The complete mitochondrial genome of Psyllaephagus populi (Hymenoptera: Encyrtidae)

Abstract

Psyllaephagus wasps are key agents in integrated pest management. Here, we sequence and characterize the complete mitochondrial genome of Psyllaephagus populi Trjapitzin 1964 (Hymenoptera: Encyrtidae). The 16,889-bp mitochondrial genome encodes 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an AT-rich control region. Nucleotide composition is strongly A + T biased (82.01%), with individual proportions of A (46.86%), T (35.15%), C (11.72%), and G (6.27%). Maximum-likelihood phylogeny using all 13 PCGs places Psyllaephagus in close association with Blastothrix. This mitogenome enriches Encyrtidae genomic resources and improves resolution of intra-family phylogenetic relationships.

Introduction

Encyrtidae (Hymenoptera: Chalcidoidea) is among the most species-rich parasitoid families and performs essential ecological functions in biological control. Globally, 5187 species in 527 genera have been described (Noyes 2023), this taxon represents a major reservoir of natural enemies for arthropod pest regulation. Ashmead (1900) erected the genus Psyllaephagus on three diagnostic characters: (1) punctiform marginal vein shorter than the stigmal vein, (2) rounded occipital margin, and (3) mid tibial spur shorter than the basitarsus. Current taxonomic records recognize 262 valid species within the genus (Noyes 2023). Females are koinobiont endoparasitoids of psyllid nymphs (Hemiptera), especially in Psyllidae, Triozidae, Liviidae, Aphalaridae, and Homotomidae. Host-parasitoid associations have been documented for 125 species (Trjapitzin 1989; Chauzat et al. 2002), underscoring the genus’s importance in suppressing psyllid populations and delivering ecosystem services in agroecosystems by reducing yield loss and phytosanitary risk.

One focal host plant is Populus euphratica (desert poplar), a deciduous tree adapted to arid environments. Po. euphratica (To avoid confusion with P. used for Psyllaephagus, for example, P. populi, we hereafter abbreviate the genus Populus as Po.; thus Po. euphratica denotes Populus euphratica.) exhibits high stress tolerance, particularly in saline-alkali soils, due to its extensive root system and water storage capacity. Its ecological functions include: (1) pioneering desert forest succession, (2) aeolian sediment stabilization, (3) microclimate modulation, and (4) pedogenesis enhancement (Zhang et al. 2005; Cao et al. 2012).

Populus euphratica sustains multiple psyllid pests: Egeirotrioza ceardi, E. gracilis, E. rufa, E. xingi, and Syntomoza unicolor (Zhang et al. 2012; Aishan et al. 2024). Psyllid injury induces leaf galls, depresses photosynthesis, accelerates senescence, and slows growth (Sun et al. 2012). These phytopathological impacts necessitate development of biological control strategies, particularly through utilization of specialized parasitoids like Psyllaephagus populi.

Psyllaephagus populi, a principal parasitoid of psyllids on Populus, was first described from steppe and desert habitats in Kazakhstan (Trjapitzin 1964). Subsequent records confirm its presence in Mongolia and Turkmenistan (Trjapitzin 1989). Despite its biocontrol value, genomic resources remain scarce. Here, we present the first record of P. populi from China, and provide the complete mitochondrial genome of P. populi, obtained by rearing galls from Po. euphratica. This study provides molecular insights into P. populi and expands mitochondrial genome resources for Psyllaephagus species, addressing a critical knowledge gap in biocontrol research.

Materials and methods

Sampling and identification

P. populi specimens were reared from Po. euphratica leaf galls (Figure 1) collected in Karamay City, Xinjiang Uygur Autonomous Region, China (85.100924°E, 45.676715°N; August 2023). Leaf galls on Po. euphratica were collected from their natural habitat by Zhulidezi Aishan. The galls were incubated in an artificial-climate incubator (Ningbo Jiangnan Instrument Factory) under standardized conditions: 28 °C constant temperature, 60% relative humidity, and 14:10 h (L:D) photoperiod. Environmental parameters were checked and calibrated daily. Morphological identification followed Trjapitzin (1989). Vouchers are deposited at the College of Life Science and Technology, Xinjiang University (contact person: Ji-Xiang Liu; email: liujixiangxuexi@163.com), with the voucher number MS_TXF.

