Land-use type acts as a key anthropogenic factor shaping the diversity of pollinator communities. Different habitats indirectly alter pollinator species richness, abundance and community structure by regulating vegetation coverage and the abundance of host and nectar plants. To clarify the mechanisms underlying habitat effects on pollinator assemblages, we conducted transect surveys to investigate pollinator communities across three typical habitats (forest, cropland and urban area) in Nanchong City. Our results revealed that pollinator alpha diversity was significantly higher in forests than in croplands and urban areas, with urban habitats harboring the lowest species diversity. Vegetation coverage and nectar plant richness were identified as the primary environmental drivers of pollinator diversity. The plant–pollinator interaction network exhibited pronounced seasonal dynamics: spring networks displayed a distinct nested architecture dominated by generalized mutualistic interactions; networks shifted gradually from generalized to specialized links in early summer; and only a small number of specialist pollinators persisted in late summer to early autumn. Accordingly, retaining wild flowering herbs adjacent to pollinator-dependent crops and conserving autumn- and winter-blooming plant species can sustain continuous floral food resources for pollinators, promote pollinator richness, stabilize pollinator communities, and enhance the ecosystem service of pollination.
Chitinase is a key enzyme involved in insect chitin metabolism, and RNA interference (RNAi) targeting the corresponding gene represents a promising strategy for eco-friendly pest management. However, the design parameters of double-stranded RNA (dsRNA), particularly fragment length and target position, remain insufficiently optimized. To investigate the effects of these parameters on the RNAi efficiency against the MsChi2 gene in Mythimna separata, we synthesized four dsRNAs of 60, 200, 500, and 1000 bp and administered them to larvae. The results indicated that 200 and 500 bp dsRNAs significantly inhibited larval growth, reduced survival, and silenced MsChi2 expression. Among these, the 200 bp dsRNA exhibited the most pronounced effects, including a 59% inhibition rate of larval body weight, 65.9% mortality, and 62% target gene silencing efficiency, accompanied by a significant reduction in chitin content and abnormalities in molting and pupation. Using 200 bp as the optimal length, five fragments targeting different coding regions of the gene were evaluated. All except one exhibited strong activity, and their RNAi efficiencies were positively correlated with the predicted numbers of effective small interfering RNAs. Collectively, these findings demonstrate that dsRNA length and target position jointly modulate RNAi efficiency, with 200 bp representing the optimal fragment length. This study provides a rational design framework for dsRNA development and establishes both theoretical and practical foundations for RNAi-based management of M. separata.
Human lice can be classified into several mitochondrial clades with distinct geographic distributions, reflecting long-term coevolution with ancient hominids and possibly with human populations. Analysis of ancient lice DNA from archaeological human remains can provide insights into the geographic origins of these clades and the timing of their dispersal. This study analyzes the COI barcode region of lice recovered from the ~1500-year-old salt-preserved mummy (Salt Man 2) at the Chehrabad salt mine in Zanjan Province, Iran. The sequences were compared with those of recent lice from Iran and other regions, available in the GenBank database, to determine clade affiliation and possible historical relationships. Our phylogenetic analysis recovered the five recognized mitochondrial clades (A–E). Ancient lice clustered within clade A but displayed novel haplotypes. Recent nits from Gilan and Kurdistan provinces were grouped within clades A and B, the only clades reported in Iran. The most complete ancient sequence (MLICE005-18) showed close similarity to sequences from Iraq, Pakistan, Egypt, and Iran. These findings, along with future research, could help determine when ancient lice lineages were established or introduced into specific regions. They could also clarify whether they represent haplotypes that have since disappeared or a lineage that still exists in understudied human populations.
