive approach and then experimentally with different isomer mixtures. beta-Carotene solutions containing high ratios of 9-cis-isomers produced more DHA, suggesting a different pathway than for the transformation of all-trans-beta-carotene to ionone and DHA. These results are important in the search for financially viable processes to produce natural carotene-derived aroma compounds.     

Crystal structure of the rabies virus nucleoprotein-RNA complex

Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.     

Distribution of Hatschekia pagellibogneravei (Copepoda: Hatschekiidae) on the gills of Pagellus bogaraveo (Teleostei: Sparidae) from Madeira, Portugal

A population of the gill parasite Hatschekia pagellibogneravei (Hesse, 1878) was studied on one of its sparid fish hosts, the blackspot seabream, Pagellus bogaraveo (Brünnich), off the coast of Madeira Island, Portugal, northeast Atlantic. Very high infection levels of this copepod were detected, with no significant seasonal differences. Abundance was negatively correlated with fish size. There were significant differences in the distribution of this copepod among the gill arches of the host, which seem to be best explained by differences in water flow within the gill habitat.     

Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.     

Synthesis of haptens and conjugates for ELISA of glycitein: development and validation of an immunological test

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta3-glycitein and Delta4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

Inheritance of the stay-green trait in tropical maize

Stay-green maize genotypes have been associated with tolerance to biotic and abiotic stresses, including tolerance to drought, and to stalk and root lodging, but there is limited information on its inheritance. Thus, this research was conducted to study the inheritance of the stay-green trait using both conventional analysis and QTL mapping of the Design III in a tropical maize population developed from two inbred lines genetically divergent for this trait. Two-hundred and fifty F₂ plants were genotyped with 177 microsatellite markers, and their backcrossed progenies to both parental inbreds were evaluated at three locations. Ten plants per plot were assessed 120 days after sowing and the plot means scores for stay-green, adjusted for days to silking emergence, were used for analysis. The additive variance was larger than the dominance variance, the genetic by location interaction variance presented a high magnitude, and the heritability coefficient on a plant-basis a low magnitude. Seventeen QTL were mapped, most of them were clustered on four chromosomes and accounted for by 73.08 % of the genetic variance. About half of the QTL interacted with location, and the average level of dominance was partial dominance. The additive effects were larger than the dominance effects; the latter were not unidirectional, so that heterosis could not be exploited in crosses. Procedures for marker-assisted selection to increase the level of stay-green are discussed and an approach is suggested for using both stable and non-stable QTL in a marker-assisted backcross program.