Characterization of the complete mitochondrial genome of five scale insects (Hemiptera: Coccoidea; Coccidae) and they phylogenetic implications

The soft scales (Hemiptera: Coccidae) represent one of the most diverse invasive groups of sap-sucking parasites, many of its members have significant impact on agriculture and forestry. Identifying soft scales based on morphology alone is extremely challenging and requires significant expertise. Accurate species identification requires the integration of both morphological and molecular data. Despite advancements in molecular techniques and the widespread application of mitochondrial genome in species identification and phylogenetic studies, only a limited number of Coccidae family representatives are available on NCBI. In this study, we sequenced the complete mitogenomes of four scale insect species for the first time and conducted a comparative analysis with existing Coccidae mitochondrial genomes. Saissetia coffeae was resequenced to improve data quality. The five newly sequenced species exhibited circular mitochondrial structures consistent with other members of Coccidae, along with a strong A+T bias and the ancestral insect gene order. Our analyses reveal conserved mitogenome organization, gene content, codon usage, AT bias, and tRNA secondary structures across the family. Phylogenetic reconstructions using both Bayesian Inference and Maximum Likelihood, based on multiple combinations of the 37 mitochondrial genes, consistently recovered the monophyly of Coccoidea. The newly sequenced species formed a stable clade in all analyses, supporting current taxonomic classifications. Characterization of the complete mitochondrial genome of five scale insects (Hemiptera: Coccoidea; Coccidae) and their phylogenetic implications Min Deng 1,2, Muhammad Asghar Hassan 1,3, Riaz Hussain 1,4 and Jichun Xing 1,5,* 1 Institute of Entomology, Guizhou University; The Provincial Special Key Laboratory for Development and Utilization of Insect Resources, Guizhou University; Guiyang, 550025 P. R. China https://orcid.org/0009-0009-4364-802X https://orcid.org/0000-0003-2590-5781 https://orcid.org/0000-0003-3864-7996 *Correspondence author: [email protected]

The soft scales (Hemiptera: Coccidae) represent one of the most diverse invasive groups of sap-sucking parasites, many of its members have significant impact on agriculture and forestry. Identifying soft scales based on morphology alone is extremely challenging and requires significant expertise. Accurate species identification requires the integration of both morphological and molecular data. Despite advancements in molecular techniques and the widespread application of mitochondrial genome in species identification and phylogenetic studies, only a limited number of Coccidae family representatives are available on NCBI. In this study, we sequenced the complete mitogenomes of four scale insect species for the first time and conducted a comparative analysis with existing Coccidae mitochondrial genomes. Saissetia coffeae was resequenced to improve data quality. The five newly sequenced species exhibited circular mitochondrial structures consistent with other members of Coccidae, along with a strong A+T bias and the ancestral insect gene order. Our analyses reveal conserved mitogenome organization, gene content, codon usage, AT bias, and tRNA secondary structures across the family. Phylogenetic reconstructions using both Bayesian Inference and Maximum Likelihood, based on multiple combinations of the 37 mitochondrial genes, consistently recovered the monophyly of Coccoidea. The newly sequenced species formed a stable clade in all analyses, supporting current taxonomic classifications. Keywords. Coccidae, scale insect, mitogenome comparison, systematics

Soft scale insects or coccoids (Coccoidea: Coccidae) are one of the third largest species-rich family in the infraorder Coccomorpha after armored scales (Diaspididae) and the mealybugs (Pseudococcidae), with approximately 1254 described species under 180 genera. They are distributed worldwide except Antarctic, and are one of the most diverse invasive groups of sap-feeders, comprises of numerous well-known pests that cause serious damage to agricultural crops and forest tress (García et al ., 2016). The morphological identification of scale insects is highly challenging and requires significant expertise. Accurate species identification requires the integration of both morphological and molecular datasets. Morphological identification is primarily based on microscopic cuticular characters in adult females. The morphology of scale insects exhibits sexual dimorphism: male adults are divided into head, thorax and abdominal segments, which most species have a pair of wings, genital sheath, and developed legs. In contract, adult females have a fused head and thorax, no wings, and an oval shaped body, typically ranging from 3-9 mm in length, the body size is small, with a few species, such as Eulecanium gigantea, reaching up to 15 mm. Due to the short lifespan of adult males, very few such individuals are collected during field investigations (Tang, 1991). Although, there have been limited research on the molecular phylogeny of scale insects. Only eight complete mitochondrial genome of the family Coccidae are currently available on NCBI. This study focuses on the comparative analysis of newly sequenced and previously known mitogenomes within the family Coccidae, including the complete mitochondrial genome of Coccus formicarii, Megalocryptes buteae, Marsipococcus maolanensis, Saissetia coffeae, Bambusaecoccus maolanensis, all from China. Furthermore, a phylogenetic analysis based on different combinations of 37 mitochondrial genes were conducted to reconstruct the relationships within Coccidae family.

