Annotated genome of Aedes japonicus japonicus using a hybrid-assembly approach

Friederike Reuss^1*Tilman Schell²Haruhiko Isawa³Shinji Kasai³Sven Klimpel^4,5,6Ruth Müller⁷Markus Pfenninger^5,8Judith Kochmann^5,8

• ¹Goethe University Frankfurt, Institute of Occupational, Social and Environmental Medicine, Frankfurt, Germany
• ²Senckenberg Research Institute, Frankfurt, Germany
• ³Department of Medical Entomology, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo, Japan
• ⁴Goethe-Universitat Frankfurt am Main Institut fur Okologie Evolution und Diversitat, Frankfurt, Germany
• ⁵Senckenberg Biodiversitat und Klima Forschungszentrum, Frankfurt, Germany
• ⁶LOEWE-Zentrum fur Translationale Biodiversitatsgenomik, Frankfurt, Germany
• ⁷Institute of Tropical Medicine, Unit of Entomology, Antwerp, Belgium
• ⁸Johannes Gutenberg Universitat Mainz Institut fur Organismische und Molekulare Evolutionsbiologie, Mainz, Germany

albopictus AalbF5 (GCF_035046485.1; Palatini et al., (2020)). Globally, Ae. aegypti is the primary vector of chikungunya virus and dengue virus (Sousa et al., 2012; Jansen et al., 2018). Aedes albopictus is considered a secondary vector to Ae. aegypti for chikungunya and dengue viruses (Jansen and Beebe, 2010; Sousa et al., 2012), however, it is the most important vector for autochthonous cases of dengue and chikungunya in Europe (Rezza et al., 2007; Gjenero-Margan et al., 2011; Succo et al., 2016). Both species, Ae. aegypti and Ae. albopictus, are an invasive species in Europe (European Centre for Disease Prevention and Control and European Food Safety Authority, 2023). Another more recent invader to North America (Kaufman and Fonseca, 2014) and Europe is Ae. japonicus japonicus while its sister species Ae. koreicus got established in Europe (European Centre for Disease Prevention and Control and European Food Safety Authority, 2023). Over the last two to three decades, Ae. j. japonicus has spread beyond its original area of distribution in East Asia via the import of used tires and trade (Kaufman and Fonseca, 2014; Koban et al., 2019) and is likely to expand its range in the future (Cunze et al., 2020). For Ae. japonicus as well as Ae. koreicus, annotated genomes (GCA_034211315.2, GCA_024533555.2) have only recently become available (Catapano et al., 2023; Nagy et al., 2024). Here, we present an annotated genome and a complete mitochondrial sequence of Ae. j. japonicus from a laboratory strain in Japan (Hoshino et al., 2010). This is the first time that individuals from the native range of this species (Kaufman and Fonseca, 2014) were sequenced. The mitochondrion of Ae. j. japonicus can help in constructing phylogenies. For example, the genus Aedes as well as the tribe of Aedini have been re-organised based on morphological analyses (reviewed in Wilkerson et al., (2015)) and molecular analyses (Zadra et al., 2021). Thus, genetic datasets are highly desirable for creating a well-founded phylogeny of Aedini or Aedes (Zadra et al., 2021). Our genome assembly can facilitate marker selection for environmental associations as well as genotype-to-phenotype-association studies. By doing so, the genomic basis of vector competence or invasion success can be identified within the species Ae. japonicus but also compared to other Aedes spp. More specifically, the created dataset allows to conduct comparative studies regarding diapause (Kreß et al., 2016; Boyle et al., 2021), thermotolerance (Kramer et al., 2023; Couper et al., 2025) and population structure (Smitz et al., 2021), all considered potential parameters influencing invasiveness (Lahondère and Bonizzoni, 2022). For example, while Ae. albopictus and Ae. aegypti are primary vectors for dengue and chikungunya viruses, Ae. japonicus is only a minor vector in the transmission of diseases agents and its vector competence is largely based on laboratory competence studies (Medlock et al., 2012; Jansen et al., 2018; Wagner et al., 2018). A trait shared between Ae. japonicus and Ae. albopictus is the ability to undergo photoperiodic diapause (Armbruster, 2016; Krupa et al., 2021), which benefits the species' survival in more temperate regions. In addition, this