(1) Background: Chironomids are biological indicators, playing an important role in monitoring and assessing the changes in water ecosystems. Mitochondrial genomes have been widely applied as a molecular marker to analyze the taxonomy and phylogeny of insects. However, knowledge of the mitogenomes of Chironomus species is scarce at present, which limits our understanding of the evolutionary relationships among Chironomus. (2) Methods: In our study, the mitogenomes and their basic structure of 12 Chironomus species and one Microchironomus species were newly sequenced. Combined with reported mitogenomes, a total of 15 mitogenomes of Chironomus were selected for a comparative mitogenomic analysis and phylogenetic reconstruction of Chironomus. (3) Results: Each mitogenome of the Chironomus species has the typical 37 genes and a control region. The basic structure of the whole mitogenomes of Chironomus species is relatively conservative, and the genetic arrangements stay the same as the ancestral mitogenome. (4) Conclusions: Our study enriches the library of mitogenomes of chironomids and provides a valuable resource for understanding the evolutionary history of Chironomus.

Tanypodinae, a highly diverse subfamily within Chironomidae (Diptera), has been poorly represented in mitochondrial genome (mitogenome) databases, with only a limited number of complete or partial sequences available. To address this gap, we present the first complete mitogenome sequences of 16 Tanypodinae species and 1 Podonominae species. By integrating these novel data with previously published chironomid mitogenomes, we conducted a comprehensive comparative mitogenomic analysis of 21 Tanypodinae species. Our results reveal that Tanypodinae mitogenomes maintain a conserved structural organization, preserving the ancestral insect gene arrangement. The nucleotide composition exhibits significant bias, with the control region demonstrating the highest A + T content among all genomic regions. Evolutionary analysis indicates that all protein-coding genes (PCGs) are subject to purifying selection, with ATP8 exhibiting the most rapid evolutionary rate. Phylogenetic reconstruction based on PCG amino acid sequences delineates seven well-supported clades within Tanypodinae, corresponding to the tribes Pentaneurini, Natarsiini, Procladiini, Tanypodini, Clinotanypodini, Macropelopiini, and Anatopyniini. These findings significantly advance our understanding of mitogenomic architecture and provide critical insights into the phylogenetic relationships within Tanypodinae.

Glyptotendipes pallens (Meigen, 1804) serves as a significant bioindicator for monitoring freshwater ecosystems. Despite the performance of COI barcode analyses on G. pallens, the mitogenome of this taxon remains unassembled and unanalyzed. In this study, we provide one newly sequenced complete mitogenome of G. pallens for the first time. The mitogenome is 16,063 bp long with high A + T content, and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a noncoding control region. The phylogenetic analysis strongly supports G. pallens and G. tokunagai as sister species.

Understanding species’ distributions beyond their native ranges is essential for monitoring non-native species occurrences. The alligator gar ( Atractosteus spatula ), native to North America and listed as a key invasive species in China’s Catalogue of Key Managed Invasive Alien Species, poses significant threats to aquatic ecosystems. To enable rapid on-site detection, we developed and validated novel molecular assays based on recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and quantitative PCR (qPCR). We designed species-specific primers and probes, optimized reaction conditions, and evaluated assay performance. Both RPA-LFD and qPCR exhibited high sensitivity, with detection limits as low as 4 copies/μL, and strong specificity for A. spatula without cross-reactivity to non-target species. Under optimized conditions, the RPA-LFD assay achieved results within 15 minutes at 38 °C. Field testing in a natural breeding pond confirmed reliable detection, and the best-performing RPA assay (RPAS1) showed comparable efficiency to the qPCR gold standard. This study provides robust, field-deployable tools that empower early detection and proactive monitoring of invasive species, with strong potential for application in citizen science and large-scale biodiversity surveillance. Graphic abstract