Sulfur dioxide (SO2) fumigations with and without CO2 were conducted in 1.9 L jars at 21 °C to determine synergism against rice weevil (Sitophilus oryzae) adults and confused flour beetle (Tribolium confusum) adults. Large-scale fumigations of 25 kg wheat in a 60 L chamber with SO2 plus 10% CO2 were conducted using an automated injection system based on SO2 concentration to demonstrate efficacy against all life stages of 3 stored product insects: confused flour beetle, granary weevil (Sitophilus granarius), and rice weevil. There was strong synergism between CO2 and SO2 against rice weevil adults and synergism was stronger at lower SO2 concentrations. Strong synergism between SO2 and 10% CO2 was also demonstrated against confused flour beetle adults with only 6.67% mortality for 428 ppm SO2 alone and 94.33% mortality for 344 ppm SO2 plus 10% CO2 in 24-h fumigations. In large-scale 24-h fumigations with 2,400 to 2,800 ppm SO2 of wheat grains under 10% CO2 atmospheres at 21 °C, complete control of adults of all 3 stored product insects were achieved. Over 95% and 99% mortalities were achieved against immature stages of confused flour beetle and the 2 weevil species, respectively. The synergistic effects of CO2 with SO2 and the system to maintain effective SO2 concentrations demonstrated in this study suggest that SO2 fumigation under CO2 enriched atmosphere has good potential for practical applications to control stored product insects.
Light trapping is a common monitoring and control method for Empoasca onukii Matsuda (Hemiptera: Cicadellidae), a major pest in tea gardens. However, in late autumn (late October), despite large populations of leafhoppers in tea gardens around 30°N latitude, the effectiveness of light trapping declines sharply. The factors causing this decline are unclear. In this study, field experiments revealed significant differences in ambient temperature and photoperiod corresponding to variation in light trapping effectiveness across different leafhopper peaks. Through a series of bioassays, we found that leafhoppers on and off of the host plant showed opposite behavioral responses to light stimulation; leafhoppers on the host exhibited negative -phototaxis, while those off the host showed positive phototaxis. Both behaviors were influenced by the light wavelength and intensity, with response intensity increasing linearly with light intensity. The phototactic rate increased with temperature within 10°C to 30°C, reaching a maximum at 30°C before decreasing. For males, the phototactic rate increased with the photophase of the rearing photoperiod extended. These results provide the first evidence that changes in photoperiod affected the -phototactic behavior of E. onukii. Low temperatures and short photoperiods caused the sharp decline in light trapping effectiveness for tea leafhoppers in late autumn, providing crucial information for the application of light trapping in leafhopper control.
The brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), is a highly invasive and polyphagous pest causing substantial economic losses in agricultural systems worldwide. Although the species has been reported in neighboring countries, information regarding its occurrence and impact on berry production in Kosovo has been lacking. This study represents the first documented occurrence of H. halys in Kosovo and aimed to investigate its population dynamics, spatiotemporal distribution, and associated damage in raspberry and blueberry plantations over a 2-year period (2024 to 2025). Insect abundance was monitored using trap-based sampling, while crop damage was assessed through damage incidence and severity index evaluations at border and center locations within plantations. The results revealed clear temporal and spatial patterns in pest occurrence and crop damage. Population abundance and damage levels varied between years, crops, and sampling locations, indicating differences in host suitability and pest distribution within plantations. Higher pest pressure was generally associated with plantation borders, suggesting colonization from surrounding habitats. Overall, the findings confirm the successful establishment of H. halys in Kosovo and demonstrate its potential to affect berry production. These results highlight the importance of implementing monitoring programs and developing targeted management strategies, particularly in border areas, to reduce the impact of this invasive pest on berry crops.
The pasture mealybug Heliococcus summervillei Brookes (Hemiptera: Pseudococcidae) is the cause of extensive pasture dieback in Australia and is an emerging biosecurity threat to pasture and crop grasses in the Americas. In this study, we present a morphological and molecular comparison of contemporary Australian specimens of H. summervillei and the primary bacterial endosymbiont Candidatus Tremblaya phenacola Gruwell (Enterobacterales: Enterobacteriaceae) with reference to historical and published specimens. Molecular analyses were conducted using three host nuclear markers (18S, 28S-D2, dynamin) and the universal bacterial marker (16S V3-V4 region). Our results identify a new, highly invasive variant of H. summervillei associated with the ongoing dieback outbreak in Australia. This work uses 16S barcoding to support host-based morphological and molecular approaches to differentiate this new variant from the type specimens.
