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

Multiplex assay for species identification and monitoring of insecticide resistance in Anopheles punctulatus group populations of Papua New Guinea

Anopheles punctulatus sibling species (An. punctulatus s.s., Anopheles koliensis, and Anopheles farauti species complex [eight cryptic species]) are principal vectors of malaria and filariasis in the Southwest Pacific. Given significant effort to reduce malaria and filariasis transmission through insecticide-treated net distribution in the region, effective strategies to monitor evolution of insecticide resistance among An. punctulatus sibling species is essential. Mutations in the voltage-gated sodium channel (VGSC) gene have been associated with knock-down resistance (kdr) to pyrethroids and DDT in malarious regions. By examining VGSC sequence polymorphism we developed a multiplex assay to differentiate wild-type versus kdr alleles and query intron-based polymorphisms that enable simultaneous species identification. A survey including mosquitoes from seven Papua New Guinea Provinces detected no kdr alleles in any An. punctulatus species. Absence of VGSC sequence introgression between species and evidence of geographic separation within species suggests that kdr must be monitored in each An. punctulatus species independently

Neighbor-joining tree showing the relationships among the human mtDNA haplotypes amplified from mosquitoes.

<p>Each symbol represents one DNA sequence amplified from one mosquito. Different shapes represent different <i>Anopheles</i> species (squares-<i>An</i>. <i>punctulatus s</i>.<i>s</i>., triangles-<i>An</i>. <i>farauti</i> 4) and is colored according to the collection site (green-Dimer, blue-Wasab, purple-Kokofine). Mixed blood meals are highlighted by boxes of the same color: for example, the two red boxes show two human mtDNA haplotypes amplified from a single <i>An</i>. <i>farauti</i> 4 mosquito collected in Kokofine.</p

Overview of the sequencing assay used to characterize blood meal composition of individual mosquitoes.

<p>(i) A first PCR amplification is performed on DNA extracted from each mosquito targeting ~140 bp of the mammalian mt 16S rRNA (gray) using primers modified with a 5’-end tail complementary to the Illumina sequencing primers (red). (ii) A second PCR amplification incorporates the Illumina adaptors and a 6-nucleotide barcode unique to each mosquito at the ends of the individual blood meal PCR products. (iii) After pooling amplification products from 96 samples together, the PCR products are simultaneously sequenced on an Illumina MiSeq to (iv) generate paired-end reads (in grey) and barcode sequences (grey box). (v) Paired-end reads are then merged to provide error-corrected consensus sequence reads. The dotted black line indicates that the 6-nucleotide barcode corresponding to each read is known but is sequenced independently.</p

Summary of the amplification range and discriminatory power predicted for the mammalian 16S rRNA primers.

<p>The table indicates, for each mammalian order, the number of species deposited in NCBI for the 16S rRNA genes, the number of species predicted to be amplified by the universal primers as well as the percentage of genera and species that would carry a unique sequence for this locus (enabling their rigorous identification).</p

Attitudes towards vaccines and intention to vaccinate against COVID-19: a cross-sectional analysis - implications for public health communications in Australia

Objective To examine SARS-CoV-2 vaccine confidence, attitudes and intentions in Australian adults as part of the iCARE Study. Design and setting Cross-sectional online survey conducted when free COVID-19 vaccinations first became available in Australia in February 2021. Participants Total of 1166 Australians from general population aged 18-90 years (mean 52, SD of 19). Main outcome measures Primary outcome: responses to question € If a vaccine for COVID-19 were available today, what is the likelihood that you would get vaccinated?'. Secondary outcome: analyses of putative drivers of uptake, including vaccine confidence, socioeconomic status and sources of trust, derived from multiple survey questions. Results Seventy-eight per cent reported being likely to receive a SARS-CoV-2 vaccine. Higher SARS-CoV-2 vaccine intentions were associated with: increasing age (OR: 2.01 (95% CI 1.77 to 2.77)), being male (1.37 (95% CI 1.08 to 1.72)), residing in least disadvantaged area quintile (2.27 (95% CI 1.53 to 3.37)) and a self-perceived high risk of getting COVID-19 (1.52 (95% CI 1.08 to 2.14)). However, 72% did not believe they were at a high risk of getting COVID-19. Findings regarding vaccines in general were similar except there were no sex differences. For both the SARS-CoV-2 vaccine and vaccines in general, there were no differences in intentions to vaccinate as a function of education level, perceived income level and rurality. Knowing that the vaccine is safe and effective and that getting vaccinated will protect others, trusting the company that made it and vaccination recommended by a doctor were reported to influence a large proportion of the study cohort to uptake the SARS-CoV-2 vaccine. Seventy-eight per cent reported the intent to continue engaging in virus-protecting behaviours (mask wearing, social distancing, etc) postvaccine. Conclusions Most Australians are likely to receive a SARS-CoV-2 vaccine. Key influencing factors identified (eg, knowing vaccine is safe and effective, and doctor's recommendation to get vaccinated) can inform public health messaging to enhance vaccination rates