# Two chromosomal reference genome sequences for the malaria mosquito, Anopheles (Nyssorhynchus) darlingi, Root, 1926 from French Guiana and Peru

Mathilde Gendrin, Katy Heu, Marta Moreno, Dionicia Gamboa, Joseph Vinetz, Carlos Tong, Jan Conn, Harriet Johnson, Haynes Heaton, Martin Wagah, et al.

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Mathilde Gendrin, Katy Heu, Marta Moreno, Dionicia Gamboa, Joseph Vinetz, et al. Two chromosomal reference genome sequences for the malaria mosquito, Anopheles (Nyssorhynchus) darlingi, Root, 1926 from French Guiana and Peru. Wellcome Open Research, 2025, 10, pp.187. 10.12688/wellcomeopenres.23989.1. hal-05422203

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DATA NOTE

# Two chromosomal reference genome sequences for the malaria mosquito, *Anopheles (Nyssorhynchus) darlingi*, Root, 1926 from French Guiana and Peru

[version 1; peer review: 3 approved]

Mathilde Gendrin<fnref n="1" />, Katy Heu<fnref n="1" />, Marta Moreno<fnref n="2" />, Dionicia Gamboa<fnref n="3" />, Joseph M Vinetz<fnref n="4" />, Carlos Tong<fnref n="5" />, Jan E Conn<fnref n="6" />,<fnref n="7" />, Harriet F Johnson<fnref n="8" />, Haynes Heaton<fnref n="9" />, Martin G Wagah<fnref n="10" />, Joanna C Collins<fnref n="10" />, Ksenia Krasheninnikova<fnref n="10" />, Sarah E Pelan<fnref n="10" />, Damon-Lee B Pointon<fnref n="10" />, James W Torrance<fnref n="10" />, Alan Tracey<fnref n="10" />, Marcela Uliano-Silva<fnref n="10" />, Jonathan M D Wood<fnref n="10" />, Katharina von Wyschetzki<fnref n="10" />, Wellcome Sanger Institute Scientific Operations: Sequencing Operations, Shane A McCarthy<fnref n="10" />,<fnref n="11" />, Mara K N Lawniczak<fnref n="10" />, Daniel E Neafsey<fnref n="12" />,<fnref n="13" />, Alex Makunin<fnref n="10" />

**V1** First published: 10 Apr 2025, **10:187**
https://doi.org/10.12688/wellcomeopenres.23989.1

Latest published: 10 Apr 2025, **10:187**
https://doi.org/10.12688/wellcomeopenres.23989.1

## Abstract

We present two genome assemblies, each generated from individual female *Anopheles (Nyssorhynchus) darlingi* (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from wild populations in French Guiana and Peru. The genome sequences are approximately

## Open Peer Review

Approval Status

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1 2 3

version 1

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10 Apr 2025

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<footnote n="1">¹Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, Cayenne, French Guiana</footnote>

<footnote n="2">²Department of Infection Biology, London School Of Hygiene and Tropical Medicine, London, England, UK</footnote>

<footnote n="3">³Laboratorio de Malaria: Parásitos y vectores, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias e Ingeniería, Universidad Peruana Cayetano Heredia, Lima District, Lima Region, Peru</footnote>

<footnote n="4">⁴Section of Infectious Disease, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA</footnote>

<footnote n="5">⁵GERESA Loreto Gerencia Regional de Salud Loreto, Unidad de Entomología, Iquitos, Peru</footnote>

<footnote n="6">⁶Wadsworth Center, New York State Department of Health, Albany, NY, USA</footnote>

<footnote n="7">⁷Department of Biomedical Sciences, College of Integrated Health Sciences, SUNY The State University of New York, Albany, New York, USA</footnote>

<footnote n="8">⁸Scientific Operations, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="9">⁹CSSE, Auburn University, Auburn, Alabama, USA</footnote>

<footnote n="10">¹⁰Tree of Life, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="11">¹¹University of Cambridge Department of Genetics, Cambridge, England, UK</footnote>

<footnote n="12">¹²Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA</footnote>

<footnote n="13">¹³Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA</footnote>
180 megabases in span. The majority of each assembly is scaffolded
into three chromosomal pseudomolecules with the X sex
chromosome assembled. The complete mitochondrial genomes were
also assembled and are both 15.4 kilobases in length. The assemblies
differ by two inversions in chromosome arm 2R.