Invertebrate pests pose one of the most significant threats to global agriculture. Trap-based surveillance is widely used to monitor the presence and abundance of pests, beneficial taxa and broader agroecosystem communities; however nonselective traps often collect hundreds or even thousands of individuals per sample, making conventional sorting and morphological identification labor-intensive and delaying the delivery of actionable information. Metabarcoding offers a scalable alternative for rapidly identifying agriculturally significant taxa in mixed trap samples, while providing more precise identifications (i.e. to species level). However, metabarcoding currently only provides semiquantitative estimates of relative abundance, rather than the accurate absolute abundance information required for many pest-monitoring and management decisions. Improving the quantitative capacity of metabarcoding is therefore an important and rapidly developing area of research across ecological, medical, microbiome, and environmental DNA research. This review summarizes progress toward quantitative metabarcoding of bulk invertebrate samples, highlighting key sources of bias, emerging correction methods, and the opportunities and challenges associated with their translation into agricultural monitoring systems. By consolidating insights from diverse ecological applications, we present a practical roadmap for improving the quantitative outputs and interpretation of metabarcoding data and integrating these novel approaches into agricultural pest monitoring and management.
DNA-based gut content analysis is a powerful tool for quantifying predation, yet dietary DNA detectability is influenced by multiple factors. Here, we systematically evaluated the effects of diet, predator traits (species, life stage, and sample type), and molecular marker properties (marker length and type) on dietary DNA detectability in ladybird beetles (Coleoptera: Coccinellidae), using Cryptolaemus montrouzieri Mulsant, 1853 as a representative species alongside other ladybird species. Dietary DNA detectability varied markedly among food types. The 50% detection success (DS50) of Ephestia kuehniella Zeller, 1879 (Lepidoptera: Pyralidae) egg DNA was extremely short, indicating that some diet items may be difficult to detect because of rapid degradation. In contrast, Brassica rapa L., 1753 (Brassicales: Brassicaceae) pollen DNA exhibited DS50 values comparable to those of the mealybug Planococcus citri Risso, 1813 (Hemiptera: Pseudococcidae) and the aphid Megoura crassicauda Mordvilko, 1919 (Hemiptera: Aphididae). Propylea japonica Thunberg, 1781 (Coleoptera: Coccinellidae) consistently exhibited shorter DS50 values than C. montrouzieri when fed on the same prey, demonstrating strong predator-dependent variation in dietary DNA detectability. Fourth-instar larvae maintained nearly 100% detection rates before pupation, and gut tissues yielded the longest DS50 among sample types. Molecular marker properties also influenced detectability: shorter mitochondrial COI fragments showed longer detection windows than longer fragments, whereas nuclear markers generally exhibited longer DS50 values than mitochondrial markers. These findings demonstrate that dietary DNA detectability is jointly shaped by diet, predator traits, and molecular marker properties, providing practical guidance for interpreting molecular dietary data and improving trophic interaction studies and biological control assessments.
Chemical ecology explores interactions among organisms and their environment, identifying critical chemical signals (semiochemicals) produced by organisms in shared habitats. Advances in this field have contributed to the development of behavioral controls for integrated pest management (IPM), such as mating disruption, mass trapping, attract-and-kill, deterrents, repellents, and push/pull strategies. Adoption of these tools and techniques could result in a paradigm shift away from a heavy reliance on insecticide applications, but commercialization and widespread adoption of behavioral controls in agricultural IPM remains relatively rare in the United States. Here, we explore potential barriers to commercialization and adoption of behavioral controls by summarizing the stages of research and development, and we discuss regulatory and market influences on commercialization.
Electrophysiological approaches are widely used to characterize insecticide target sites, whereas behavioral symptoms associated with different modes of action remain less quantitatively explored. Here, we developed a computer vision-based framework to quantify insecticide-induced behavioral responses in the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae). Convolutional neural networks were trained on 253 video recordings of aphids treated with neonicotinoid, organophosphate, or pyrethroid insecticides. A top-down pose-estimation approach enabled reliable detection of 11 body parts, from which 66 behavioral features were extracted. Feature selection and machine-learning classification were used to evaluate differentiation among insecticide classes. Among 31 tested algorithms, bagging ensemble trees showed the highest performance, achieving 74.36% accuracy and a macro-averaged Area Under the Curve (AUC) of 0.895 on the independent test set. Classification of additional compounds within trained classes showed moderate predictive consistency, whereas compounds with untrained modes of action produced dispersed prediction patterns across classes. These findings demonstrate that automated behavioral profiling can capture class-associated patterns under controlled conditions and may provide a quantitative complement to conventional toxicological assays.