dataset provides data to study candidate genes not only related to vector competence but also insecticide resistance. It also provides genomic resources for marker identification, e.g., to be used in eDNA approaches for a more rapid species' detection in the field (Wittwer et al., 2024), genetic control measures such as gene drives, Wolbachia-based methods (Verkuijl et al., 2025; Wang et al., 2025) or RNA interference (Müller et al., 2023). Article types Data Report Methods 3.1 Origin of biological material, DNA isolation For DNA and RNA isolation, the offspring of ten females Ae. j. japonicus was collected in the egg stage from the "Narita" laboratory strain (Hoshino et al., 2010) and raised to the desired stages (Figure 1A) for DNA and RNA isolation. A pool of five sisters in the adult stage was used for DNA MinION long read and Illumina short read sequencing, while a single adult female (another sister) was used for PacBio DNA sequencing. DNA was isolated using the protocol 'HMW gDNA Extraction from Single Insects' (10x Genomics, Pleasanton, CA, USA). The fragment size distributions and DNA concentrations were assessed using TapeStation (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer measurements using the DNA BR kit (ThermoFischer Scientific, Waltham, MA, USA). 3.2 DNA sequencing data The Illumina sequencing provider (BGI Hong Kong) handed over already filtered, so called clean, reads in eight pairs. These paired-end read files were adapter-trimmed using autotrim 0.6.1 (Waldvogel et al., 2018) and its dependencies FastQC, Trimmomatic 0.39 (Bolger et al., 2014) and MultiQC (Ewels et al., 2016). After a quality-check, one file pair was additionally cropped to 140 bp length using Trimmomatic 0.39. All trimmed reads were combined into one forward, one reverse (both paired-end), and one unpaired fastq-file. Illumina reads were classified in Kraken 2 (paired-end files with the additional option -paired) using a customized database consisting of the Kraken 2-databases 'bacteria', 'archaea', 'human', and UniVec-Core'. MinION library preparation followed the manufacturer's protocol for the 1D-Ligation Kit (SQK-LSK109) of Oxford Nanopore Technologies (ONT). In total, eight flow cells in three runs were used. ONT-basecalling from fast5-files was conducted with Guppy 3.4.5 (available via registering at https://nanoporetech.com/support) using default settings and these specifications: the flowcell ID, the name of the kit used for library preparation (SQK-LSK109) and the device (--device auto). For the single female, one run on the PacBio Sequel II in CCS mode was performed. The Guppy-basecalling includes adapter trimming and Q-score-filtering. 3.3 RNA sequencing For RNA extractions, 100 eggs, 15 L2 larvae, eight L4 larvae, four pupae, two adult males and two adult females were used, respectively (Figure 1A). Tissue samples were collected in Trizol and extracted using the Zymo RNA Kit (Zymo Research). Eggs, larvae and pupae were pooled for an immature pool. The fragment size distributions and RNA concentrations per pool were assessed using TapeStation (Agilent Technologies) and Qubit Fluorometer with the Qubit RNA HS Kit measurements (ThermoFischer Scientific). Library construction and sequencing on a BGISEQ-500 Illumina platform were done at BGI Hong Kong. Raw RNA Illumina reads were quality-checked and adapter-trimmed using autotrim 0.6.1 (Waldvogel et al., 2018) and its dependencies FastQC, Trimmomatic 0.39 (Bolger et al., 2014) and MultiQC (Ewels et al., 2016). HISAT2 (Kim et al., 2019) was used to map the RNA sequencing reads to the genome assembly. 3.4 Mitochondrial genome Raw PacBio circular consensus sequencing (CCS) reads with adapters were used in NOVOPlasty 4.2 (Dierckxsens et al., 2016) to assemble the mitochondrion of Ae. j. japonicus. For annotations, GeSeq (Tillich et al., 2017) and MITOS2 Galaxy 2.0.6 (Al Arab et al., 2017; Donath et al., 2019) were used. Using Geneious Prime 2021.2.2 (Biomatters Limited), the origin was manually set, the sequence was circularised and the annotations curated manually. 