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¹ Laboratorio de Investigación en Genética Evolutiva - LIGE., Universidad Pedagogica y Tecnologica de Colombia, Tunja, Boyaca, Colombia
² Grupo de Estudios en Genética y Biología Molecular - GEBIMOL, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Boyacá, Colombia
³ Grupo de Investigación en Sistemática Molecular - GSM, Universidad Nacional de Colombia, Medellín, Colombia
⁴ Centro de Investigación La Selva, Corporación Colombiana de Investigación Agropecuaria, AGROSAVIA, Rionegro, Antioquia, Colombia
⁵ Universidad Nacional de Colombia, sede Bogotá, Facultad de Ciencias, National University of Colombia Natural Sciences Institute, Bogotá, Bogota, Colombia

Andrés Gómez-Palacio
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Sandra Uribe-Soto
Roles: Conceptualization, Funding Acquisition, Investigation, Project Administration, Supervision, Writing – Review & Editing

Carolina Ortiz-Muñoz
Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Dimitri Forero
Roles: Conceptualization, Data Curation, Funding Acquisition, Investigation, Resources, Supervision, Writing – Review & Editing

Specimens of the assassin bug genus Apiomerus Hahn (Hemiptera: Reduviidae) were collected in Colombia between 2018 and 2025 through active daytime manual searches on vegetation in the departments of Antioquia, Casanare, and Santander, at elevations ranging from 184 to 2369 m a.s.l. Geographic coordinates were recorded for all sampling localities.

Morphological identification was based on external diagnostic characters, particularly hemelytral colour patterns, together with examination of male and female genitalia. Species determinations followed the taxonomic treatments and original descriptions of Forero and Weirauch,¹² Gil-Santana et al., ¹³ and Carl Stål,^14,15 and were performed by Dr Dimitri Forero.

The ethanol-preserved specimen used for the mitogenome of Apiomerus ochropterus (voucher UNAL:MEFLG_Apio093; female) was collected on 13 January 2025 in Reserva Forestal El Centello, Jardín, Antioquia, Colombia (5°30′25.729″N, 75°50′50.262″W; 2369 m; cloud forest), and is deposited in the Francisco Luis Gallego Museum, Universidad Nacional de Colombia.

Additional dry-pinned specimens processed for genomic analyses included: Apiomerus nitidicollis (voucher ICN:ICN111295; female), collected on 4 December 2024 in Reserva Natural de la Sociedad Civil San Juan de Tinije, vereda Palmarito, Maní, Casanare (4.8508°N, 72.3719°W; 184 m; gallery forest edge), and deposited in the Instituto de Ciencias Naturales (ICN), Universidad Nacional de Colombia; Apiomerus luctuosus (voucher ICN:ICN111161; female), collected on 8 December 2024 in vereda Las Atalayas, hacienda Pajonales, sitio Cunaviche, Aguazul, Casanare (5.0851°N, 72.5040°W; 242 m; piedmont terra firme forest), and deposited in ICN; and Apiomerus sp. (voucher IAvH:IAvH-E-206069), collected on 21 February 2018 in vereda La Belleza, Carmen de Chucurí, Santander (6°34′11.5″N, 73°34′12.3″W; 847 m; sandy forest), and deposited in the Instituto Humboldt (IAvH).

Fresh specimens were euthanized by freezing and preserved either dry or in 96% ethanol. For each specimen, one leg was dissected and used for DNA extraction. Voucher specimens are curated in publicly accessible institutional collections, ensuring long-term preservation and traceability of the genomic data.

Prior to DNA extraction, each dissected leg was briefly surface-cleaned to reduce potential contamination from external tissues and environmental material. Total genomic DNA was extracted from a single leg of each adult specimen using the DNeasy Blood & Tissue Kit (Qiagen, Germany; cat. no. 69504), following the manufacturer’s instructions with minor modifications for insect tissue. Each extraction included 180 μL Buffer ATL and 20 μL Proteinase K, followed by digestion at 56 °C. For Apiomerus sp., A. luctuosus, and A. nitidicollis, the digestion step lasted 3 h, and DNA was eluted in 100 μL Buffer AE. For A. ochropterus, digestion was extended to 4 h, and DNA was recovered through two sequential elutions of 25 μL each to maximize yield.

DNA concentration and purity were quantified using a NanoDrop 2000/2000c spectro-photometer (Thermo Fisher Scientific, USA; cat. No. ND-2000) and verified by 1% agarose gel electrophoresis in 1× TAE buffer (Invitrogen, USA; cat. No. 15558–042) with GelRed stain (Biotium, USA; cat. No. 41003). Only samples with concentrations ≥1 ng μL⁻¹ and 260/280 and 260/230 ratios between 1.8 and 2.0 were used for sequencing.

High-quality DNA extracts were selected for shallow whole-genome sequencing (approximately 7× coverage). Paired-end libraries (2 × 150 bp, ~350 bp insert size) were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, USA; cat. No. E7645L) according to the manufacturer’s protocol, including size selection with AMPure XP magnetic beads (Beckman Coulter, USA; cat. No. A63881). Library concentrations were determined using a Qubit 4 Fluorometer and the Qubit dsDNA HS Assay Kit (cat. No. Q32854). Libraries were pooled equimolarly and sequenced on an Illumina NovaSeq X platform (Illumina, USA) at Macrogen Inc. (Seoul, South Korea), generating approximately 8–10 Gb of raw paired-end data per sample, corresponding to an estimated genomic coverage of ~7 ×.