The horn fly (Haematobia irritans) (L.) is an extensive economic pest to cattle and annual high infestations warrant the development of sampling procedures to inform management practices for cattle producers. There is little research on developing a sampling plan for the horn fly and a binomial sequential sampling (BSS) plan offers a potentially efficient method for the classification of pest density. Analyses of horn fly counts within and among cattle across different pastures were quantified from 2023 to 2024. Taylor's power law (TPL) regressions indicated that the spatial distribution of horn flies is highly aggregated on cattle, and density estimation models based on the proportion of animals infested above selected tally thresholds revealed strong predictive relationships. A tally threshold of 350 horn flies/animal resulted in the strongest relationship for all herd types (R2 = 0.92). These relationships were validated with independent field data, and Wald's sequential probability ratio test was used to develop upper and lower sequential sampling stop lines based on ratios of infested cattle equating to estimated economic thresholds (ETs). Sampling plans were further validated, using resampling validation software, and average sample numbers along with operating characteristics among ETs were calculated. Sampling requirements for management decisions were low, indicating a high probability of adoption for our sampling program. This study is the first to develop and validate a BSS plan for H. irritans on cattle in pasture settings and implementation of this efficient plan has the potential for major economic returns from horn fly management.
Strawberry sap beetles, Stelidota geminata (Say), are a pest problem in strawberry production in the northeast United States. Both adults and larvae reduce the quality and yield as the result of feeding directly on ripe, overripe, and decaying berries. This study (i) examined the seasonal activity and feeding injury of sap beetle populations in the matted row and plasticulture production systems, (ii) compared the yield and fruit quality associated with the sap beetle feeding in both production systems, and (iii) evaluated the effects of mulching and the timing of renovation in the matted row system on sap beetle populations. Results provide clear evidence that certain cultural management practices of each production system can significantly reduce the risk of sap beetle injury. The lack of straw mulch in the matted strawberry rows resulted in minor reductions in damaged fruit and sap beetle emergence; however, this is not a common practice in commercial production. The heavy mulch treatment provided no additional protection for sap beetles or any decrease in fruit damage compared to the normal mulched strawberries. The timing of renovation immediately after the last harvest resulted in 60% fewer adults emerging compared to the emergence in plots renovated 10 d later. This practice reduced the number of beetles that leave the strawberry field to infest other host crops, as well as lowering the risk to strawberries the following year. This study provides the first evidence that the annual plasticulture system can significantly reduce the risk of sap beetle feeding injury. Strawberries grown in this system are at less risk to sap beetles because of the physical restrictions of the plastic mulch, along with earlier ripening and less accessibility of berries hanging higher on plants above the mulch layer.
The Old World date mite, Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae), is one of the most economically important pests of date palm (Phoenix dactylifera L.) throughout the Middle East and North Africa. Reliable estimation of mite density is essential for integrated pest management (IPM), but the aggregated spatial distribution of this species reduces the efficiency of conventional fixed-sample-size sampling. This study developed and validated cultivar-specific fixed-precision sequential sampling plans for O. afrasiaticus on the commercially important date palm cultivars Sayer and Barhi using field data collected from commercial orchards in southwestern Iran during 2020 and 2021. Taylor's power law adequately described the variance-mean relationship for both cultivars (R² > 0.93), with aggregation parameters significantly greater than unity (b = 1.380 for Sayer and 1.460 for Barhi), confirming aggregated distributions. Green's fixed-precision sequential sampling model was used to construct stop lines at precision levels of D = 0.25, 0.15, and 0.10. Required sample size decreased with increasing mite density and increased as higher precision was required. Validation using the Resampling for Validation of Sampling Plans (RVSP) procedure with 1,000 iterations and 10 independent datasets showed excellent agreement between target and achieved precision. Mean achieved precision ranged from 0.101 to 0.251 for Sayer and from 0.099 to 0.249 for Barhi, with deviations from target precision below 0.01 for all sampling plans. The proposed sequential sampling plans provide statistically robust and operationally efficient tools for estimating O. afrasiaticus populations and improving pest monitoring and decision-making in date palm IPM programs.