Floral volatile organic compounds (VOCs) play a key role in chemical communication between plants and pollinators. Given the limited understanding of the chemical signals involved in the interactions among Passiflora edulis Sims (Passifloraceae), Poincianella pyramidalis (Tul.) L.P. Queiroz (Fabaceae), and Xylocopa (Neoxylocopa) nigrocincta Smith (Hymenoptera: Apidae), this study aimed to identify the floral VOCs emitted by these plant species and evaluate their detection by the antennae of X. nigrocincta. Floral VOCs were collected using dynamic headspace sampling, adsorbed in Porapak Q, and eluted with 500 µL of hexane. The resulting extracts were analyzed by gas chromatography with flame ionization detection (GC-FID), gas chromatography–mass spectrometry (GC–MS), and gas chromatography coupled with electroantennographic detection (GC–EAD). A total of 23 compounds were identified, six of which were shared by both plant species. The chemical profiles differed markedly: P. edulis was characterized mainly by methoxylated ethers and esters, whereas P. pyramidalis showed a predominance of terpenes. 1,4-dimethoxybenzene, the major compound in P. edulis, and (E)-β-ocimene, the most abundant compound in P. pyramidalis, elicited electroantennographic responses in X. nigrocincta, demonstrating that both compounds are detected by the olfactory system of these bees. These findings reveal differences in the floral VOC profiles of the two plant species and identify compounds detected by X. nigrocincta, providing a basis for future behavioral studies to investigate the role of these volatiles in plant–pollinator interactions.
Genome-scale searches for insect olfactory genes are vulnerable to rapid receptor divergence, tandem duplication, fragmented gene models, circular labels, and evolutionary leakage between training and test data. Sequence foundation models trained on DNA or proteins may extend homology-based annotation, but available evidence does not support their use as autonomous annotators or direct predictors of sensory function. This critical review organizes the problem around annotation failures rather than model catalogues. We distinguish six inference levels, from candidate locus detection to organism-level function, and define task-specific high-confidence reference criteria. We also propose a benchmark using biological hard negatives, sequence-cluster and taxonomic holdouts, conventional baselines, calibration, abstention, and workload-aware retrieval metrics. Evidence integration is defined operationally as a pipeline that retains model scores alongside similarity, domains, topology, phylogeny, expression, and assay evidence, without promoting a weak signal to a stronger biological claim. A published ESM-2-supported analysis of remote insect chemoreceptor homology illustrates both the potential and the current evidence boundary. A convincing benefit will require an incremental gain over transparent baselines under frozen biologically realistic splits, with reliable uncertainty and a clear route from ambiguous predictions to review or experiment.
Fopius arisanus (Hymenoptera: Braconidae) is an important parasitoid species in tephritid fruit fly biological control and host–parasitoid interaction research. Quantitative real-time PCR (qRT-PCR) is widely used to analyze gene expression, which requires stable reference genes for accurate normalization. However, no systematic evaluation of suitable reference genes for qRT-PCR analysis across F. arisanus tissues has been conducted. We evaluated the expression stability of ten candidate reference genes (Actin, α-tubulin, β-tubulin, RPL37a, EF1-α, EF1-δ, RPL4, RPS18, RPL13, and GAPDH) across five adult tissues from females and males. The stability of these genes was analyzed using the ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder, and the optimal number of reference genes was determined by geNorm pairwise variation analysis. The results revealed that the optimal reference genes differed between the female and male tissue panels: Actin and RPL13 were the most stable combination for female tissues, whereas RPL13 and RPS18 were recommended for male tissues. Notably, β-tubulin and GAPDH showed the poorest stability in female tissues, whereas α-tubulin and β-tubulin were the least stable in male tissues. The suitability of the stable and unstable reference genes was further validated by normalizing the expression of the target genes vitellogenin (Vg) and dual-specificity phosphatase CDC14A (CDC14A). Our findings represent a systematic evaluation of reference genes for F. arisanus and establish a reliable normalization strategy for future gene expression studies in this parasitoid wasp.
