Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50=13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step towards the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp telomeric repeat unit (TRU) and are confirmed by analysing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step towards resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.

Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up-to-47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here we report the development of a marker-assisted-mapping (MAM) strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using MAM, we mapped a transgene that was inserted in a > 100 Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. The identification of the causal gene of the sex-linked re mutation provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods. To facilitate genome-wide forward genetics in Ae. aegypti, we generated and compiled a number of lines with markers throughout the genome. Thus, by overcoming the challenges presented by the vast recombination deserts and the scarcity of markers, we have shown that effective forward genetic analysis is increasingly feasible in this important arboviral vector species.

Flavin-containing Trx reductase (TrxR) of Thermoplasma acidophilum (Ta), a thermoacidophilic facultative anaerobic archaeon, lacks the structural features for the binding of 2′-phosphate of nicotinamide adenine dinucleotide phosphate (NADPH), and this feature has justified the observed lack of activity with NADPH; NADH has also been reported to be ineffective. Our recent phylogenetic analysis identified Ta-TrxR as closely related to the NADH-dependent enzymes of Thermotoga maritima and Desulfovibrio vulgaris, both being anaerobic bacteria. This observation instigated a reexamination of the activity of the enzyme, which showed that Ta-TrxR is NADH dependent; the apparent Km for NADH was 3.1 μM, a physiologically relevant value. This finding is consistent with the observation that NADH:TrxR has thus far been found primarily in anaerobic bacteria and archaea.

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Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up-to-47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here we report the development of a marker-assisted-mapping (MAM) strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using MAM, we mapped a transgene that was inserted in a >100 Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. This study provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods by improving upon the current re-based genetic sexing strains. MAM does not require densely populated markers and can be readily applied throughout the genome to facilitate the mapping of genes responsible for insecticide- and viral-resistance. By enabling effective forward genetic analysis, MAM bridges a significant gap in establishing Ae. aegypti as a model system for research in vector biology. As large regions of suppressed recombination are also common in other plant and animal species including those of economic significance, MAM will have broad applications beyond vector biology.