Keywords

Anopheles darlingi, South American malaria vector, genome
sequence, chromosomal

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This article is included in the **Tree of Life**
gateway.

1. Craig Wilding ID, University College Dublin School of Biology and Environmental Science (Ringgold ID: 123708), Dublin, Ireland

2. Renee Ali ID, Johns Hopkins University Bloomberg School of Public Health, Baltimore, USA

3. **Komal Kumar Bollepogu Raja** ID, Baylor College of Medicine, Houston, USA

Any reports and responses or comments on the
article can be found at the end of the article.

**Corresponding authors:** Mara K N Lawniczak (mara@sanger.ac.uk), Alex Makunin (am60@sanger.ac.uk)

**Author roles:** **Gendrin M:** Investigation; **Heu K:** Investigation; **Moreno M:** Investigation; **Gamboa D:** Investigation; **Vinetz JM:** Investigation; **Tong C:** Investigation; **Conn JE:** Investigation; **Johnson HF:** Investigation, Methodology; **Heaton H:** Investigation, Methodology; **Wagah MG:** Visualization; **Collins JC:** Data Curation; **Krasheninnikova K:** Methodology; **Pelan SE:** Data Curation; **Pointon DLB:** Data Curation; **Torrance JW:** Data Curation; **Tracey A:** Data Curation; **Uliano-Silva M:** Investigation, Methodology; **Wood JMD:** Data Curation; **von Wyschetzki K:** Methodology; **McCarthy SA:** Investigation, Methodology; **Lawniczak MKN:** Conceptualization, Funding Acquisition, Investigation, Supervision, Writing - Review & Editing; **Neafsey DE:** Conceptualization, Funding Acquisition; **Makunin A:** Formal Analysis, Methodology, Project Administration, Visualization, Writing - Original Draft Preparation

**Competing interests:** No competing interests were disclosed.

**Grant information:** This work was supported by a Bill & Melinda Gates Foundation Award (INV-009760) to ML. The Wellcome Sanger Institute is funded by the Wellcome Trust (220540), which supports ML. SAM is supported by the Wellcome Trust Grant (WT207492). This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Grant Number U19AI110818 to the Broad Institute (DEN). The Peruvian Anopheles (Nyssorhynchus) darlingi colonization project was funded by US Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases Cooperative Agreement U19AI089681 (Amazonian ICEMR) to JMV and NIH R01AI54139-02 to JEC.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

**Copyright:** © 2025 Gendrin M *et al*. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**How to cite this article:** Gendrin M, Heu K, Moreno M *et al*. Two chromosomal reference genome sequences for the malaria mosquito, *Anopheles (Nyssorhynchus) darlingi*, *Root, 1926 from French Guiana and Peru* [version 1; peer review: 3 approved]
Wellcome Open Research 2025, **10**:187 https://doi.org/10.12688/wellcomeopenres.23989.1

**First published:** 10 Apr 2025, **10**:187 https://doi.org/10.12688/wellcomeopenres.23989.1
## Species taxonomy

Animalia; Arthropoda; Insecta; Diptera; Culicidae; Anophelinae; Anopheles; *Anopheles darlingi*; Root, 1926 (NCBI txid:43151).

## Background

The *Anopheles darlingi* mosquito is the most important Neotropical malaria vector, spanning an enormous geographic range from southern Mexico to northern Argentina¹,². It belongs to a subgenus (*Nyssorhynchus*) that exhibits considerable evolutionary divergence from other anopheline subgenera³ harbouring familiar malaria vectors from Africa and Asia, resulting in the proposal to recognize *Nyssorhynchus* as a genus-level classification¹. Given the importance of *An. darlingi* as a neotropical vector, the first short-read genome assembly was published in 2013, based on a collection of male and female mosquitoes from the Brazilian Amazon⁵. The resulting assembly provided a first understanding of this divergent lineage of mosquitoes, but was highly fragmented (over 8000 scaffolds, N50 scaffold length = 81 kb) due to heterozygosity in the sequencing template from many individual mosquitoes and the inherent difficulty of assembling highly contiguous genomes from short reads.