(1) Background: Mosquitoes represent major vectors of human pathogens, and their control remains a global public health priority. However, the widespread use of synthetic insecticides has resulted in environmental contamination, adverse effects on non-target organisms, and the rapid emergence of insecticide resistance. These limitations have intensified the search for sustainable botanical alternatives. (2) Methods: The present study investigated the phytochemical composition and larvicidal efficacy of flower extracts from Chrysanthemum cinerariaefolium and Oenothera biennis, alongside their nanoemulsion formulations, against the third-instar larvae of Culex pipiens. Larvicidal assays demonstrated that nanoemulsion formulations markedly enhanced bioactivity compared with conventional extracts. (3) Results: After 48 h of exposure, C. cinerariaefolium nanoemulsion exhibited the strongest toxicity (LC50 = 26.63 ppm), followed by methanol (44.46 ppm), hexane (73.90 ppm), and aqueous extracts (209.99 ppm). Corresponding LC50 values for O. biennis were 31.89, 80.15, 140.69, and 393.22 ppm, respectively. Biochemical analyses indicated that C. cinerariaefolium treatments strongly inhibited detoxification and digestive enzymes (esterases, GABA-T, amylase, and lipase) and suppressed antioxidant defenses (SOD, GST, CAT, and GSH). This was accompanied by a significant elevation of oxidative stress biomarkers, including lipid peroxidation and protein carbonylation. In contrast, O. biennis induced comparatively moderate biochemical responses. The phytochemical profiling utilizing GC-MS revealed the identification of forty-nine and fifty volatile components from the C. cinerariaefolium (CW-Hex) and O. biennis (BOF) n-hexane extracts, respectively, primarily consisting of oxygenated hydrocarbons, fatty acids, and their esters. Furthermore, UPLC-MS/MS-assisted profiling of methanol, ethyl acetate, and aqueous extracts identified thirty-eight metabolites for CW and thirty-one for BOF. The CW extracts were predominant in flavonoids and fatty acids while also containing cinnamic acid derivatives, phenolic acids, diterpenoids, triterpenoids, pyrethrins, phenyl ethanoids, steroids, aurones, and tannins. BOF extracts were particularly rich in flavonoids, followed by fatty acids, cinnamic acid derivatives, tannins, and steroids. (4) Conclusions: These findings demonstrate that plant-derived formulations of C. cinerariaefolium, particularly in nanoemulsion form, exert potent larvicidal activity through the disruption of metabolic and antioxidant pathways, offering a promising eco-friendly strategy for vector control.
In efforts to survey biting flies, there is a need for a multi-species trap that can capture diverse families of hematophagous flies, thus functioning as a more robust and representative trapping method. The current study evaluated the efficiency of the baited P38T BioFlyTrap with other standard traps, namely the H-trap used in South Africa, the Vavoua (West/Central Africa), and the Nzi (East Africa), in informing decision-making to undertake enhanced surveillance and control interventions against biting flies in South Africa. A comparative study was done using a 4 × 4 Latin Square Design (LSD) approach over a period of 16 days in northeastern KwaZulu-Natal. The H-trap was most efficient for Glossinidae, while the Nzi trap caught the most diverse species dominated by Tabanidae. The P38T BioFlyTrap showed moderate performance across all categories (Glossinidae, Tabanidae, Muscidae (Stomoxyini)) of biting flies. The Vavoua trap was the least effective trap for Glossinidae and Tabanidae, but was relatively effective for Stomoxyini. In this environment, baited P38T BioFlyTrap would be useful for broader spectrum surveillance, while baited H-trap and Nzi remain specifically more performant, respectively for Glossinidae and Tabanidae.
Insects lack adaptive immunity and rely exclusively on innate immune system for pathogen defense. However, the specific role of BmToll9-2, a key Toll receptor in the silkworm (Bombyx mori), in mediating immune responses, particularly against bacterial challenges in the larval fat body, remains incompletely understood. To address this gap, this study employed a combination of molecular approaches, including RNA interference (RNAi) targeting BmToll9-2, bacterial challenges with heat-inactivated Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), and quantitative real-time PCR (qPCR) to assess the transcriptional changes of BmToll9-2 and immune-related genes in the fat body, a key immune tissue. Quantitative analysis showed that after BmToll9-2 RNAi, bacterial challenges significantly upregulated BmToll9-2 expression in the fat body at 12 h post-challenge, whereas BmToll9-2 silencing alone notably downregulated most downstream signaling genes of the Toll pathway by 53.07–83.14%, as well as 11 immune effector genes, including antimicrobial peptide genes, by 64.48–93.40%. Importantly, feeding bacteria post-RNAi reversed these downregulations: E. coli induced a stronger upregulation of both signaling and effector genes compared to S. aureus. This study provides transcriptional evidence that BmToll9-2 may act as a positive regulator of the Toll pathway signaling and antimicrobial peptides in silkworm larval fat body, facilitating robust and rapid immune signaling. This study deepens our understanding of Lepidopteran innate immunity by elucidating key molecular mechanisms, thereby providing a solid foundation for refining RNAi-based pest control strategies.