Raw paired-end Illumina reads were quality-filtered and adapter-trimmed using fastp v0.23.4.¹⁶ The resulting reads were initially assembled de novo with SPAdes v3.15.5¹⁷ using default parameters for short-read data. Contigs of putative mitochondrial origin were identified by BLASTN comparison against the reference mitogenome of Agriosphodrus dohrni (Harpactorinae; NCBI Reference Sequence NC_015842.1). These SPAdes-derived mitochondrial reconstructions were retained as an independent assembly for comparison with the organelle-specific assemblies described below.

The filtered paired-end reads were subsequently reassembled using GetOrganelle v1.7.7.1¹⁸ in animal mitochondrial mode (-F animal_mt), with the A. dohrni mitogenome supplied as the seed sequence. Assemblies were generated using k-mer sizes of 21, 45, 65, 85, and 105. Sample-specific word sizes and extension-round limits were used to accommodate differences in mitochondrial read coverage and assembly-graph complexity: a word size of 80 and a maximum of 30 extension rounds were used for A. ochropterus; a word size of 100 and 10 extension rounds for A. nitidicollis; a word size of 90 and 20 extension rounds for A. luctuosus; and an automatically estimated word size and 10 extension rounds for Apiomerus sp. Assembly graphs were examined to distinguish closed circular paths from unresolved or linear scaffolds.

The selected GetOrganelle assemblies were compared reciprocally with the original SPAdes/BLASTN-derived mitochondrial reconstructions using BLASTN. Comparisons included nucleotide identity, query coverage, alignment continuity, total assembly length, and the presence of duplicated, additional, or unresolved terminal regions.

The original filtered paired-end reads were independently mapped to the SPAdes/BLASTN-derived and GetOrganelle assemblies using Minimap2 v2.28-r1209.¹⁹ Alignment files were processed using SAMtools v1.18,²⁰ and assembly support was evaluated from coverage breadth at ≥1× and ≥10×, read-depth distributions, depth uniformity, and the number of properly paired reads. For each putatively circular GetOrganelle assembly, reads were additionally mapped across a reconstructed circular-junction reference to test whether coverage and paired-read support continued uninterrupted across the sequence boundary. The A. luctuosus assembly was retained as a non-circular scaffold and was therefore not subjected to circular-junction validation. An assembly was classified as circular only when GetOrganelle recovered a closed path without ambiguous bases and the reconstructed junction was supported by continuous read coverage and properly paired reads.

Initial mitochondrial genome annotation was performed with MITOS2²¹ through the Galaxy server. Annotations were subsequently refined by comparison with previously published reduviid mitogenomes and by manual curation of gene boundaries, start and stop codons, and tRNA predictions. Transfer RNA annotations were independently evaluated using tRNAscan-SE v2.0.12²² in organellar mode, and discrepant predictions were resolved through comparison of genomic coordinates, anticodons, predicted secondary structures, and conserved mitochondrial gene order. Circular mitochondrial genome plots and comparative figures were generated in R²³ using circlize v0.4.17²⁴ package.

Phylogenetic relationships were inferred using the concatenated amino acid sequences of the 13 mitochondrial protein-coding genes (PCGs: atp6, atp8, cox1–3, cob, nad1–6, and nad4l) from four newly assembled mitogenomes of Apiomerus and 22 representative Harpactorinae species retrieved from GenBank. To ensure balanced taxonomic representation and avoid redundancy, a single complete mitochondrial genome accession was selected per species based on BLAST screening and manual curation of available records. The final dataset included representative taxa such as Epidaus famulus (NC_085748.1), Polididus armatissimus (NC_069625.1), Rhynocoris altaicus (PQ613804.1), R. fuscipes (MZ440304.1), Scipinia horrida (NC_037744.1), Sclomina erinacea (ON116856.1), and S. guangxiensis (ON116892.1). The Triatominae species Triatoma infestans (KY640305.1) was included as outgroup.

Annotated GenBank records were downloaded using NCBI Entrez Direct utilities, and the 13 mitochondrial PCGs were extracted based on annotated coding sequence (CDS) features using a custom Python script. Gene names were standardized across annotations (e.g., cox1/coi, cob/cytb, nad/nd synonyms), and only the first occurrence of each PCG per genome was retained. Amino acid translations were generated from extracted CDS sequences and aligned independently for each gene using MAFFT v7.525^25,26 under the automatic algorithm selection strategy.

Individual gene alignments were concatenated into a supermatrix using a custom Python script, and a partition file was generated assigning each PCG as an independent data block. The final amino acid matrix comprised 27 taxa (four Apiomerus species, 22 Harpactorinae representatives, and one outgroup). Maximum likelihood analyses were conducted in IQ-TREE v2.0.3²⁷ under partitioned model selection (MFP+MERGE). Branch support was assessed with 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT replicates.

