whole-mount-fluorescence-situ-hybridization-to-study-spermatogenesis

Whole-Mount Fluorescence <em>In Situ</em> Hybridization to Study Spermatogenesis in the <em>Anopheles</em> Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Virginia Tech

Anopheles sinensis (Diptera: Culicidae) is an important vector of Plasmodium vivax in Southeast Asia. To facilitate population genetic and genomic studies of An. sinensis, we developed a standard cytogenetic photomap for this species. The polytene chromosomes were straightened and divided into 39 numbered divisions and 116 lettered subdivisions. The chromosomal localizations of 13 DNA probes were determined by fluorescent in situ hybridization. A comparison of the physical map for An. sinensis with the genome map for Anopheles gambiae revealed a whole-arm autosomal translocation between the two species. Specifically, the 2R arm of An. gambiae corresponds to the 3R arm of An. sinensis and the pattern of correspondence of the other chromosome arms remains regular. We mapped the breakpoints of the polymorphic paracentric chromosomal inversion 3Ra to subdivisions 28A and 31A. The standard cytogenetic map developed in this study will be useful for detailed comparative genome mapping and population genetic studies of An. sinensis.

A standard cytogenetic map for Anopheles sinensis and chromosome arm homology between the subgenera Anopheles and Cellia.

Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors.

Structural divergence of chromosomes between malaria vectors Anopheles lesteri and Anopheles sinensis.

Anopheles sinensis is a dominant natural vector of Plasmodium vivax in China, Taiwan, Japan, and Korea. Recent genome sequencing of An. sinensis provides important insights into the genomic basis of vectorial capacity. However, the lack of a physical genome map with chromosome assignment and orientation of sequencing scaffolds hinders comparative analyses with other genomes to infer evolutionary changes relevant to the vector capacity.

Comparative physical genome mapping of malaria vectors Anopheles sinensis and Anopheles gambiae.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of , , and . We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes.

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  assembly of the New World malaria mosquito,  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 toward 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 analyzing the termini of long reads and through both chromosomal  hybridization and a 31 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 toward 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.

Article	Year
A standard cytogenetic map for Anopheles sinensis and chromosome arm homology between the subgenera Anopheles and Cellia. Medical and veterinary entomology\| PubMed ID: 25171604	2014
Structural divergence of chromosomes between malaria vectors Anopheles lesteri and Anopheles sinensis. Parasites & vectors\| PubMed ID: 27887641	2016
Comparative physical genome mapping of malaria vectors Anopheles sinensis and Anopheles gambiae. Malaria journal\| PubMed ID: 28583133	2017
Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes. Cells\| PubMed ID: 32024176	2020
The Beginning of the End: A Chromosomal Assembly of the New World Malaria Mosquito Ends with a Novel Telomere. G3 (Bethesda, Md.)\| PubMed ID: 32883756	2020

Jiangtao Liang

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