Aggregated pest populations in crop fields pose a significant challenge for efficient detection and economic threshold decisions. Stink bugs are one common pest group that tends to be spatially aggregated and cause localized crop injury within fields. Understanding elements of spatial distribution is important to provide scouting guidance that limits false positive and negative rates when monitoring stink bug populations. The objectives of this study were to characterize the distribution of stink bug species in North Carolina and Virginia soybean fields and to estimate the optimal sample size required for accurate and precise sampling. A standardized sweep sampling protocol was used to measure stink bug populations in 154 commercial soybean fields over three years (2022-2024). We used Taylor's Power Law to characterize the within-field spatial distribution of stink bugs and found a strong spatial aggregation for stink bug species life stages. Furthermore, the estimated aggregation patterns differed among the common species. We used the spatial aggregation parameters to calculate the optimal sample size required to estimate stink bug density at four precision levels. Results showed that increased sampling improved accuracy of density estimations for a given precision, which is an expected outcome of pest sampling analysis focused on aggregated pests. This information on spatial aggregation and sample size will be helpful to improve stink bug scouting in soybeans in the Southeast United States.
The Bemisia tabaci (Gennadius) species complex comprises numerous cryptic species that differ significantly in invasiveness, host range, virus transmission, and insecticide resistance. Accurate and rapid identification of high-risk cryptic species is therefore essential for biosecurity surveillance and diagnostic responses. In Australia, the Mediterranean (MED) and Asia II-1 cryptic species are not present but pose significant incursion risks due to their global spread and biological characteristics. In this study, a novel qPCR assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) region was developed for specific detection of cryptic species Asia II-1 and was multiplexed with an existing MED-specific qPCR assay into a single-tube reaction. The multiplex assay was optimized and evaluated using synthetic gBlock DNA and extensively validated against a broad panel of whitefly specimens, including 13 B. tabaci cryptic species and 10 nontarget whitefly species present in Australia. Both assays demonstrated high sensitivity, efficiency, and specificity in singleplex and multiplex formats, with no cross-reactivity detected in nontarget species. Assay robustness was further confirmed through an interlaboratory ring test conducted across two independent diagnostic laboratories, which achieved complete concordance in species identification and minimal variation in Ct values. This multiplex qPCR assay provides a rapid, reliable, and transferable diagnostic tool for simultaneous detection of two high-risk exotic B. tabaci cryptic species in Australia. Its strong specificity, reproducibility, and compatibility with portable qPCR platforms make it well suited for routine diagnostics and biosecurity surveillance, enabling faster decision-making in response to potential incursions.
Insects rely heavily on olfactory cues to locate, evaluate, and discriminate among potential host plants. Stink bugs are emerging as globally important pests whose success is closely linked to their ability to exploit plant volatile cues during host selection. The brown marmorated stink bug, Halyomorpha halys (Stål, 1855), and the southern green stink bug, Nezara viridula (Linnaeus, 1758), are important invasive pests of crops and fruit orchards, causing serious economic losses worldwide. Tomato is one of their preferred host plants, although the 2 species differ in their preferences for fruit ripening stages. To investigate the olfactory basis of host selection, we collected headspace volatiles from tomato fruits at 3 ripening stages. We analyzed their composition by gas chromatography-mass spectroscopy, with principal component analysis revealing clear differences among stages. Antennally active components within these blends were then identified by gas chromatography-electroantennographic detection using antennae of female and male H. halys and N. viridula, and their activity was subsequently confirmed with electroantennogram recordings with synthetic standards. Several compounds, including 2-carene, α-phellandrene, 3-carene, limonene, and β-phellandrene, consistently elicited antennal responses in both species and sexes. Among these, α- and β-phellandrene elicited the strongest responses. Our results provide the first identification of tomato volatile cues detected by these stink bug species and suggest that these compounds may serve as potential olfactory signals involved in host finding.
