Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing

Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources.     

Response to acetylcholine and myosin content of isolated canine airways

Contractility of tracheal smooth muscle strips and spiral strips of fourth to fifth generation bronchi was studied in organ baths. The relationship among contractility, airway smooth muscle myosin, and smooth muscle thickness was also examined. The trachea was divided into three segments, each consisting of 12-14 rings. Smooth muscle strips from each of the three regions (top, middle, and bottom of the trachea) and from fourth to fifth generation bronchi were studied. Acetylcholine (ACh) sensitivity (-log EC50) was 8.1, 7.1, 7.9, and 6.1 for the top, middle, and bottom of the trachea and the bronchi, respectively. At P = 0.01, the EC50 ACh value of the top of the trachea differed from the EC50 value of the bronchi. Maximal tension (Tmax) generated in bronchi (3.2 g) was lower (P less than 0.01) than in the top (10.4 g), middle (7.1 g), and bottom of the trachea (5.1 g). Differences between trachea and bronchi disappeared when Tmax was corrected for smooth muscle myosin content. Thickness of smooth muscle in bronchi was less (P less than 0.01) than in the three regions of trachea. Tmax was significantly correlated with airway smooth muscle thickness (r = 0.56; P less than 0.05). These results suggest that in mongrel dogs sensitivity to ACh shows a gradient from the top of the trachea to the bronchi and that Tmax is greater in the trachea than in the bronchi and is significantly correlated with thickness of smooth muscle.     

Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.     

Relationships between Presence of the Eye Cup and Maintenance of Lens-Forming Capacity in Larval Xenopus laevis

The lentectomized eye of larval Xenopus laevis can regenerate a lens by a process of lens-transdifferentiation of the cornea and pericorneal epidermis. These tissues can form the lens only when they become in direct communication with the environment of the vitreous chamber (neural retina) indicating that the eye cup plays a fundamental role in this process.
In this work the role of the eye cup in the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis was studied by allowing these tissues to cover the enucleated orbit for different periods, and then implanting them into the vitreous chamber of the contralateral eye. Under these experimental conditions the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis showed no significant correlation with the time from enucleation to implantation.     

Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells

To assess whether and how specifically contact influences the functioning of differentiated cells, we have studied the secretion of adult pancreatic B-cells as a function of aggregation to either homologous B-cells or other heterologous endocrine islet cell types, all present in a mixed cell suspension. Using an immunological plaque assay for insulin, we have quantitated the proportion of single and aggregated B-cells inducing the formation of a hemolytic plaque (a reflection of the size of the secreting cell population) and the area of these plaques (a reflection of the hormonal output of individual cells or aggregates) after a 30-min stimulation by 16.7 mM glucose. By taking into account the number of B-cells within the aggregates, we have calculated from these data the insulin output on a per B-cell basis. We show here that the homologous contact between companion B-cells promotes the recruitment of secreting B-cells and increases their individual secretion of insulin twofold over that of single B-cells. By contrast, heterologous B- to non-B-cell contact was not effective in enhancing the recruitment of secreting B-cells and in promoting their individual secretion. These findings show that a highly differentiated cell function, such as insulin secretion, is controlled specifically by homologous cell to cell contacts.     

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

Chrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers-exposed plants were analyzed by dot-blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.     

Chromosome break-induced DNA replication leads to nonreciprocal translocations and telomere capture.

In yeast, broken chromosomes can be repaired by recombination, resulting in nonreciprocal translocations. In haploid cells suffering an HO endonuclease-induced, double-strand break (DSB), nearly 2% of the broken chromosome ends recombined with a sequence near the opposite chromosome end, which shares only 72 bp of homology with the cut sequence. This produced a repaired chromosome with the same 20-kb sequence at each end. Diploid strains were constructed in which the broken chromosome shared homology with the unbroken chromosome only on the centromere-proximal side of the DSB. More than half of these cells repaired the DSB by copying sequences distal to the break from the unbroken template chromosome. All these events were RAD52 dependent. Pedigree analysis established that DSBs occurring in G1 were repaired by a replicative mechanism, producing two identical daughter cells. We discuss the implications of these data in understanding telomerase-independent replication of telomeres, gene amplification, and the evolution of chromosomal ends.