Mangroves support a diverse insect fauna that plays essential roles in ecosystem functioning; however, the diversity of bark and ambrosia beetles (Curculionidae: Scolytinae) in the mangroves of the Mexican Pacific remains poorly understood. This study aimed to characterize the diversity and community structure of Scolytinae associated with two mangrove ecosystems in the state of Colima, western Mexico: Palo Verde Estuary and Juluapan Lagoon. Beetles were sampled using ethanol-baited traps (70% ethanol). A total of 1869 individuals belonging to 19 species and 10 genera were collected. Hypothenemus was the most species-rich genus, whereas Xyleborus volvulus was the dominant species. Five species are reported for the first time in Colima: Ambrosiodmus obliquus, Corthylus comatus, Premnobius cavipennis, Hypothenemus dolosus, and Hypothenemus indigens. Palo Verde Estuary showed higher beetle abundance, whereas Juluapan Lagoon exhibited greater species diversity and community evenness. Although the community shared several genera with mangrove ecosystems from other regions of Mexico and Brazil, it differed in species composition and dominance patterns. This study provides the first baseline on the Scolytinae fauna associated with the mangroves of Colima and establishes a reference for future ecological monitoring and phytosanitary surveillance programs.
The melon fly, Zeugodacus cucurbitae, and the oriental fruit fly, Bactrocera dorsalis, are economically important tephritid pests whose sympatric distribution and overlapping host ranges precipitate intense interspecific competitive interactions. To explore the interspecific competition during larval development between these two species, we conducted a comparative study under controlled laboratory conditions. Larvae were evaluated on five host fruits—pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), winter melon (Benincasa hispida), bitter gourd (Momordica charantia), and guava (Psidium guajava)—across six larval density gradients (4.0, 1.33, 0.8, 0.57, 0.44, and 0.36 larvae g−1 of host tissue). In mixed-species treatments, Z. cucurbitae exhibited significantly higher survival rates and greater adult dry weights than B. dorsalis on all cucurbit hosts except guava, indicating a consistent competitive advantage. Under high-density conditions, mixed-species treatments displayed stronger competitive effects than single-species treatments. Survival rate and adult dry weight of both species were significantly affected by the interactive effects of host plant species and larval density, with these factors jointly determining competitive outcomes. These findings improve our understanding of interspecific competition in tephritid fruit flies and provide a scientific basis for developing more effective management strategies for these economically important pests.
Short-term high temperature is a critical environmental factor affecting the population dynamics of invasive insect pests. Krüppel-homolog 1 (Kr-h1) is a key transcription factor involved in insect reproduction. In this study, we investigated the role of ZcKr-h1 in female reproduction and lifespan under normal and short-term high-temperature conditions in the melon fly, Zeugodacus cucurbitae (Coquillett). RNA interference (RNAi)-mediated knockdown of ZcKr-h1 markedly reduced female fecundity, shortened the oviposition period, delayed ovarian development, and decreased adult lifespan. Moreover, ZcVgR (vitellogenin receptor) expression was consistently suppressed following ZcKr-h1 knockdown at both 25 °C and 45 °C, while ZcVg3 expression was upregulated at 25 °C but showed no significant change at 45 °C. These findings demonstrate that ZcKr-h1 is essential for female reproduction and survival under both normal and short-term high-temperature conditions, and that VgR is the primary downstream effector mediating its reproductive functions. This study provides a potential molecular target for environmentally friendly pest management during hot seasons.