Consequently, improved genome assemblies of *An. darlingi* were produced as part of the Anopheles Reference Genomes Project (PRJEB51690). Here we present two chromosomally complete genome sequences for *Anopheles darlingi*, based on single female specimens from Macouria, French Guiana and Cahuide, Loreto Department, Peru. The latter is derived from the first sustained colony of *Anopheles (Nyssorhynchus darlingi)* established in 2013 from 135 wild-caught female mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department⁶, for which maintenance of heterozygosity was demonstrated. This colony had no significant evidence of a bottleneck, decrease in total alleles, or increase in inbreeding compared with field specimens (founder population). Low-moderate differentiation between field and laboratory populations was detected⁷. The genome assembly for the specimen from French Guiana has been annotated by NCBI RefSeq to support its use as an updated reference for the species.

## Genome sequence report for specimen from French Guiana

The genome was sequenced from a single female *Anopheles darlingi* collected from Macouria, French Guiana (5.015, -52.528). A total of 32-fold coverage in Pacific Biosciences single-molecule HiFi long reads (N50 11.387 kb) were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from a female sibling. Manual assembly curation corrected 13 missing joins or misjoins, reducing the scaffold number by 5.8%.

The final assembly has a total length of 182 Mb in 66 sequence scaffolds with a scaffold N50 of 94.952 Mb (Table 1). 98.89% of the assembly sequence was assigned to three chromosomal-level scaffolds, representing two autosomes

(numbered and oriented against the *An. albimanus* STECLA strain assembly (8; GCA_013758885.1)), and the X sex chromosome (Figure 1–Figure 4; Table 2).

The assembly has a BUSCO 5.3.2⁹ completeness of 97.2% using the diptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

## Genome sequence report for specimen from Peru

The genome was sequenced from a single female *Anopheles darlingi* from an established mosquito colony that originated from Cahuide, Peru (-4.230, -73.460). Unfortunately, this colony no longer exists and is therefore not available for further research. A total of 68-fold coverage in Pacific Biosciences single-molecule HiFi long reads (N50 10.925 kb) and 113-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from a female sibling. Manual assembly curation corrected 26 missing joins or misjoins and removed 1 duplicated haplotig.

The final assembly has a total length of 182 Mb in 54 sequence scaffolds with a scaffold N50 of 95.091 Mb (Table 3). 98.92% of the assembly sequence was assigned to three chromosomal-level scaffolds, representing two autosomes and the X sex chromosome (Table 4). Synteny between this assembly and that of *An. albimanus* AalbS3 showed identical chromosome arms, with numerous rearrangements within the arms (Figure 5). Compared to the assembly generated from a female from French Guiana, this assembly has two large scale inversions in chromosome arm 2R (Figure 6; Table 5).

The assembly has a BUSCO 5.3.2⁹ completeness of 97.0% using the diptera_odb10 reference set.

## Chromosome arms delineation

To facilitate the delineation of chromosome arms in both assemblies, we predicted most common tandem repeats using TRASH¹⁰. In autosomes, largest blocks of tandem repeats coincided with expected centromere locations and were mostly composed of repeats with 353 base long monomers (Figure 7, Figure 8). We used outermost copies of this repeat to annotate chromosome arms (Table 6). In the X chromosome, no large tandem repeat blocks were observed on the centromere adjacent side, i.e. at the end of the chromosome sequence. In both assemblies, the majority of chromosome arms ended with tandem repeats with 281 base long monomers, suggesting that these repeats act as telomeres – non-canonical satellite repeats are characteristic for other *Anopheles* species⁸.