UDP-glycosyltransferases (UGTs) are phase II enzymes. They catalyze sugar conjugation of endogenous and xenobiotic compounds. Through this activity, UGTs contribute to insect detoxification and physiology. Our previous work showed that disruption of either SlUGT40D20 or SlUGT40D22 increased susceptibility of Spodoptera litura larvae to Bacillus thuringiensis insecticidal proteins. Here, we used CRISPR/Cas9 mutagenesis to generate 3 additional UGT40 mutant strains: a SlUGT40F25 mutant in the wild-type background, a SlUGT40D20/D22 dual-mutant and a SlUGT40D20/F25 dual-mutant in the SlUGT40D20-KO background. Diet-overlay bioassays showed that all mutant strains were more susceptible than the WT strain to Cry1Ac. The SlUGT40D20/D22 strain had the lowest LC50, although its confidence interval overlapped that of SlUGT40D20-KO; therefore, this combined effect should be interpreted as a trend rather than statistically confirmed additivity. The SlUGT40D20/F25 strain did not differ from SlUGT40D20-KO, consistent with a nonframeshift SlUGT40F25 deletion that retained predicted transmembrane helices. Leaf-dip bioassays indicated no significant changes in responses to most chemical insecticides, except increased mortality of SlUGT40D20/D22 larvae exposed to emamectin benzoate. These findings associate the susceptibility change of S. litura to Cry1Ac and emamectin benzoate with knockout of single or double SlUGT40 genes and support a potential role of UGT40 genes in Cry1Ac tolerance and suggest compound-specific effects on insecticide susceptibility.
Neonicotinoids are a widely utilized class of insecticides that bind to nicotinic acetylcholine receptors as their primary mode of action causing paralysis and death. Neonicotinoids have also been shown to affect various aspects of insect physiology and behavior. Studies were conducted in 3 coleopteran species to determine the effects of neonicotinoids on ecdysis. Neonicotinoids were topically applied to pupae of Tenebrio molitor Linnaeus, Tribolium castaneum Herbst, and Leptinotarsa decemlineata Say at doses that did not have direct paralytic effects. Topical neonicotinoid applications at least 1 d before adult emergence led to arrested ecdysis in all 3 beetle species. Adults developed beneath the pupal cuticle and initiated normal pre-ecdysis movements, including initiating shedding of the abdominal cuticle and tracheal linings. However, the process stalled at this stage; the remaining pupal cuticle and tracheae were not shed, wings remained unexpanded, and the head and thoracic ecdysial lines usually did not break. Despite these impairments, the adults remained alive, as indicated by movements of their antennae and legs. Topical application of neonicotinoids to last-instar larvae of T. molitor and L. decemlineata and penultimate instar of L. decemlineata also caused arrested pupal ecdysis and arrested larval ecdysis, respectively. Data obtained using 4 neonicotinoids suggests nicotinic acetylcholine receptors are involved in the regulation of coleopteran ecdysis. Combined with results of previous research demonstrating neonicotinoids cause arrested ecdysis in Lepidoptera, this suggests that neonicotinoids could be disrupting the function of crustacean cardioactive peptide producing neurons.
Cochliomyia hominivorax (Diptera: Calliphoridae) is an obligate parasitic species and a source of major public health concern as an agent of myiasis in humans, livestock, and wildlife systems, causing millions of dollars in agricultural losses annually. A major approach for screwworm control and eradication is the sterile insect technique (SIT) using gamma radiation. Electron beam (eBeam) radiation is a safer and potentially less expensive alternative ionizing radiation source being explored for SIT. To test this method, late-stage pupae of Cochliomyia macellaria, a sister species of C. hominivorax, were exposed to low energy electron beam (LEEB), high energy electron beam (HEEB), and Cs-137 radiation. Treated pupae were allowed to develop to adulthood, and male flies had their testes dissected and analyzed using flow cytometry to evaluate differences in ploidy impacts of each radiation type. Results indicated that the ploidy and abundance of highly polyploid nuclei, most notably 32C nuclei, significantly increased in flies irradiated with HEEB and Cs-137. Flies treated with Cs-137 also had increased amounts of underreplicated nuclei that were not typically seen in control flies or in flies treated with LEEB or HEEB. Results from a small sample size of frozen C. hominivorax adults treated with similar doses of Co-60 indicated similar trends of increased 32C and underreplicated nuclei, but more sampling is needed to investigate species and/or radiation type effects. These results indicate that eBeam can induce ploidy changes in treated flies and highlight the potential of flow cytometry as a biological dosimetry tool given further testing.