3.5 Genome size estimations We used two in silico genome size estimation methods based on k-mers and read mapping. Jellyfish 2.3.0 (Marçais and Kingsford, 2011) was used to count k-mers in the Ae. j. japonicus Illumina paired-end reads processed by Kraken 2 v2.0.8 (Wood et al., 2019), which were returned as unclassified. The online version of GenomeScope 2.0 (Ranallo-Benavidez et al., 2020) was used to estimate a k-mer based genome size (Supplementary material: Supplementary figure 1). backmap.pl v0.5 (Schell et al., (2017); Pfenninger et al., (2022)); dependencies: bwa 0.7.17-r1188, minimap2 2.29-r1283, samtools 1.20, qualimap 2.2.1, bedtools 2.28.0, multiqc 1.9) was used to estimate the fraction of assembled reads via the mapping rate and for genome size estimation with the ModEst method (Pfenninger et al., 2022). Flow cytometry was used as a sequencing-free method for genome size estimation. Genome sizes for Ae. j. japonicus and Ae. koreicus were estimated following a flow cytometry protocol with propidium iodide-stained nuclei (Hare and Johnston, 2012) in the modification of Männer et al., (2024). We included Ae. koreicus here, since no flow cytometric genome size estimate exists for this species (Supplementary material: Supplementary table 1). One whole adult mosquito was used per suspension and chopped with a razor blade in a petri dish. Two adults per species (one male and one female each, collected as sympatrically occurring pupae on the graveyard Wiesbaden-Kloppenheim on May 27, 2025, and lab-reared to adults) were measured on three consecutive days to minimise instrumental errors. 3.6 Genome assembly, scaffolding, and gap closing A de novo genome was assembled with PacBio CCS reads with the Flye 2.8 assembler (Kolmogorov et al., 2019). We identified the mitochondrial sequence in the Flye assembly using blastn 2.10.0 (Altschul et al., 1990) and respective contigs (>90% target sequence identity and all blast hits per contig >70% contig length) were removed to ensure that the mitochondrion was removed but nuclear mitochondrial DNA segments (NUMTs) kept in the nuclear genome. Subsequently, several rounds of scaffolding and gap closing were conducted (Supplementary material: Supplementary figure 2): The MinION long reads were used to scaffold the Flye assembly using SLR (Luo, 2014). TGS-GapCloser 1.0.1 (Xu et al., 2020) was applied to close gaps by using, first, the PacBio CCS reads, and second, constructed continuous long reads ("CLR" reads) together with Illumina reads. The latter used for polishing the nely added "CLR"-gap sequence inside TGS-GapCloser. "CLR" reads are all PacBio subreads, which were not involved in the generation of a CCS read. They were filtered for the longest per zero-mode waveguide. After this sequence extension, SSPACE (Boetzer et al., 2011) was used to scaffold again using the "CLR" reads, followed by another two step gap closing with TGS-GapCloser using CCS reads as well as "CLR" and Illumina reads as described above. This workflow allowed incorporation of all generated sequencing data (MinION long reads, Illumina short reads, PacBio CCS reads) into the genome assembly (Supplementary material: Supplementary figure 2). Every step of the genome assembly was evaluated regarding quality using QUAST 5.0.2 (Gurevich et al., 2013) and regarding completeness using BUSCO 5.4.6 with the diptera_odp10 gene set in genome mode. The process of gap-closing and scaffolding (Supplementary material: Supplementary figure 2) was checked to ensure that the quality of the resulting assembly did not decrease. 3.7 Structural annotation A reference-based annotation of the Ae. j. japonicus genome was produced using the GeMoMa 1.9 software (Keilwagen et al., 2019), own RNA sequencing data and the Ae. albopictus and Ae. aegypti annotations for reference (GCF_035046485.1, GCF_002204515.2). The annotation of Ae. koreicus (GCA_024533555.2) was additionally included as a third reference in a second GeMoMa run (Supplementary material: Supplementary table 3). In addition, an annotation with BRAKER 3.0.3 (Stanke et al., 2008; Li et al., 2009; Barnett et al., 2011; Lomsadze et al., 2014; Buchfink et al., 2015; Hoff et al., 2016a, 2016b; Brůna et al., 2021) with RNA sequencing data as evidence was computed. BRAKER and