## Methods

### Sample acquisition and nucleic acid extraction

*Anopheles darlingi* offspring were reared from a wild caught gravid female collected from Macouria, French Guiana (5.015, -52.528) by Katy Heu and Romuald Carinci in April 2019. Individuals from the resulting brood were shipped
<table><caption>**Table 1.** Genome data for *Anopheles darlingi* from French Guiana, *idAnoDarIMG_H_01*.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>idAnoDarIMG_H_01</td></tr><tr><td>Species</td><td>Anopheles darlingi</td></tr><tr><td>Specimen</td><td>idAnoDarIMG-H_01</td></tr><tr><td>NCBI taxonomy ID</td><td>43151</td></tr><tr><td>BioProject</td><td>PRJEB53253</td></tr><tr><td>BioSample ID</td><td>ERS10527351</td></tr><tr><td>Isolate information</td><td>female, whole organism</td></tr><tr><th colspan="2">Raw data accessions</th></tr><tr><td>PacificBiosciences SEQUEL II</td><td>ERR9439499</td></tr><tr><td>Hi-C Illumina</td><td>ERR9356785, ERR9356786</td></tr><tr><td>PolyA RNA-Seq Illumina</td><td>ERR9356791, ERR9356792, ERR9356793</td></tr><tr><th colspan="2">Genome assembly</th></tr><tr><td>Assembly accession</td><td>GCA_943734745</td></tr><tr><td>Accession of alternate haplotype</td><td>GCA_943734775</td></tr><tr><td>Span (Mb)</td><td>181.653</td></tr><tr><td>Number of contigs</td><td>96</td></tr><tr><td>Contig N50 length (Mb)</td><td>19.203</td></tr><tr><td>Number of scaffolds</td><td>66</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>94.952</td></tr><tr><td>Longest scaffold (Mb)</td><td>94.952</td></tr><tr><td>BUSCO* genome score</td><td>C:97.2%[S:96.7%,D:0.5%],F:0.6%,M:2.2%,n:3285</td></tr></tbody></table>

on dry ice to the UK. From this brood, a single female *idAnoDarIMG-H_01* was used for Pacific BioSciences and its sibling female *idAnoDarIMG-H_08* was used for Arima Hi-C.

*Anopheles darlingi* offspring from Peru were reared from a colony in April 2019. The original collection to create this colony used wild females collected from Cahuide, Loreto, Peru (–4.230, –73.460) by the Amazonian International Center of Excellence in Malaria Research (Amazonian ICEMR) team in a long-term project led by Dionicia Gamboa, Marta Moreno, Jan Conn and Joseph Vinetz⁶. A single mated female from

this colony was used to rear a brood to be used for sequencing. From this brood, female sibling *idAnoDarJC-H15_27* was used for Pacific BioSciences and 10x linked reads, and female sibling *idAnoDarJC-H15_22* was used for Arima Hi-C.

For both reference genomes, high molecular weight (HMW) DNA extraction used one whole insect disrupted by manual grinding with a blue plastic pestle in Qiagen MagAttract lysis buffer and then extracted using the Qiagen MagAttract HMW DNA extraction kit with two minor modifications¹¹. The quality of the DNA was evaluated using an Agilent FemtoPulse to

<footnote>* BUSCO scores based on the diptera_odb10 BUSCO set using BUSCO 5.3.2. C=complete [S=single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/idAnoDarIMG_H_01/dataset/CALSDZ01/busco.</footnote>
<figure><img src="image_9.png" /><figcaption>**Figure 1.** Snail plot summary of assembly statistics for *Anopheles darlingi* assembly idAnoDarIMG_H_01. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 181,652,731 bp assembly. The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (94,951,917 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (94,951,917 and 71,270,736 bp), respectively. The pale grey spiral shows the cumulative sequence count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoDarIMG_H_01/dataset/CALSDZ01/snail.</figcaption></figure>

ensure that most DNA molecules were larger than 30 kb, and preferably > 100 kb. In general, single mosquito extractions range in total estimated DNA yield from 200 ng to 800 ng, with an average yield of 500 ng. Low molecular weight DNA was removed using a 0.8X AMPure XP purification. For the specimen from Peru, a small aliquot (less than ~5% of the

total volume) of HMW DNA was set aside for 10X Linked Read sequencing. For PacBio sequencing, DNA was sheared to an average fragment size of 12–20 Kb using a Diagenode Megaruptor 3 at speeds ranging from 27 to 30. Sheared DNA was purified using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the
<figure><img src="image_10.png" /><figcaption>**Figure 2.** Blob plot of base coverage in idAnoDarIMG_H_01 PacBio HiFi reads against GC proportion for *An. darlingi* assembly idAnoDarIMG_H_01. Chromosomes are coloured by phylum. Circles are sized in proportion to chromosome length. Histograms show the distribution of chromosome length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoDarIMG_H_01/dataset/CALSDZ01/blob.</figcaption></figure>

DNA sample. The concentration and quality of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer with the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sheared and cleaned sample on the FemtoPulse system once more. The median DNA fragment size for *Anopheles* mosquitoes was 15 kb and the median yield of

sheared DNA was 200 ng, with samples typically losing about 50% of the original estimated DNA quantity through the process of shearing and purification.