Figure 1.

(a) The side view of Psyllaephagus populi (Trjapitzin, 1964) head (photo by Jin-Ling Wang). (b) The lateral view of Psyllaephagus populi. (c) The frontal view of the fore wing. (d) Collected photos of the insect gall. The specimen was reared from galls on leaf of Populus sp. at Karamay City, Xinjiang Uygur Autonomous Region, China (85.100924E, 45.676715N).

DNA extraction and sequencing

Genomic DNA was extracted from a mixture of six fresh specimens using CTAB (cetyltrimethylammonium bromide) method (Doyle 1987) under liquid nitrogen conditions. DNA integrity and quantity were evaluated on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). DNA libraries were constructed using the Illumina TruSeq^® DNA PCR-Free HT Kit and sequenced on the Illumina HiSeq platform (San Diego, CA) to generate 150 bp paired-end reads. Mitogenome assembly achieved 51–8002× read depth coverage (Supplementary Figure S1). Raw reads were adapter-trimmed and quality-filtered with fastp v0.20.0 (Chen et al. 2018).

Assembly and annotation

De novo assembly used MitoZ v3.6 (Meng et al. 2019), with gap closure by NOVOPlasty v4.3.3 (Dierckxsens et al. 2017). The draft was curated in BioEdit v7.7.1 (Hall et al. 2011) to remove redundant overlapping sequences in the control region and circularized to yield a complete mitogenome. Annotation was performed in MITOS (Bernt et al. 2013) using the invertebrate mitochondrial genetic code (transl_table = 5) and the RefSeq39 + MiTFi tRNA model as reference. All tRNA gene structures were predicted using ARWEN v1.2.3 (Laslett and Canback 2008). Annotation accuracy was verified through comparative analysis with published Encyrtidae mitogenomes. The circular map of the mitogenome was created with OrganellarGenomeDRAW (OGDRAW) v1.3.1 (Lohse et al. 2007).

Phylogenetic analysis

The phylogenetic matrix comprised 18 Encyrtidae species as ingroups and two Aphelinidae species as outgroups (Supplementary Table S1). The 13 mitochondrial protein-coding genes (PCGs) were processed using PhyloSuite v1.2.3 (Zhang et al. 2020). Sequence alignment was conducted using MACSE v2.07 (Ranwez et al. 2018), followed by gap removal with Gblocks v0.91b (Talavera and Castresana 2007). Maximum-likelihood (ML) trees were inferred in IQ-TREE v1.6.9 (Nguyen et al. 2015) with the GTR + F + ASC + R4 model selected by ModelFinder (Kalyaanamoorthy et al. 2017). Node support was assessed with 1000 bootstrap replicates (Hoang et al. 2018) and 1000 SH-aLRT tests (Guindon et al. 2010). The phylogenetic tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Results

Mitochondrial genome structure

The mitogenome of P. populi comprises 16,889 bp in a circular configuration. Base composition is strongly A + T-biased (82.01%), with individual proportions of 46.86% A, 35.15% T, 11.72% C, and 6.27% G. The genome encodes 37 genes: 13 PCGs, 22 tRNAs, and two rRNAs, and a 1020 bp non-coding control region (Figure 2). The 82.01% A + T content lies within known ranges for hymenopteran mitogenomes (75.64–87.42%) (Aydemir and Korkmaz 2020).

Figure 2.

Circular map of the P. populi mitochondrial genome. Different colors indicate different types of genes and regions.

Gene order and nucleotide composition

Relative to other Encyrtidae (Xing et al. 2022), P. populi shows a distinct gene rearrangement while retaining partial synteny with two Encarsia mitogenomes (Zhu et al. 2018). All 13 PCGs initiate with typical ATN codons: six genes (nad2, nad3, cox2, nad5, nad4l, and nad6) with ATT, six genes (cox3, atp6, cox1, nad4, cob, and nad1) with ATG, and atp8 with ATA. Termination codons comprised TAG (nad1 and nad3) and TAA (remaining PCGs). Twenty-two tRNA genes (62–83 bp) in P. populi are distributed throughout the coding regions. The two rRNA genes span 2190 bp in total. The 1020 bp control region contains two 351 bp tandem repeats.