2.1 Specimen material, genomic DNA extraction, and sequencing Scale insects are suitable for growing in an environment with warm and humid climate, diverse landform, complex surface composition and soil types, and rich vegetation types. In nature, the external body morphology, colour, size and shape of soft scales can be quite distinct from other insects varydepending on the specific group (Hodgson, 1994). Both nymphs and adult soft scales feed on plants and are found in almost all parts of the host, however they are most commonly found on leaves and stems (García et al ., 2016). Different developmental stages of both males and females of Coccus formicarii were found under carton coverings made by the ant Crematogaster subnuda nigrosubnuda Özdikmen, 2010 (Hymenoptera: Formicidae) on the stem nodes of Callicarpa bodinieri Léveillé, 1912 (Angiospermae: Lamiaceae). The ants, which were feeding on the honeydew excreted by the scale insects, constructed a tent-like carton cover to protect them from predators and parasites. Adult females of Coccus formicarii, Megalocryptes buteae and were collected on January 7, 2024 at Maolan National Nature Reserve, (25°32′67″N, 107°98′03″E, 741.10 m altitude, collected by Min Deng, Shitao Meng and Qing Liu) in Yongkang Town, Libo County, Guizhou Province, China; adult females of Bambusaecoccus maolanensis were collected on January 7, 2024 at Maolan National Nature Reserve, (25°29′23″N, E:107°93′76″, 725.2 m altitude, collected by Min Deng and Shitao Meng) in Yongkang Town, Libo County, Guizhou Province, China; adult females of Saissetia coffeae were collected on January 7, 2024 in Libo County, 25°41′91″N, E: 107°88′43″, 410.7m altitude, collected by Min Deng and Shitao Meng) in Libo County, Guizhou Province, China; adult females and males of Marsipococcus maolanensis were collected on July 11, 2024 at Maolan National Nature Reserve, (25°20′73″N, E:107°94′72″, 706.7 m altitude, collected by Min Deng and Shangmi Hu) in Libo County, Guizhou Province, China (Figure 1). One to twelve intact female adult samples were collected and sent to Beijing Qingke Biological Technology Co. for extraction and sequencing of mitogenome. The remaining samples were stored in anhydrous ethanol and maintained at the temperature of -20°C. The quantity and quality of the extracted genomic DNA was detected by using Qubit and Agilent 5400, after that, Using TruSeq® Nano DNA Library Prep Kit to constructed library(Illumina, USA). Qubit 3.0 was utilized for primary quantification of the constructed library, and the library was then diluted. Library insert fragment were characterized by using Agilent 2100 Bioanalyzer. Following confirmation of expectations, the library’s effective concentration was precisely determined using quantitative Polymerase Chain Reaction (QPCR). The qualified libraries were sequenced using Illumina NovaSeq. After sequencing, quality control was executed on the results, and the next generation analysis was carried out after passing the test. Raw sequencing reads were processed using Trimmomatic to remove adapter sequence (Bolger et al ., 2014). The complete mitogenome of C. formicarii was sequenced using the Illumina Novaseq 6000 sequencing platform with an average insert size of 350 bp and a paired-end 150 bp in length. The average insert length was determined to be 350 base pairs, and a total of 6 GB of high-quality clean data was generated. 