For Hi-C data generation, separate sibling mosquito specimens were used as input material for the Arima V2 Kit according to the manufacturer's instructions for animal tissue. This approach
<figure><img src="image_11.png" /><figcaption>**Figure 3.** Cumulative chromosome length for *An. darlingi* assembly idAnoDarIMG_H_01. The grey line shows cumulative length for all chromosomes. Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule. The interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoDarIMG_H_01/dataset/CALSDZ01/cumulative.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 4.** Genome assembly of *An. darlingi*, idAnoDarIMG_H_01: Hi-C contact map. Visualised in HiGlass. Chromosomes are arranged in size order from left to right and top to bottom. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/I/?d=Pap61sCkTXaWekXO6tp-Wg.</figcaption></figure>
<table><caption>**Table 2.** Chromosomal pseudomolecules in the genome assembly of *An. darlingi* from French Guiana, idAnoDarIMG_H_01.</caption><thead><tr><th>INSDC accession</th><th>Chromosome</th><th>Size (Mb)</th><th>Count</th><th>Gaps</th></tr></thead><tbody><tr><td>OX030911.1</td><td>2RL</td><td>94.951</td><td>1</td><td>10</td></tr><tr><td>OX030912.1</td><td>3RL</td><td>71.271</td><td>1</td><td>5</td></tr><tr><td>OX030913.1</td><td>X</td><td>13.401</td><td>1</td><td>15</td></tr><tr><td>OX030914.1</td><td>MT</td><td>0.015</td><td>1</td><td>0</td></tr><tr><td></td><td>2 Unlocalised</td><td>0.030</td><td>1</td><td>0</td></tr><tr><td></td><td>X Unlocalised</td><td>1.957</td><td>60</td><td>0</td></tr><tr><td></td><td>Unplaced</td><td>0.026</td><td>1</td><td>0</td></tr></tbody></table>

<table><caption>**Table 3.** Genome data for *Anopheles darlingi* from Peru, idAnoDarIJC-H15_27.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>ASM3899477v1</td></tr><tr><td>Species</td><td>Anopheles darlingi</td></tr><tr><td>Specimen</td><td>idAnoDarIJC-H15_27</td></tr><tr><td>NCBI taxonomy ID</td><td>43151</td></tr><tr><td>BioProject</td><td>PRJNA830873</td></tr><tr><td>BioSample ID</td><td>SAMN33940061</td></tr><tr><td>Isolate information</td><td>female, whole organism</td></tr><tr><td colspan="2"><strong>Raw data accessions</strong></td></tr><tr><td>PacificBiosciences SEQUEL II</td><td>SRX26935726</td></tr><tr><td>10X Genomics Illumina</td><td>SRX26935727, SRX26935728, SRX26935729, SRX26935730</td></tr><tr><td>Hi-C Illumina</td><td>SRX26803935</td></tr><tr><td colspan="2"><strong>Genome assembly</strong></td></tr><tr><td>Assembly accession</td><td>GCA_038994775</td></tr><tr><td>Span (Mb)</td><td>182.384</td></tr><tr><td>Number of contigs</td><td>86</td></tr><tr><td>Contig N50 length (Mb)</td><td>13.497</td></tr><tr><td>Number of scaffolds</td><td>54</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>95.091</td></tr><tr><td>Longest scaffold (Mb)</td><td>95.091</td></tr><tr><td>BUSCO* genome score</td><td>C:97.0%[S:96.3%,D:0.7%], F:0.7%,M:2.3%,n:3285</td></tr></tbody></table>