Phylogenetic analysis

Phylogenetic analysis revealed that P. populi forms a well-supported clade with Psyllaephagus sp. (100% bootstrap support). The phylogeny confirmed the placement of P. populi within Encyrtinae, with Psyllaephagus showing a close phylogenetic relationship to Blastothrix (100% bootstrap support; Figure 3).

Figure 3.

The maximum-likelihood (ML) phylogenetic tree of Encyrtidae based on concatenated 13 mitochondrial protein coding genes. All species involved in the tree have scientific names with accession number on right side. GenBank or GenBase accession numbers are shown in Table S1.

Discussion and conclusions

The P. populi mitochondrial genome combines conserved features typical of Encyrtidae with lineage-specific characteristics. The 82.01% AT content aligns with typical hymenopteran mitogenomes, confirming evolutionary constraints on nucleotide composition in this insect order. The gene repertoire (13 PCGs, 22 tRNAs, and two rRNAs) maintains the ancestral arthropod mitochondrial architecture, whereas the 1020 bp control region with two 351 bp tandem repeats may encompass regulatory elements for replication/transcription.

The observed gene rearrangement distinguishes P. populi from other Encyrtidae species reported by Xing et al. (2022), while partial synteny with Encarsia (Zhu et al. 2018) points to conserved mechanisms within Chalcidoidea. The codon usage pattern follows conserved mitochondrial translation mechanisms, while the exclusive ATA start codon for atp8 may reflect functional optimization.

The phylogenetic reconstruction conclusively places P. populi within Encyrtinae, with 100% bootstrap support for its clustering with congeners and sister relationship to Blastothrix. This robust nodal support validates mitochondrial PCGs as reliable markers for resolving chalcidoid systematics.

These data furnish a genomic baseline for taxonomy, molecular identification of biological control agents, and comparative evolution across Chalcidoidea. The tandem repeats in the control region are promising markers for population-level analyses, while the conserved PCG codons enable comparative evolutionary studies across Chalcidoidea.

Our findings also extend the known range of P. populi. P. populi, previously recorded from Central Asia, is here reported for the first time from China based on material reared from galls on Po. euphratica leaves. Our discovery extends its known range almost 1200 km eastward from Central Asian steppes (Trjapitzin 1964). This biogeographical distribution pattern likely suggests co-adaptation between the parasitoid and psyllid-plant complexes across arid landscapes. While historical records associate P. populi with Egeirotrioza intermedia (Trjapitzin 1989), this psyllid was not detected during our surveys in Xinjiang. The gall-rearing experiments exclusively yielded four psyllid candidates: E. gracilis, E. rufa, E. xingi, and Syntomoza unicolor (Aishan et al. 2024). This discrepancy may arise from either (1) geographic variation in host specificity across the expanded range or (2) earlier taxonomic misassignments. Integrative DNA barcoding of both parasitoids and psyllid hosts will be decisive in resolving host associations.

Funding Statement

This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region under Grant Number 2024D03002 and the Beijing Natural Science Foundation under Grant Number 5244031.

Author contributions

The study was conceptualized and designed by Ning Huangfu. Zhulidezi Aishan contributed field sampling and revised the final version of the paper. Ji-Xiang Liu performed data analysis and wrote the manuscript. Qing-Song Zhou provides guidance on experiments and data analysis. Wen Zhong revised the manuscript. Ning Huangfu reviewed and approved the manuscript. All authors participated in editing and finalizing the manuscript, and approved the final version for publication.

Ethical approval

The sampling site is located outside of any protected area, and ethical approval is not necessary.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data supporting the findings of this study are openly available in the GenBank under accession number PP846803. The associated BioProject, BioSample, and SRA numbers are PRJNA1117190, SAMN41561227, and SRR29202825, respectively.