2.2 Mitochondrial genome sequence Prediction, assembly, annotation, and analyses Clean sequence mitogenome of 4 species ( Coccus formicarii, Megalocryptes buteae Bambusaecoccus maolanensis, Marsipococcus maolanensis ) were assembled with the reference mitogenome of Didesmococcus koreanus (NC057479) and 1 species ( Saissetia coffea ) were assembled with the reference mitogenome of Coccus hesperidum (NC085772), using the NOVOPlasy v2.7.0 (Dierckxsens et al., 2017) and GENEIOUS (v. 11.2.3) (Kearse et al., 2012). A comparison with the published mitotic genome of the family Coccidae confirmed the presence of two ribosomal RNA (rRNA) and 13 protein-coding genes (PCGs) of 5 species in the parasite. Using the MITOS web server to annotated the assembled sequences (http://mitos. bioinf. unileipzig.de/index.py; Bernt et al., 2013). The location and secondary structure of 22 tRNA genes were retrieved from the MITOS web server and which were then graphically illustrated in Adobe Photoshop CS 6.0 and Adobe Illustrator 2023. The genomic annotation and circular map were generated using Proksee (https://proksee.ca/projects/new; Grant & Stothard, 2008). Molecular Evolutionary Genetics Analysis v. 7.0 is used to compute base composition. AT and GC skew were calculated using the formulas of AT-skew = [(A − T)/(A + T)] and GC-skew = [(G − C)/(G + C)](Perna & Kocher, 1995). 2.3 Molecular phylogenetic analyses The phylogenetic relationships within the family Coccidae were analyzed using BI and ML analysis methods. by using different combinations of 37 mitochondrial genomes. This analysis included 11 species belonging to four subfamilies available on NCBI, withnewly sequenced data of 5 species as ingroup, and two outgroup species belonging to the family Aclerdidae (Table 1). All complete mitogenomes of ingroup and outgroup were obtained using PhyloSuite (v1.2.2; Zhang et al., 2020). The nucleotide and amino acid sequences of the 13 PCGs were arranged using Multi-Sequence Comparison by Log-Extraction MUSCLE (Edgar, 2004) in Molecular Evolutionary Genetics Analysis MEGA 8.0 (Kumar et al., 2016), with stop codons were carefully removed. RNA gene sequences were alignment with the help of Multiple Alignment using Fast Fourier Transform MAFFT and then trimmed with trimAl. After that, we use PhyloSuite to assemble the permutation sequences of each gene to create into four datasets: (1) Amino acid sequences of 13 PCGs; (2) Nucleotide sequences of 13PCGs; (3) Nucleotide sequences of 13PCGs and two rRNA genes; (4) 37 mitochondrial genes. The aligned sequences then converted into phylip and nexus formats using PhyloSuite (v1.2.3) for subsequent ML and BI analyses. The phylogenetic analysis was performed using the ML and BI methods. ML analyses was conducted in IQ-TREE (Nguyen et al., 2015) with 5000 ultrafast bootstrap replicates as implemented on the webserver (http://iqtree.cibiv.univie.ac.at/), and the BI analyses was performed using MrBayes v.3.2.6 (Ronquist et al., 2012) via PhyloSuite software with various partition schemes and best-fitting models determined by PartitionFinder (Lanfear et al., 2017; Tables S1–S9). The BI analyses were based on MCMC (Markov Chain Monte Carlo) sampling, with four simultaneous runs of 2 million generations and discarding the initial 25% generations as burn-in. All trees were visualized and edited using the Interactive Tree of Life (iTOL v) (Letunic & Bork, 2021). The mitogenome organization, gene composition, codon usage, AT content, and the secondary structures of tRNA in Coccus formicarii and Marsipococcus maolanensis were thoroughly analyzed and reported. Furthermore, phylogenetic trees inferred from concatenated sequences based on different combinations of 37 mitochondrial genes and reconstruct the relationships within the family Coccidae.