The increasing demand for sustainable protein sources has stimulated interest in edible insects such as Tenebrio molitor (yellow mealworm) larvae. While probiotics supplementation has been explored to improve insect growth performance, its impact on protein quality and downstream processing remains poorly understood. This study investigated the combined effects of probiotic-enriched diet supplementation during larval rearing and post-harvest ultrasound-assisted extraction (UST) on the protein and lipid fractions of yellow mealworm larvae. Three probiotic strains, namely, Pediococcus pentosaceus (PP), Bacillus velezensis (BV), and Bacillus coagulans (BC), were incorporated into larval diets and compared with a standard diet (without probiotics). Protein extraction was performed by both alkaline solubilization–isoelectric precipitation, which served as a control, and ultrasound application for 5, 15, and 25 min. The results from the trial found effects of both probiotics and UST, as well as their interaction, on the protein yield, although an improvement in yield was not obtained with increased sonication, and the BV group produced the highest yield among the probiotic treatment groups. In addition to this, the study did not find any effects associated with ultrasound pretreatment or probiotics on proximate composition and color, except fat % and increased lipid co-extraction, especially at longer durations. Probiotics, on the other hand, showed an influence on the fatty and amino acid profile, while they interacted with UST to affect the extracts’ content. This study did not find any effects on particle size, but the zeta potential was affected by probiotic diet enrichment. Similarly, the free SH content was influenced by sonication, while both probiotics and UST played a role in hydrophobicity. Regarding the solubility, it was found to be pH-dependent and ranged from 0 to ≈90%. Importantly, this study found that probiotics did not have any effect on protein solubility; however, UST had an effect at pH 2 and 7 but not at pH 11. In terms of hydrophobicity, both factors and their interactions were found to have an effect. Moreover, this study could not determine any effects on the inherent fluorescence absorbance of protein molecules. Apart from this, FTIR analysis revealed β-sheet-dominated secondary structures (≈41–47%), with treatment-dependent shifts in α-helix and β-turn contributions. The heat enthalpies associated with denaturation and melting temperature were influenced by sonication, where probiotics could not confer an effect. Overall, this study might demonstrate the strain-specific improvement in protein yield and modulation of the physicochemical functionality, as well as the nutritive value of the protein. Both biological factors and ultrasound-assisted extraction processes can act together to tailor the physicochemical and functional properties of biofractions, but more studies are needed for process optimization.
Thirty-two Chinese Anastatus species (Hymenoptera: Chalcidoidea: Eupelmidae) are studied, comprising 20 previously described species and 11 new species. Ten of the new species belong to the subgenus A. (Anastatus): A. (Anastatus) nozdormusp. nov., A. (Anastatus) kryptos sp. nov., A. (Anastatus) napoleonissp. nov., A. (Anastatus) yalini sp. nov., A. (Anastatus) battutai sp. nov., A. (Anastatus) beagleisp. nov., A. (Anastatus) neltharionsp. nov., A. (Anastatus) tigrinussp. nov., A. (Anastatus) huijuanaesp. nov., A. (Anastatus) ashrafisp. nov. One belongs to the subgenus A. (Cladanastatus): A. (Cladanastatus) alexstraszasp. nov. One additional species is newly recorded from China: A. (Anastatus) mantoidae Motschulsky rec. nov. A new synonymy is also proposed: A. (Anastatus) flavaeratus Peng & Tang, 2020 syn. nov. under A. (Anastatus) gastropachae Ashmead, 1904. Morphologically similar species were assigned to three informal species groups (formosanus, fulloi, and japonicus groups) based on morphology. Of the already-described species of the Chinese Anastatus, 15 were newly recorded from different provinces of China; this was true for all species except A. colemani, A. bifasciatus, A. polikiarkoudus, A. taibaiensis, and A. zdenekbouceki. Molecular analyses based on the mitochondrial cytochrome c oxidase subunit I (COI) gene were conducted on a total of 34 species (26 species derived from the specimens collected from China and eight species’ sequences downloaded from the BOLD and NCBI). Multivariate morphometric analyses (PCA, LDA, MANOVA) were employed to distinguish morphologically similar species. A comprehensive diagnostic key to all Chinese Anastatus species is also provided, accompanied by photographs of diagnostic characters.
