Background: Hylurgus ligniperda, a major international forestry quarantine pest, was recently found to have invaded and posed a serious threat to the Pinus forests of the Jiaodong Peninsula in China. Continuous monitoring and vigilance of the early population is imperative, and rapid molecular detection technology is urgently needed. We focused on developing a single-gene-based species-specific PCR (SS-PCR) method. Results: We sequenced and assembled the mitochondrial genome of H. ligniperda to identify suitable target genes. We identified three closely related species for detecting the specificity of SS-PCR through phylogenetic analysis based on 13 protein-coding genes (PCGs). Subsequently, we analyzed the evolution of 13 PCGs and selected four mitochondrial genes to represent slow-evolving gene (COI) and faster-evolving genes (e.g. ND2, ND4, and ND5), respectively. We developed four species-specific primers targeting COI, ND2, ND4, and ND5 to rapidly identify H. ligniperda. The results showed that the four species-specific primers exhibited excellent specificity and sensitivity in the PCR assays, with consistent performance across a broader range of species. This method demonstrates the ability to identify beetles promptly, even during their larval stage. The entire detection process can be completed within 2–3 h. Conclusions: This method is suitable for large-scale species detection in laboratory settings. Moreover, the selection of target genes in the SS-PCR method is not affected by the evolutionary rate. SS-PCR can be widely implemented at port and forestry workstations, significantly enhancing early management strategies and quarantine measures against H. ligniperda. This approach will help prevent the spread of the pest and effectively preserve the resources of Chinese pine forests.

Platygaster robiniae is economically important as a highly specific parasitoid of the invasive pest Obolodiplosis robiniae which was introduced into the Euro-Asia region in the last decade. Despite being a critical and specific parasitoid of the invasive pest O. robiniae and its use as an effective biocontrol agent, the absence of sequence information from P. robiniae have limited its genetic applications for pest management in forests. Mitochondrial (mt) genomes generally contain abundant nucleotide information and thus are helpful for understanding species history. Here, we sequenced the complete mt genome of P. robiniae using next generation sequencing, and annotated 13 protein-coding, 22 tRNA, and 2 rRNA genes and a 702 bp noncoding region. Comparative analysis indicated that this mt genome has a normal A + T content and codons use, however possessed both the expected and unique rearrangements. Ten tRNAs at four gene blocks COII-ATP8, COIII-ND3, ND3-ND5 and the A + T-rich region-ND2 were rearranged, including gene shuffles, transpositions and inversions. Notably, two genes tRNA^Ser(UCN) and tRNA^Leu(CUN) had undergone long-range inversions, which is the first record of this rearrangement type in the superfamily Platygastroidea. The D-loops of both tRNA^Ile and tRNA^Leu(CUN) were absent from the tRNA secondary structure, which has not been reported from hymenopteran previously. Phylogenetic analysis based with the maximum likelihood and Bayesian methods showed that P. robiniae grouped with other species of Platygastridae, and that the superfamily Platygastridea is sister to the other Proctotrupomorpha superfamilies. Our tree strongly supports the monophyly of the five superfamilies of Proctotrupomorpha. This study discovered some unique characters of P. robiniae, and contributes to our understanding of genome rearrangements in the order Hymenoptera.

Insect infestations cause substantial changes in the leaves they attack, but the effects of insect infestation on photosynthesis and gene expression in the whole infestation period are rarely reported. In this study, the effects of poplar aphid (Chaitophorus populialbae Boyer de Fonscoloube) on photosynthetic rate and gene expression of Populus alba var. pyramidalis were studied, from 2 to 36 days after low-density aphid inoculation (DAI). The results revealed a dynamic change of photosynthesis in poplar after aphid infestation: compensatory increased at 8 DAI, was inhibited at 17 DAI, but repaired after 21 DAI. Aphid infestation promoted the expression of the majority of differentially expressed genes (DEGs) at 4, 6, 8, and 21 DAI. The DEGs were enriched into a plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway, suggesting a significant but transient resistance to disease or other stresses. Photosynthesis-related DEGs were downregulated at 8 DAI, which might result in photosynthetic inhibition at 17 DAI. The expression of flavonoid biosynthesis-related DEGs dynamic changed from all downregulated at 2 DAI to almost all upregulated at 21 DAI, suggesting a persistent resistance to insect infestation. This study reveals dynamic changes in photosynthesis, resistance to pathogens, and insects in poplar leaves after aphid infestation.

Agrilus mali stands as a significant wood-boring pest prevalent in Northeast Asia. Identifying this pest beetle is often hindered by insufficient efficient, rapid, on-site discrimination methods beyond examining adult morphological features. As a result, an urgent need arises for developing and implementing a rapid and accurate molecular technique to distinguish and manage the beetle. This study presents a straightforward, swift, highly specific, and sensitive method built upon recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD). This method demonstrates the capability to promptly identify the beetle, even during its larval stage. RPA primers and probes were designed using the internal transcribed spacer 1 region. Through probe optimization, false-positive signals were successfully eliminated, with an accompanying discussion on the underlying causes of such signals. The RPA-LFD assays exhibited remarkable specificity and sensitivity, requiring as little as 10⁻³ ng of purified DNA. Furthermore, the extraction of crude DNA was achieved through immersion in sterile distilled water, thus streamlining the assay process. Achievable at temperatures ranging from 30 to 50 °C, the RPA-LFD assay can be executed manually without specialized equipment. By merging the RPA-LFD assay with DNA coarse extraction, A. mali can be detected within just 30 min. This current study effectively demonstrates the immense potential of RPA-LFD in quarantine and pest management. Additionally, it presents a universal technique for the rapid on-site diagnosis of insects, showcasing the wide applicability of this method.

Agrilus mali Matsumura is a wood-boring beetle that aggressively attacks species of the genus Malus, that has recently caused serious damage to the wild apple tree M. sieversii (Lebed.) in the western Tianshan Mountains in Xinjiang. It was first detected there in the early 1990s and spread rapidly, being thus considered a regional invasive pest. To explore the possible outbreak mechanism of the local population and characterize the genetic differentiation of A. mali across different regions of China, we used three mitochondrial genes (COI, COII, and CytB) to investigate the genetic diversity and genetic structure of 17 A. mali populations containing 205 individuals collected from five Chinese provinces. Among them, nine populations were from the western Tianshan Mountains. Ultimately, of the 136 pairwise F_st comparisons, 99 showed high genetic differentiation among overall populations, and Tianshan populations exhibited significant differentiation with most of the non-Tianshan populations. Furthermore, A. mali populations represented relatively abundant haplotypes (54 haplotypes). Nine populations from the Tianshan Mountains showed 32 haplotypes (26 of which were unique), displaying relatively high genetic diversity. Additionally, the Mantel test revealed population genetic differentiation among either overall populations or the Tianshan Mountains populations, likely caused by geographical isolation. Phylogenic relationships showed that all populations clustered into three clades, and Tianshan Mountains populations, including CY, occupied one of the three clades. These results suggest that A. mali in the western Tianshan region has possibly been present in the area for a long period, and may not have been introduced recently. Highly frequent gene flows within Tianshan populations are possibly caused by human activities and may enhance the adaptability of A. mali along the western Tianshan Mountains, leading to periodic outbreaks. These findings enhance our understanding of jewel beetle population genetics and provide valuable information for pest management.