GeMoMa annotations for Ae. j. japonicus were compared regarding contiguity statistics calculated with a custom script by author TS (named "contiguity statistics" in Table 1, Supplementary material: Supplementary table 2, Supplementary table 3) and regarding BUSCO 5.4.6 statistics using the protein sequences as input (Supplementary material: Supplementary table 2). Complete and single-copy BUSCO gene IDs unique to the GeMoMa-annotation were extracted and merged with the BRAKER annotation's BUSCO IDs using gff-merge and gff3_to_fasta of the GFF3toolkit 2.1.0. (Chen et al., 2019). Since the merging did not improve the BRAKER-annotation substantially (Supplementary material: Supplementary figure 4, Supplementary Table 2), the latter alone was used for subsequent analyses. 3.8 Functional annotation and detection of integrated virus sequences InterProScan 5.61.93 (Jones et al., 2014) was run with the options [-f tsv -iprlookup -pa -goterms -dp -cpu 54], blastp 2.14.0 with options [-num_threads 70 -max_hsps 1 -max_target_seqs 1 -outfmt 6] against the Swiss-Prot database (The UniProt Consortium et al., 2025), Pannzer2 web version (Törönen and Holm, 2022) and GhostKOALA web version (Kanehisa et al., 2016) were run to functionally annotate the amino acid file of the Ae. j. japonicus BRAKER annotation as well as the annotations of Ae. albopictus and Ae. aegypti for comparison (Supplementary material: Supplementary table 4, Supplementary figure 5). Integration of viral sequences was checked using a published database for endogenous viral elements (Palatini et al., (2020); their additional file 4) identified (tblastn 2.14.0 with options [-max_hsps 1 - max_target_seqs 1 -outfmt 6]; (Altschul et al., 1990)) in the respective Aedes-amino acid file (Supplementary material: Supplementary table 4, Supplementary figure 5). Data analysis 4.1 Mitochondrion The mitochondrial genome is available under the GenBank accession-number MZ566802 and NCBI accession-number NC_081591.1. The total length is 16,848 bp. As of June 25th, 2025, there are seven additional complete mitochondrial sequences of the species available (OP373191.1, OR668893-4.1, PQ588181.1, PV094741-3.1) generated from mosquitoes originating from Italy, Germany, the Netherlands, and Hawaii, USA. Thus, this is the first Ae. j. japonicus mitochondrion from the species' native range (Japan). 4.2 Assembly and genome size estimates An Ae. j. japonicus-assembly was obtained with a total length of 1.2 Gb, a contig N50 of 677 kb, a scaffold N50 of 712 kb, and a total number of 6,029 scaffolds (Figure 1B, Table 1A). The BUSCO protein set was 92.9% complete, with only 1.7% fragmented BUSCOs (Figure 1B). Flow cytometric genome size estimates were 1.3 Gb for Ae. j. japonicus as well as for Ae. koreicus (Supplementary material: Supplementary table 1). The latter being in line with the size of the Ae. koreicus genome (1.1 Gb; Supplementary material: Supplementary table 1; Nagy et al., (2024)). The k-mer-based estimate of Ae. j. japonicus was 695 Mb length and the mapping-based estimate was best performing, regarding peak shape, with mapped CCS reads. The mapping-based genome size estimate was 1.2 Gb (Supplementary material: Supplementary figure 3). This compilation of genome size estimates can facilitate calculations for genome coverage and sequencing costs for further projects. 4.3 Structural and functional annotations The annotation with BRAKER resulted in 23,878 predicted protein-coding genes with a median length of 2,027 bp. Protein sequences of the predicted genes showed a BUSCO completeness of 91.4% (Table 1B). Among the protein-coding genes, 99% (28,458 genes) could be functionally annotated with at least one of the applied methods but GO terms could be found for 60% of the sequences (Supplementary material: Supplementary table 4). 