<table><caption>**Table 4.** Chromosomal pseudomolecules in the genome assembly of *An. darlingi* from Peru, idAnoDarIJC-H15_27.</caption><thead><tr><th>INSDC accession</th><th>Chromosome</th><th>Size (Mb)</th><th>Count</th></tr></thead><tbody><tr><td>CM077074.1</td><td>2RL</td><td>95.091</td><td>1</td></tr><tr><td>CM077075.1</td><td>3RL</td><td>71.801</td><td>1</td></tr><tr><td>CM077073.1</td><td>X</td><td>13.508</td><td>1</td></tr><tr><td>CM077077.1</td><td>MT</td><td>0.015</td><td>1</td></tr><tr><td></td><td>X Unlocalised</td><td>1.974</td><td>52</td></tr></tbody></table>

of using a sibling was taken in order to enable all material from a single specimen to contribute to the PacBio data generation given we were not always able to meet the minimum suggested guidance of starting with > 300 ng of HMW DNA from a specimen. Samples proceeded to the Illumina library prep stage even if they were suboptimal (too little tissue) going into the Arima reaction.

To assist with annotation for assembly of specimen from French Guiana, generated by NCBI RefSeq¹² and available through VectorBase¹³ under accession AdarGF1, RNA was extracted from separate whole sibling and unrelated insect specimens (idAnoDarIMG-G_01, idAnoDarIMG-G_02, and idAnoDarIMG-H_11) using TRIzol, according to the manufacturer's instructions. RNA was then eluted in 50 µl RNAse-free water, and its concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay. Samples were not always ideally preserved for RNA, so qualities varied but all were sequenced anyway.

## Sequencing

We prepared libraries as per the PacBio procedure and checklist for SMRTbell Libraries using Express TPK 2.0 with low DNA input. Every library was barcoded to support multiplexing. Final library yields ranged from 20 ng to 100 ng, representing only about 25% of the input sheared DNA. Libraries from two specimens were typically multiplexed on a single 8M SMRT Cell. Sequencing complexes were made using Sequencing Primer v4 and DNA Polymerase v2.0. Sequencing was carried out on the Sequel II system with 24-hour run time and 2-hour pre-extension. A 10X Genomics Chromium read cloud sequencing library was also constructed according to the manufacturer's instructions (this product is no longer available). Only 0.5 ng of DNA was used and only 25-50% of the gel emulsion was put forward for library prep due to the small genome size. For Hi-C data generation, following the Arima HiC V2 reaction, samples were processed through Library Preparation using a NEB Next Ultra II DNA Library Prep Kit and sequenced aiming for 100x depth. RNA libraries were

<footnote>* BUSCO scores based on the diptera_odb10 BUSCO set using BUSCO 5.3.2. C=complete [S=single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison.</footnote>
<figure><img src="image_13.png" /><figcaption>**Figure 5.** Synteny between genome assemblies of *An. albimanus* AalbS3 and *An. darlingi*, idAnoDarIMG_H_01. Chromosome arms arrangements are identical, while within arms there are numerous rearrangements.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 6.** Synteny between genome assemblies of *An. darlingi* from French Guiana, idAnoDarIMG_H_01, and from Peru, idAnoDarJC-H15_27. Coordinates of two inversions are provided in Table 5.</figcaption></figure>
created using the directional NEB Ultra II stranded kit. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (10X and Hi-C), or Illumina HiSeq 4000 (RNAseq).

## Genome assembly

Assemblies were carried out with Hifiasm¹⁴; haplotypic duplications were identified and removed with purge_dups¹⁵. For

the specimen from Peru, one round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes¹⁶. The assembly was then scaffolded with Hi-C data¹⁷ using SALSA2¹⁸. The assembly was checked for contamination as described previously¹⁹. Manual curation was performed using gEVAL²⁰, HiGlass²¹ and Pretext²². The mitochondrial genome was assembled using MitoHiFi²³, which performs annotation using MitoFinder²⁴. The genome was analysed and BUSCO scores were generated within the BlobToolKit environment²⁵. Synteny analyses were performed with D-GENIES²⁶. Tandem repeats were annotated with TRASH¹⁰. Table 7 contains a list of all software tool versions used, where appropriate.