The neonicotinoid imidacloprid can cause lethal and sublethal effects in non-target organisms, including impaired behavior, reduced fertility, disrupted locomotion, and reduced predatory performance in arthropods. Spiders are among the most abundant arthropod predators in terrestrial ecosystems, including agricultural ecosystems, yet the effects of neonicotinoids on the predatory function of jumping spiders remain poorly understood. The functional response of a predator describes how predation rates change with prey density and is key to understanding predator-prey dynamics. We tested whether acute exposure to a commercial formulation of imidacloprid at field-recommended concentrations alters the functional response of the Aussie bronze jumping spider, Helpis minitabunda (L. Koch, 1880). We compared the predation of treated (imidacloprid) and control (distilled water) spiders across 5 prey densities over a 9-h observation period. Both treated and control spiders exhibited a Type II functional response. However, treated spiders showed more than double the handling time compared to controls, with no difference in attack rate, indicating impaired prey processing efficiency rather than reduced prey detection ability. Overall, treated spiders killed fewer prey than control spiders, and the mean realized predation rate at the highest prey density was more than halved. Control spiders killed more prey as prey density increased over the early observation periods (up to 5 h), whereas treated spiders did not. Our findings demonstrate that acute exposure to field-relevant concentrations of imidacloprid impairs the predatory performance of the jumping spider H. minitabunda, which has potential implications for natural pest suppression by spiders in ecosystems where neonicotinoids are applied.
The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Erebidae), is a highly polyphagous invasive pest that poses increasing economic and ecological threats to woody plants in agricultural, forestry, and peri-urban landscapes. Spatially explicit risk assessment is crucial to support targeted monitoring and management, yet the ecological drivers of its invasion risk often vary geographically. Here, we compiled 671 occurrence records from national pest surveys, GBIF, and literature to model the invasion risk of H. cunea in China using 14 climatic, topographic, vegetation, and anthropogenic predictors. Four non-spatial ensemble machine-learning algorithms were evaluated using 5-fold spatial cross-validation. The optimal model (LightGBM) was interpreted using SHAP and GeoShapley to quantify global, model-level driver importance across the China-wide dataset and geographically varying driver effects. High-risk areas were highly concentrated in the North China Plain, Liaodong Peninsula, Shandong Peninsula, and the Beijing-Tianjin region. Elevation, mean temperature of the warmest quarter, and human population density emerged as the most influential predictors. Notably, GeoShapley revealed that the impacts of elevation and population density are not uniform but are significantly amplified within specific regional hotspots. These findings indicate that uniform, "one-size-fits-all" management strategies may be inefficient. Instead, our geographically explicit framework provides a transparent spatial decision-support tool for plant protection authorities to prioritize targeted surveillance, optimize quarantine deployment, and implement early interventions against H. cunea.
Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant. Although its toxicity to aquatic organisms is well known, research on terrestrial arthropods-especially soil-dwelling ants-remains limited. The red imported fire ant (RIFA), (Solenopsis invicta Buren) (Hymenoptera: Formicidae), frequently inhabits soil and is broadly distributed, making it likely to encounter PFOA. This study examined the behavioral, lethal, and physiological effects of PFOA on large and medium RIFA workers. Contact and stomach toxicity experiments showed that PFOA significantly reduced aggregation, climbing, grasping, and crawling over 72 h, with notable mortality at higher concentrations. In contact trials, mortality reached 68.33% (large workers) and 62.50% (medium workers) at 10 mg/g after 48 h, and excavation behavior was markedly inhibited at 0.061-0.61 mg/g. The median lethal concentration (LC50) of PFOA for large workers decreased sharply over time (1660 mg/L on day 3 → 531 mg/L on day 4 → 217 mg/L on day 5). Sublethal colony-level effects emerged by day 21 (28.58%, 30.94%, and 42.55% mortality at 10, 100, and 500 mg/L, respectively). Catalase (CAT) activity increased dose-dependently, indicating oxidative stress (eg from 451 to 2412 U/g in large workers after contact exposure). Despite PFOA exposure (up to 250 mg/L), RIFA maintained higher attack initiation and more intense aggression than Carebara diversa, which suffered 90% to 100% mortality after confrontation in 180 min. Collectively, RIFA exhibits considerable tolerance to PFOA. While PFOA induces sublethal physiological and behavioral impairments, it does not overturn RIFA's competitive dominance, suggesting that environmental contamination alone may not significantly mitigate this ant's confrontation.
