Andrew F. Cockburn

Background: To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia. Methods: Subgingival plaque samples were obtained from ten individuals at least 70 years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples. Results: More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size. Conclusions: The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.

Reports of mosquito baculoviruses are extremely uncommon and epizootics in field populations are rarely observed. We describe a baculovirus that was responsible for repeated and extended epizootics in field populations of Culex nigripalpus and C. quinquefasciatus over a 2 year period. These mosquito species are important vectors of St Louis and Eastern equine encephalitis in the United States. Our initial attempts to transmit this baculovirus to mosquitoes in the laboratory were unsuccessful. A salt mixture similar to that found in water supporting infection in the field was used in laboratory bioassays and indicated that certain salts were crucial to transmission of the virus. Further investigations revealed conclusively that transmission is mediated by divalent cations: magnesium is essential, whereas calcium inhibits virus transmission. These findings represent a major advancement in our understanding of the transmission of baculoviruses in mosquitoes and will allow characterization of the virus in the laboratory. In addition, they can explain, in great part, conditions that support epizootics in natural populations of mosquitoes that vector life-threatening diseases of man and animals.

A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.

We analyzed variation in mitochondrial DNA (mtDNA) of two neotropical mosquitoes, Anopheles rangell (n = 181) and A. trinkae (n = 45), with very different distribution patterns in Latin America, to assess species boundaries for these putative sister taxa and to examine population genetic structure. Phylogenetic analyses revealed (1) support for the monophyletic origin of each species; (2) diagnostic restriction site differences between the species; (3) geographic partitioning of haplotypes by country in A. rangell from Bolivia, Ecuador, and Venezuela compared with considerable overlap in haplotypes of A. trinkae from Bolivia and Ecuador; and (4) similar levels of mean haplotype and nucleotide diversity in both species, but lower levels of mean nucleotide divergence in A. trinkae compared with A. rangell. We hypothesize that higher maternal gene flow and lower divergence in A. trinkae are most likely due either to a distinctive matrilineal history or to a smaller effective population size, which may have been influenced by a smaller, essentially linear geographic range along the eastern flank of the Andes. In the cladistic analysis of A. rangell, the Bolivian haplotypes appear to be more derived than those from Ecuador or Venezuela, yet there is no evidence to support the hypothesis of a recent range expansion from Ecuador into Bolivia.

Two Hsp82 genes were isolated from the malaria vector Anopheles albimanus in a single lambda phage clone. The two genes are in a head-to-head arrangement separated by approx. 0.9 kbp. Northern hybridizations and 5' RACE demonstrate that both genes are transcribed, have moderate levels of constitutive transcription, and are also heat-inducible with maximum transcript accumulation occurring after 40°C heat shocks. Both genes have typical heat-shock promoters and conserved intron boundaries in the untranslated leaders. The open reading frames are 99.6% identical differing in only nine silent nucleotide positions in the 2166 bp ORFs. However, precisely outside the ORFs, the flanking DNA of the two genes shows no evidence of common derivation. The high degree of identity between the two ORFs appears to be a result of gene conversion occurring by a process similar to that previously suspected in the A. albimanus Hsp70 genes and several D. melanogaster genes arranged as palindromes. This process probably involves a stem-loop intermediate and is restricted in extent by flanking sequence divergence. These Hsp82 genes clearly demonstrate the extreme precision with which gene conversion can lead to proteincoding-region homogeneity yet allow flanking DNA divergence.

Sequence variation of the ribosomal DNA internal transcribed spacer 2 (ITS2) was examined for populations of the malaria vector Anopheles nuneztovari collected in Colombia, Venezuela, Bolivia, Suriname, and Brazil. Mosquitoes from Colombia and Venezuela had identical ITS2 sequences and were distinguished from sequences in other populations by three insertion/deletion events (indels) and by one transversion. The length of the ITS2 was 363-369 bp, and it had a G+C content of 55.3%-55.7%. Variation in the length of the ITS2 between and within populations was due to indels in simple repeats. ITS2 consensus sequences were similar or identical for samples from the following three groups: (1) Colombia, Bolivia, and Venezuela; (2) Suriname and northern Brazil; and (3) eastern and central Brazil. The presence of two different consensus sequences from a single location near Manaus, Brazil, suggests that populations from eastern Brazil and those from Suriname converge in this region of the Amazon Basin. These data show that putative cryptic species of An. nuneztovari are distinguished by very minor differences in DNA sequence of the ITS2 region.

The complete sequence (15 455 bp) of the mitochondrial DNA of the mosquito Anopheles quadrimaculatus species A is reported. This genome is compact and very A + T rich (77.4% A + T). It contains genes for 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement is the same as in Drosophila yakuba, except that the positions of two contiguous tRNAs are reversed and a third tRNA is transcribed from the complementary strand. Protein-coding genes, rRNAs, and most tRNAs were similar to D. yakuba. Two tRNAs had nonstandard secondary structures comparable with those of nematode mitochondrial tRNAs. The very small putative control region (625 bp) contains no sequence motifs similar to those used in vertebrates and other insects for initiation of transcription and replication.Key words: mitochondrial DNA, Anopheles quadrimaculatus.

Four Hsp70 genes of the malaria vector Anopheles albimanus were isolated from a genomic DNA library as two non‐overlapping clones each containing a pair of divergently transcribed genes having 75% DNA sequence similarity to the protein‐coding regions of the Drosophila metanogaster Hsp70genes. The clones were assigned to two loci on chromosome 2R by in situ hybridization. These clones hybridize strongly to heat‐shock but only weakly to non‐shocked mosquito RNA. The Hsp70 gene family of A. albimanus is undergoing concerted evolution probably by gene conversion. The general arrangement of the genes suggests that divergently transcribed pairs of genes at two loci is an ancient Dipteran arrangement predating the Nematocera/Cyclorrapha divergence.