3.1 Mitochondrial genome structure and nucleotide composition In this study, we sequenced, annotated, assembled and analyzed the complete mitochondrial genome of 5 species including the mitogenome organization, gene composition, codon usage, AT content, and the secondary structures of tRNA. The mitochondrial genomes of the five scale insect species range in length from 15,479 to 17,089 base pairs (bp), and all consist of 37 mitochondrial genes and a control region (Table 2 − 6). The complete circular mitogenome map with gene arrangement of the five scale insect species are shown in Figure 2. The nucleotide composition (A+T content) of the mitochondrial genome for each species is as follows: C. formicari i, 83.5% (A=51.90%, T=31.50%); M. buteae, 86.5% (A=44.10%, T=42.40%); S. coffeae, 80.7% (A=49.40%, T=31.30%); B. maolanensis, 83.4% (A=51.90%, T=31.50%); and M. maolanensis, 90.5%. Correspondingly, the G+C content is 16.5% (C=5.50%, G=11.00%) for C. formicarii, 13.5% (C=9.20%, G=4.30%) for M. buteae, 19.3% (C=11.30%, G=8.00%) for S. coffeae, 16.6% (C=12.50%, G=4.10%) for B. maolanensis, and 9.5% for M. maolanensis . The genomic nucleotide composition in isolated PCGs, tRNAs, rRNAs, and control region all exhibits an A + T content above 80% (Table 7). The secondary structures of tRNAs for Coccus formicarii and Marsipococcus maolanensis are presented in Figure 3. The taxonomic status and phylogenetic relationships, along with the gene order of each species are presented, including a comparison of mitochondrial gene arrangements among Coccidae and two outgroup species (Figure 4 and 5). The four gene clusters of all species (cox1-trnL2-cox2-trnK-trnD, atp8-atp6, nad5-trnH-nad4-nad4L, and nad1-rrnL-trnV-rrnS) were relatively conserved. Most of the PCGs use TAA as termination codon except for nad4 and nad5 ending with codon AAT. Codon usage and the relative synonymous codon usage (RSCU) of four adult females and one adult male were calculated and summarized in Figure 6. Comparison of Coccidae the mitochondrial gene arrangement among Coccidae and three outgroup in Figure 7. 3.2 Phylogenetic analyses We discussed the internal phylogentic relationships in the family Coccidae based on 76 species as ingroup: Psyllidae (5 spp.), Aleyrodidae (5 spp.), Aphididae (4 spp.), Margarodidae (1 spp.), Pseudococcidae (1 spp.), Coccidae (11 spp.), Aclerdidae (2 spp.), Peloridiidae (3 spp.), Cicadellidae (3 spp.), Delphacidae (2 spp.), Issidae (1 spp.), Fulgoridae (1 spp.), Cicadidae (1 spp.), Cercopidae (2 spp.), Aradidae (5 spp.), Veliidae (1 spp.), Gerridae (4 spp.), Saldidae (1 spp.), Reduviidae (1 spp.), Anthocoridae (1 spp.), Miridae (2 spp.), Nabidae (4 spp.), Cydnidae (1 spp.), Pentatomidde (5 spp.), Rhyparochromidae (1 spp.), Lygaeidae (1 spp.), Malcidae (2 spp.), Alydidae (1 spp.), Coreidae (3 spp.), and three species as outgroup: Thripidae (1 spp.), Stenurothripidae (1 spp.), Aeolothripidae (1 spp.). The complete newly mitochondrial genome of 5 species including the newly sequenced data of fives scale insects were retrieved from NCBI. The phylogenetic trees were reconstructed using four datasets and two analysis methods. Eight phylogenetic trees were obtained based on BI and ML analyses. Among these, the best tree was selected with identical topologiesand high nodal support values for the subfamily Eulecaniinae as the basal clade and sister to the remaining Coccidae subfamilies. Our phylogenetic analysis found that the four subfamilies within the three clades are recovered as monophyletic with high support values in both BI and ML analyses, which are usually in accordance with previously published studies (Figures 4 and 5; Hou et al ., 2023). The topological structure and species position of the phylogenetic tree might be affected by high A+T content and poorly conserved PCGs. The trees generated from the following database: PCG_AA、13 PCGs、PCG123-2rRNA、and PCG123-2rRNA-22tRNA were identical, which clustered into monophyletic clades with high support values.