<table><caption>**Table 5.** Inversions between genome assemblies of *An. darlingi* from French Guiana and Peru.</caption><thead><tr><td>INSDC accession</td><td>Chromosome</td><td>Start</td><td>End</td></tr></thead><tbody><tr><td>OX030911.1</td><td>2RL</td><td>6,114,428</td><td>12,943,365</td></tr><tr><td>OX030911.1</td><td>3RL</td><td>18,970,078</td><td>21,986,145</td></tr></tbody></table>

## Ethics/compliance issues

The genetic resources accessed and utilised under this project were done so in accordance with the UK ABS legislation (Nagoya Protocol (Compliance) (Amendment) (EU Exit) Regulations 2018 (SI 2018/1393)) and the national ABS legislation within the country of origin, where applicable.

<figure><img src="image_15.png" /><figcaption>**Figure 7.** Tandem repeats annotation for genome assembly of *An. darlingi* from French Guiana, *idAnoDarIMG_H_01*.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 8.** Tandem repeats annotation for genome assembly of *An. darlingi* from Peru, *idAnoDarIJC-H15_27*.</figcaption></figure>

<table><caption>**Table 6.** Chromosome arms annotation in genome assemblies of *An. darlingi*.</caption><thead><tr><th>Chromosome arm</th><th>idAnoDarIMG_H_01 start</th><th>idAnoDarIMG_H_01 end</th><th>idAnoDarIJC-H15_27 start</th><th>idAnoDarIJC-H15_27 end</th></tr></thead><tbody><tr><td>2R</td><td>1</td><td>52,837,268</td><td>1</td><td>52,904,211</td></tr><tr><td>2L</td><td>53,277,590</td><td>94,951,917</td><td>53,898,552</td><td>95,090,923</td></tr><tr><td>3R</td><td>1</td><td>35,602,462</td><td>1</td><td>35,719,087</td></tr><tr><td>3L</td><td>36,556,652</td><td>71,270,736</td><td>36,907,605</td><td>71,801,356</td></tr></tbody></table>

<table><caption>**Table 7.** Software tools used.</caption><thead><tr><th>Software tool</th><th>Version</th><th>Source</th></tr></thead><tbody><tr><td>hifiasm</td><td>0.14</td><td>14</td></tr><tr><td>purge_dups</td><td>1.2.3</td><td>15</td></tr><tr><td>longranger align</td><td>2.2.2</td><td>27</td></tr><tr><td>freebayes</td><td>1.3.1</td><td>16</td></tr><tr><td>SALSA2</td><td>2.2-4c80ac1</td><td>18</td></tr><tr><td>MitoHiFi</td><td>2</td><td>23</td></tr><tr><td>gEVAL</td><td>N/A</td><td>20</td></tr><tr><td>HiGlass</td><td>1.11.6</td><td>21</td></tr></tbody></table>

<table><thead><tr><th>Software tool</th><th>Version</th><th>Source</th></tr></thead><tbody><tr><td>PretextView</td><td>0.1.x</td><td>22</td></tr><tr><td>BlobToolKit</td><td>3.4.0</td><td>28</td></tr><tr><td>BUSCO</td><td>5.3.2</td><td>9</td></tr><tr><td>D-GENIES</td><td>1.4</td><td>26</td></tr><tr><td>TRASH</td><td>1.2</td><td>10</td></tr></tbody></table>

## Data availability

European Nucleotide Archive: PRJEB53253, https://identifiers.org/bioproject/PRJEB53253; PRJNA830873, https://identifiers.org/bioproject/PRJNA830873. The genome sequences
are released openly for reuse. The *Anopheles darlingi* genome sequencing initiative is part of the Anopheles Reference Genomes project PRJEB51690. All raw sequence data and the assemblies have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 3.

## Author information

Members of the Wellcome Sanger Institute Scientific Operations: Sequencing Operations collective are listed here: https://doi.org/10.5281/zenodo.4790456.