4.4 Comparisons to other Aedes-genomes In comparison to other Aedes-genomes, the size of the nuclear genome assembly of Ae. j. japonicus is comparable to others within Aedes (Supplementary material: Supplementary table 1). The Ae. j. japonicus-assembly has slightly better statistics compared to the publicly available assembly (GCA_034211315.2) regarding continuity and BUSCO completeness (Table 1A). The GC-content is the same as the GCA_034211315.2 assembly and comparable to the sister species Ae. koreicus (Table 1A). For the three Aedes-species, a comparable amount (60% to 70%) of integrated virus sequences could be detected (Supplementary material: Supplementary table 4, Supplementary figure 5). The slightly lower number of viruses that could be recovered in the Ae. japonicus annotation is explainable by the lower quality of the scaffold-level Ae. j. japonicus-genome compared to the chromosome-level genomes of Ae. albopictus and Ae. aegypti or the selection of the input virus database. A biological reason could be species-specificity of viral integrations. Dataset usage and availability 5.1 Dataset re-use potential The dataset presented here could be used in subsequent analyses regarding phylogeny, evolution of diapause and invasiveness, adaptation to non-native habitats, and the search for genetic targets of vector control measures. It is the first time, that individuals from the native range of Ae. j. japonicus were sequenced (nuclear and mitochondrial genomes) allowing comparative studies regarding differences between native and invasive populations of the species. Differences could occur due to adaptation to the new environment during the invasion process. Important phenotypic traits such as diapause, heat tolerance or insecticide resistance could be altered during invasion. The dataset presented here also fills a gap of knowledge regarding comparative studies between well-studied primary (Ae. aegypti, Ae. albopictus) and understudied secondary (Ae. j. japonicus, Ae. koreicus) vector species regarding their different competences for arboviral transmission. 5.2 Dataset availability All data of this project was made publicly available: This project was registered under the BioProject number PRJNA1085103 at NCBI. There, raw data, the assembly, and the RNA sequencing data can be found. The mitochondrial genome is available under GenBank accession-number MZ566802. The genome annotation with the corresponding assembly, and the amino acid sequence file can be found at the Goethe University Data Repository (GUDe; https://gude.uni-frankfurt.de/home) under the DOI https://doi.org/10.25716/gude.12xf-dt1*. 5.3 Figures and tables 5.3.1 Figure 1 caption (A) Biological material for DNA and RNA isolation. We used closely related (offspring of one female) individuals for DNA isolation to minimise variation. (B) Snail plot of statistics of the Ae. j. japonicus assembly. 5.3.2 Table Genome assembly (A) and annotation statistics (B) of selected Aedes spp.-genomes. Calculations of contiguity statistics by a custom script. CDS: coding exon regions. Total gene space: sum of all nucleotides that are annotated as a gene. Single CDS mRNA: Number of mRNAs that only have a single coding exon. (A) Assembly statistics Aedes-species j. japonicus This study japonicus GCA_0342 11315.2 koreicus GCA_0245335 55.2 albopictus GCF_035046 485.1 aegypti GCF_0022045 15.2 Quast No. scaffolds 6,029 25,235 6,100 1,497 2,310 1,185,987,502 1,389,713,0 1,100,040,858 1,344,164,50 1,278,732,104 712,605 118,241 329,610 450,188,506 409,777,670 999.63 199.05 2.82 125.87 1.79 6,744 25,703 6,127 6,007 2,539 1,174,131,623 1,386,947,0 1,100,009,795 1,342,452,19 1,278,709,169 677,340 112,964 329,031 1,015,000 11,758,062 39.44 39.50 39.67 40.33 38.18 92.9 92.4 84.0 95.7 96.7 83.5 78.8 70.7 90.7 93.4 9.4 13.6 13.3 5.0 3.3 1.7 2.4 2.7 1.6 1.6 5.4 5.2 13.3 2.7 1.7 23,878 21,377 23,630 18,293 No. mRNA 28,836 22,580 33,058 28,304 No. CDS 120,432 87,069 171,395 173,240 Mean mRNAs/gene 1.21 1.06 1.40 1.55 Mean CDSs/mRNA 4.18 3.86 5.18 6.12 Median gene length 2,027 2,068 4,392 5,172 Median mRNA length 2,223 2,081 14,453 29,581 Median CDS length Total gene space 407,892,850 144,700,806 738,863,314 683,632,137 Total mRNA space 407,892,850 144,698,715 702,543,701 669,443,925 Total CDS space 28,457,713 24,159,712 38,510,628 24,237,954 Single CDS mRNA 4,854 3,931 5,908 2,031 %BUSCO (n=3,285) Complete 91.4 81.2 98.5 99.4 Single-copy 62.3 48.7 61.8 60.5 Duplicated 29.1 32.5 36.7 38.9 Fragmented 2.8 2.8 0.2 0.2 Missing 5.8 16.0 1.3 0.4