The expansion of invasive mosquito species of the genus Aedes has become a growing public health concern in Europe due to their role in the transmission of arboviruses such as dengue, Zika, and chikungunya. Early detection and surveillance of these vectors are essential to prevent the establishment of local transmission cycles. This study presents a review of surveillance reports and rapid risk assessments issued by the Health Alerts and Emergencies Coordination Centre of the Spanish Ministry of Health, complemented by relevant scientific literature on the presence and spread of Aedes albopictus, Aedes aegypti, and Aedes japonicus in Spain. The analysis describes the evolution of vector detection between 2004 and 2026, identifies geographical patterns of expansion, and examines the surveillance and response mechanisms implemented by Spanish health authorities. Particular attention is given to environmental, climatic, and socioeconomic factors that may facilitate the establishment of these invasive species. The results highlight the importance of strengthening entomological surveillance systems, improving coordination between national and regional health authorities, and enhancing early warning mechanisms to reduce the risk of arbovirus transmission in Spain. Unlike previous European reviews, this study integrates surveillance reports, grey literature, citizen science contributions, and scientific evidence to provide a comprehensive assessment of invasive Aedes surveillance in Spain and identify priority actions for strengthening arbovirus preparedness.
It has been suggested that hypoxia-inducible factor 1α (HIF1α) serves as a key regulatory factor in insect metabolism remodelling under hypoxia, in which lipid metabolism appears to play an important role. However, the underlying mechanism remains incompletely understood. In this paper, the function of HIF1α in regulating lipid metabolism under hypoxia in Callosobruchus chinensis (Coleoptera: Bruchidae), a typical stored-product insect, was investigated. Based on the amplification and cloning, the sequence of the CcHIF1α gene was analysed and found to be highly conserved throughout evolution. Then HIF1α expression in the adult C. chinensis was knocked down using RNA interference (RNAi) via feeding methods. After HIF1α knockdown, insects under hypoxic conditions exhibited higher mortality, along with decreased activities of four typical antioxidant enzymes (CAT, POD, GST, and SOD) and increased levels of two peroxide products (ROS and LPO), compared to those of the control. Moreover, comprehensive lipidomic analysis using UHPLC-QE–MS/MS revealed that lipid metabolism was reprogrammed, with significant changes in the levels of lipids such as SM, LPC, PI, SQDG, MAG, PE, ACar, and TAG. In particular, the increase in ceramide (Cer) or Cer-related lipids was observed to be associated with the production of peroxides. ELISA assays further verified the effect of HIF1α on changes in these characteristic lipids, such as the increase in PA and Cer and a decrease in TAG, PC and SM. Correspondingly, the expressions of the key genes (i.e., SPTLC1, KDSR, SMPD3, SGPL1, DGAT) involved in sphingolipid metabolism changed following HIF1α knockdown. These results are consistent with the possibility that HIF1α-mediated regulation of lipid metabolism may be important for insects facing hypoxic environments, and these findings could provide an in-depth understanding of insect hypoxia adaptation, thereby potentially contributing to the development of innovative pest management strategies.
The Amphinemura sinensis species group is distributed in China and Vietnam and is characterized by the horn-shaped lateral processes of the dorsal sclerite and the pointed ventral sclerite of the male epiproct. Species in the group are often difficult to distinguish because of their similar morphology and the limited number of reliable diagnostic characters. In this study, we examined the A. sinensis species using morphological and molecular data. COI sequences were obtained for 16 of the 34 recognized species, including the four new species described herein. Detailed examination of male genital structures and comparisons with previously described species support the recognition of four new species. The four new species are described and illustrated. With these additions, the A. sinensis species group now comprises 34 species.