With the advancement of science and technology, high-throughput sequencing (HTS) has become more prevalent. This has resulted in a significant number of researchers providing high-throughput sequencing data for insect mitochondrial genomes, which has laid a good foundation for the study of insect phylogeny and provided more scientific reference value (Cameron, 2014). With this good background, we apply modern technology to our research, in this study, we increasing the scale insect mitogenomes available in GenBank, and newly sequenced the mitogenome of 5 species, which belonging to the tribe Coccini in the subfamily Coccinae. Consistent with other scale insects, the 5 species mitogenome contains a typical set of 37 genes, comprising 13 PCGs, 22 tRNAs and two rRNAs, this result is in agreement with those obtained by predecessors, no special arrangement was added (Lu et al ., 2020; Lu et al ., 2023; Hou et al ., 2023 ). Cameron (2014), Li et al . (2017), Zhang et al . (2021), and Hou et al . (2023) in previous published mitogenomic studies of scale insects with the conclusion that nucleotide bias is a common phenomenon, is consistent in that we found a significant A+T bias in the 5 species. Oliveira et al . (2008) and Lu et al . (2023) point out that the high A+T in scale insects may be related tothe nature of their host plants and their parasitic lifestyle in their study, there may be other complex causes affecting it that have yet to be discovered and confirmed. Codon usage analysesin scale insects from our study found that TTA (Leu 2 ) was the most frequently use codon, while CTT (Leu 1 ) was the least used. Regarding tRNA secondary structures in scale insects, some exhibit a typical clover leaf structure, while some lack DHU or T-arms, resulting in truncated structures, presenting an incomplete clover structure. This phenomenon is consistent with previous research on the mitochondrial genomic studies of scale insects (Lu et al. 2023; Xu et al. 2023). Based on previous studies combined with our research results, we hypothesize that DHU or T arm absence may be a common trait among tRNAs in the subfamily Coccinae. Despite limitations in predicting tRNA secondary structures, truncated forms do not necessarily imply impaired functionality. However, tRNA of scale insect often deviate from the conventional Watson-Crick pairing rules, it does not fully comply with this pairing rule; notably, in scale insects, in addition to the typical A-U and G-C coordination, the most common nucleotide mismatch is G-U. It can be concluded that this unique pairing structure may play a critical role in maintaining the stability of the tRNA secondary structure of scale insects (Wu et al ., 2022; Hou et al ., 2023). Further studies on tRNA structure of scale insects may yield significant results.

This study provided 5 new mitogenome sequences for scale insects from China. The five mitogenomes range in length from 15,479 to 17,089 bp, all characterized by a high A+T content (ranging from 80.7% to 90.5%) and a correspondingly low G+C content (ranging from 9.5% to 19.3%). It includes 37 typical mitochondrial genes including 13 PCGs, 22 tRNAs, and two rRNAs. Here in, we included 76 ingroup and 3 outgroup species, and reconstructed the phylogenetic analysis by using 4 different datasets i.e. Nucleotide sequences of 13PCGs; Amino acid sequences of 13 PCGs; Nucleotide sequences of 13PCGs and two rRNA genes; 37 mitochondrial genes. Among the 22 tRNA genes, except for 7 genes with the common clover secondary structure, the remaining 15 tRNAs lacked DHU arms, TψC arms, or both. All described scale insect mitogenome had the same structure, A+T content, and base composition. Evaluation of relative synonymous codon usage (RSCU) revealed a significant bias, with different coccid species showing preferences for distinct codons. The most frequently used codon across these species was TTA (Leu). At the same time, this finding serves as a good confirmation. Specifically, our phylogenetic study confirmed the monophyly of the genera Coccus, Marsipococcus, Megalocryptes, Bambusaecoccus, and Saissetia . This research significantly expand the mitochondrial genome database for scale insects, laying the groundwork for future phylogenetic and analysis of scale insects.

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The first author is grateful to Feng-E Li (Guizhou University, Guiyang, China) for helping in reconstructing the phylogenetic trees by using different datasets of the complete mitochondrial genome of Coccidae. Funding information This work was supported by the Ministry of Science and Technology of the People’s Republic of China (Grant no. 2005DKA21402), Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (Grant no. 2019HJ2096001006). Conflict of Interest statement The authors declare no conflict of interest. Data Availability statement All the required data are uploaded as supplementary material. I confirm that the Data Availability Statement is included in the main file of my submission, and that access to all necessary data files is provided to editors and reviewers. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 192views 71downloads Citations Download citation Min Deng, Muhammad Asghar Hassan, Hussain Riaz, et al. Characterization of the complete mitochondrial genome of five scale insects (Hemiptera: Coccoidea; Coccidae) and they phylogenetic implications. Authorea. 22 December 2025. DOI: https://doi.org/10.22541/au.176640889.91045720/v1 DOI: https://doi.org/10.22541/au.176640889.91045720/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.