Sequencing generated 14.3–21.1 million raw paired-end reads per specimen (Table 1). After quality filtering and adapter trimming, 14.1–20.9 million reads were retained, corresponding to retention rates of 98.87–99.05%. A. luctuosus yielded the largest number of clean reads, whereas A. nitidicollis yielded the fewest. The filtered reads were subsequently used for SPAdes-based de novo assembly and GetOrganelle mitochondrial genome reconstruction.

Reciprocal whole-sequence comparisons showed that the shared regions of the original SPAdes/BLASTN-derived reconstructions and the GetOrganelle assemblies had 95.02–99.97% nucleotide identity. Relative to the earlier sequences, the GetOrganelle assemblies were 1,503 bp longer for A. ochropterus, 2,422 bp longer for A. nitidicollis, and 3,302 bp longer for Apiomerus sp., whereas the A. luctuosus scaffold was 994 bp shorter ( Figure 1a). The GetOrganelle assemblies contained 93.76–100% of the earlier sequences, whereas the earlier reconstructions represented only 80.65–100% of the GetOrganelle assemblies. Most differences therefore involved terminal, control-region, or previously unresolved portions rather than extensive divergence across the shared mitochondrial sequence.

Read mapping supported the selected GetOrganelle assemblies. Coverage breadth ranged from 99.28% to 100% at ≥1× and from 97.24% to 100% at ≥10× (Figure 1b). Read depth was more uniform in the GetOrganelle assemblies of A. nitidicollis, A. luctuosus, and Apiomerus sp., whereas A. ochropterus showed a modest increase in depth variability. Properly paired-read support increased for the GetOrganelle assemblies of A. ochropterus, A. nitidicollis, and Apiomerus sp., but decreased for the shorter A. luctuosus scaffold.

The three circular assemblies showed uninterrupted read coverage across their reconstructed junctions, with no zero-coverage positions within the evaluated windows ( Figure 1c). Mean junction depths were 250.9× for A. ochropterus, 2,816.0× for A. nitidicollis, and 136.8× for Apiomerus sp. The corresponding junctions were supported by 31, 1,506, and 77 properly paired reads, respectively. Because the A. luctuosus assembly remained non-circularized, it was evaluated through terminal-coverage analysis rather than circular-junction validation.

The GetOrganelle assemblies selected after comparative and read-based validation measured 17,410 bp in A. ochropterus, 19,405 bp in A. nitidicollis, 14,943 bp in A. luctuosus, and 16,944 bp in Apiomerus sp., with A+T contents of 72.36%, 73.27%, 70.62%, and 70.25%, respectively (Table 2; Figure 2a). Complete circular mitogenomes were recovered for A. ochropterus, A. nitidicollis, and Apiomerus sp., whereas A. luctuosus was represented by a non-circularized scaffold in which the control region was incompletely recovered. Nevertheless, all four assemblies contained 13 protein-coding genes, 22 tRNA genes, and two rRNA genes.

After annotation refinement and manual curation, the four assemblies showed complete conservation of mitochondrial gene content, gene order, and transcriptional orientation, with no detected rearrangements of protein-coding genes, rRNAs, or tRNAs (Figure 2b). Differences among the assemblies were therefore associated primarily with genome length, control-region recovery, and intergenic spacing rather than with changes in mitochondrial gene organization.

Phylogenetic relationships inferred from concatenated mitochondrial PCGs recovered Apiomerus as a monophyletic group (Figure 2c). Within the genus, A. ochropterus and Apiomerus sp. were recovered as sister taxa, A. nitidicollis was sister to that clade, and A. luctuosus occupied the basal position among the sampled Apiomerus taxa. The broader tree also recovered the other highlighted harpactorine genus-level clades shown in Figure 2c. Bootstrap values were generally high, although support for the A. ochropterus + Apiomerus sp. node was moderate.

We thank all members of the AGROSAVIA and Universidad Nacional de Colombia field teams who assisted with sample collection.

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Gómez-Palacio A, Uribe-Soto S, Ortiz-Muñoz C and Forero D. Mitochondrial Genome Assemblies of Four “Bee-Assassin” (Hemiptera: Reduviidae: Apiomerus) Recovered Using Shallow Whole-Genome sequencing [version 2; peer review: 2 approved, 2 approved with reservations]. F1000Research 2026, 15:921 (https://doi.org/10.12688/f1000research.179613.2)

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Current Reviewer Status: ?

VERSION 2

PUBLISHED 26 Aug 2026

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Feiyun Tu, Hainan Normal University, Haikou, Hainan, China 

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Carlos Eduardo Almeida, Universidade Federal de São João del Rei, Campus Centro-Oeste “Dona Lindu”, Divinópolis, Brazil 

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PUBLISHED 12 Jun 2026

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Carlos Eduardo Almeida, Universidade Federal de São João del Rei, Campus Centro-Oeste “Dona Lindu”, Divinópolis, Brazil 

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Rui Alves, University of Lleida, Lleida, Spain 

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Annabel Whibley, The University of Auckland, Auckland, Auckland, New Zealand 

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