Timely and accurate termite detection is essential for effective termite control. To address the challenges posed by the small size of termite individuals and the susceptibility of target features to background texture interference under complex backgrounds, this study proposes ESD-YOLO, a fine-grained feature-enhanced object detection model. Using YOLO11n as the baseline, ESD-YOLO redesigns the feature extraction, deep feature aggregation, and multi-scale feature fusion stages to improve the representation of small-scale termite targets under complex backgrounds. Specifically, the Efficient Multi-scale Attention (EMA) mechanism is incorporated into the C3k2 module to enhance feature discriminability between termite individuals and the background. A Spatial Pyramid Pooling-Fast with Dual Global Pooling (SPPF-DGP) module is employed to supplement deep features with global contextual information and salient response information. In addition, the DySample dynamic upsampling module is introduced to improve spatial alignment during multi-scale feature fusion and enhance boundary representation for small targets. Experimental results show that ESD-YOLO achieves Precision, Recall, mAP@0.5, and mAP@0.5:0.95 values of 95.39%, 96.33%, 97.85%, and 65.92%, respectively, with 2.67 M parameters and 6.68 G FLOPs. Compared with Faster R-CNN, RetinaNet, RT-DETR, and several YOLO-series models, ESD-YOLO demonstrates strong small-target detection and localization performance under the controlled complex-background conditions established in this study, providing a methodological reference for automated termite detection in practical settings.
The genus Sigara Fabricius, 1775 (Hemiptera: Corixidae) is an important freshwater bioindicator, but its conservative morphology complicates traditional species identification. This study combines DNA barcoding, traditional taxonomy, and climatic factors within an integrative framework to provide new insights into species delimitation. We analyzed cytochrome c oxidase subunit I (COI) sequences from 501 Sigara specimens collected from 15 countries, including 95 newly generated COI barcodes. Our integrated approach included phylogenetic analyses, genetic distance assessments, and ecological niche modeling. The results revealed remarkable diversity, with sequences clustering into 46 Barcode Index Numbers (BINs). COI genetic distances showed a pronounced bimodal pattern, with most intraspecific divergences below 1% and interspecific divergences mainly ranging from 8–16%, while the few intermediate divergences (4–8%) suggested potential cryptic diversity or recent species divergence. Most morphologically defined species corresponded to distinct molecular lineages, supporting traditional taxonomy, whereas some morphospecies exhibited genetic discordance potentially associated with species divergence. Integrative analyses revealed that climatic factors, particularly isothermality and seasonal temperature and precipitation, are major drivers of Sigara MOTU distributions, highlighting the important role of climate in shaping their geographic patterns. These findings indicate that although COI barcodes are not sufficient to define the species boundaries of Sigara alone, they provide a key entry point for the accurate division of species, which in turn promotes the development of molecular tools for freshwater ecological monitoring and conservation.
Aphids are important agricultural pests that interact with diverse bacterial taxa living within or associated with their bodies. Some bacteria, such as the obligate symbiont Buchnera aphidicola, provide essential nutritional functions and are predominantly inherited vertically, whereas other aphid-associated bacteria may have more variable distributions and ecological roles. In this study, we investigated patterns of association between aphid evolutionary relationships and selected bacterial taxa. Aphids were identified using morphological characteristics and mitochondrial cytochrome c oxidase subunit I (COI) sequences, and host phylogenetic relationships were reconstructed using mitochondrial DNA data. We then compared the host phylogeny with that of the obligate symbiont Buchnera aphidicola based on 16S rRNA sequences and screened aphid specimens for selected aphid-associated bacteria (Wolbachia, Pantoea, and Arsenophonus) using diagnostic PCR assays. The host and Buchnera phylogenies showed significant global congruence, consistent with a strong long-term host-associated evolutionary relationship and predominantly vertical inheritance of Buchnera. In contrast, the selected aphid-associated bacteria showed heterogeneous detection patterns across the sampled aphid taxa. Pantoea was detected relatively frequently and across a broad range of sampled hosts, whereas Wolbachia and Arsenophonus showed more variable and restricted detection patterns. Given the small and uneven sample sizes among aphid species, these findings are interpreted as descriptive screening patterns rather than species-level prevalence estimates. Overall, the results highlight contrasting patterns of host-associated evolution between Buchnera and other aphid-associated bacteria and provide a basis for further investigation of the ecological and evolutionary processes underlying these associations.