## Acknowledgments

We acknowledge Romuald Carinci (Institut Pasteur de la Guyane) for assistance with field sample collection, and Nithya Swaminathan (Broad Institute) for support with project management and data submission. We are grateful to the Ministerio de Agricultura, Dirección General Forestal y de Fauna Silvestre and the Dirección de Salúd, Gobierno Regional de Loreto Gobierno Regional de Loreto under the auspices of Resolución Directoral Number 0425-2012-AG-DGFFS-DGEFFS for permission to carry out these studies of Anopheles darlingi in Peru.

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# Open Peer Review

Current Peer Review Status:

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<figure><img src="image_18.png" /></figure>

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## Version 1

Reviewer Report 10 May 2025

https://doi.org/10.21956/wellcomeopenres.26465.r122270

© 2025 Raja K. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Komal Kumar Bollepogu Raja

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Baylor College of Medicine, Houston, USA

The manuscript by Gendrin et al presents two high quality genome assembly sequences for the species *Anopheles darlingi* from French Guiana and Peru. The authors have achieved 182 Mb of assemblies with N50 of about 95 Mb and BUSCO completeness of >97% suggesting good quality of the genome sequences. This assembly is a substantial improvement over the fragmented 2013 version of the genome. The authors have sufficiently introduced the biology and evolutionary context of the species including colony origin. The experimental and bioinformatics methodology is robust. The use of TRASH for centromeric and telomeric repeat annotation is commendable. Overall, this manuscript represents a significant advance in *An. darlingi* genomics and will be a valuable resource for malaria vector research.

**Minor comment**

Please be consistent between 'idAnoDarIMG-H_01' and 'idAnoDarIMG_H_01'

**Is the rationale for creating the dataset(s) clearly described?**

Yes

**Are the protocols appropriate and is the work technically sound?**

Yes

**Are sufficient details of methods and materials provided to allow replication by others?**

Yes

**Are the datasets clearly presented in a useable and accessible format?**

Yes

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** Single cell genomics, bioinformatics, genomics and Neurobiology
**I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.**

Reviewer Report 10 May 2025

https://doi.org/10.21956/wellcomeopenres.26465.r122268

© 2025 Ali R. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Renee Ali

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Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA

A good addition to the genomic data available for this important neotropical vector *Anopheles darlingi*. The methods were well explained and reproducible. It would be nice to produce another assembly or compare to the first version if possible to observe whether there were also inversions on the chromosome arm 2R.

**Is the rationale for creating the dataset(s) clearly described?**

Yes

**Are the protocols appropriate and is the work technically sound?**

Yes

**Are sufficient details of methods and materials provided to allow replication by others?**

Yes

**Are the datasets clearly presented in a useable and accessible format?**

Yes

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** Genetics, mosquito populations, understudied vectors

**I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.**

Reviewer Report 26 April 2025

https://doi.org/10.21956/wellcomeopenres.26465.r122272

© 2025 Wilding C. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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**Craig Wilding**

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University College Dublin School of Biology and Environmental Science (Ringgold ID: 123708),
Dublin, Leinster, Ireland

In this article, the authors describe the generation and annotation of chromosomal-scale genome
sequences from two separate *Anopheles darlingi* individuals collected from geographically isolated
(French Guiana and Peru) populations. Both genomes are similarly high quality (in terms of
scaffold-length and BUSCO completeness) and were generated using a combination of PacBio HiFi
and Hi-C scaffolding for genome sequencing, and Illumina for RNASeq (on a specimen from
French Guiana).

Having two assemblies has permitted the authors to identify two major chromosomal inversions
on chromosome 2R.

The methods utilized are reported clearly although syntenic comparison for the French Guianan
sample was reported to be undertaken versus the "*An. albimanus* STECLA strain assembly
(GCA_013758885.1)" whilst synteny for the Peruvian sample is reported as generated against "*An.
albimanus* AalbS3". Whilst I think these are the same, for clarity, it would be better to be consistent
in reference to the *An. albimanus* strain. Note, I believe that it should be the STELCA strain (not
STECLA).

**Is the rationale for creating the dataset(s) clearly described?**

Yes

**Are the protocols appropriate and is the work technically sound?**

Yes

**Are sufficient details of methods and materials provided to allow replication by others?**

Yes

**Are the datasets clearly presented in a useable and accessible format?**

Yes

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** Evolutionary genetics/genomics

**I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.**