Characterisation of the complete mitochondrial genome and 13 microsatellite loci through next-generation sequencing for the New Caledonian spider-ant Leptomyrmex pallens

The complete mitochondrial genome and a set of polymorphic microsatellite markers were identified by 454 pyrosequencing (1/16th of a plate) for the New Caledonian rainforest spider-ant Leptomyrmex pallens. De novo genome assembly recovered the entire mitochondrial genome with mean coverage of 8.9-fold (range 1-27). The mitogenome consists of 15,591 base pairs including 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The genome arrangement is typical of insect taxa and very similar to the only other published ant mitogenome from the Solenopsis genus, with the main differences consisting of translocations and inversions of tRNAs. A total of 13 polymorphic loci were also characterized using 41 individuals from a single population in the Aoupinié region, corresponding to workers from 21 nests and 16 foraging workers. We observed moderate genetic variation across most loci (mean number of alleles per locus = 4.50; mean expected heterozygosity = 0.53) with evidence of only two loci deviating significantly from Hardy-Weinberg equilibrium due to null alleles. Marker independence was confirmed with tests for linkage disequilibrium. Most loci cross amplified for three additional Leptomyrmex species. The annotation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the colony structure, population genetic patterns, and dispersal strategy of L. pallens in the context of rainforest fragmentation in New Caledonia. Furthermore, this paper confirms a recent line of evidence that comprehensive mitochondrial data can be obtained relatively easily from small next-generation sequencing analyses. Greater synthesis of next-generation sequencing data will play a significant role in expanding the taxonomic representation of mitochondrial genome sequences.

Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialiswere collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.