Genetic characterization of 2 Ceutorhynchus (Coleoptera: Curculionidae) weevils with mitogenomes and insights into the phylogeny and evolution of related weevils.

The rape stem weevil (Ceutorhynchus asper Roel.) and its close relatives primarily breed on cruciferous plants and cause severe damage to rapeseed production. However, their genetic and molecular information is still scarce. Here, we generated mitogenomes for both C. asper and Ceutorhynchus albosuturalis. The lengths of the 2 mitochondrial genomes are 14,207 bp (C. asper) and 15,373 bp (C. albosuturalis), and both weevils exhibit identical numbers of protein-coding genes with the absence of trnI. A + T contents for both mitogenomes are high (80% and 79.9%, respectively). Haplotype and genetic distance analyses showed that the genetic differentiation of C. asper populations in northwestern China is low. Based on 5 datasets from mitogenomes, phylogenetic analyses with maximum-likelihood and Bayesian methods show that both species (C. asper and C. albosuturalis) fall in the CCCMS clade (Curculioninae, Conoderinae, Cossoninae, Molytinae, and Scolytinae) of Curculionidae and belong to clades H and I of the genus Ceutorhynchus, respectively. Larvae of the clade H weevils mainly are borers in petioles or stems of cruciferous plants, while larvae of the clade I weevils mainly inhabit the fruits of the same plants, suggesting that ecological niche specialization can play a critical role in the diversification of Ceutorhynchus species. This study generates baseline molecular and genetic information for future research of Ceutorhynchus-related taxa and provides insights into the phylogeny and evolution of Curculionidae.

The impact of climate change and the conservation of the keystone Asian honeybee using niche models and systematic prioritization.

Global warming has seriously disturbed the Earth's ecosystems, and in this context, Asian honeybee (Apis cerana) has experienced a dramatic decline in recent decades. Here, we examined both direct and indirect effects of climate change on A. cerana through ecological niche modeling of A. cerana, and its disease pathogens (i.e., Chinese sacbrood virus and Melissococcus plutonius) and enemies (i.e., Galleria mellonella and Vespa mandarinia). Ecological niche modeling predicts that climate change will increase the potential suitability of A. cerana, but it will also cause some of the original habitat areas to become unsuitable. Outbreak risks of Chinese sacbrood disease and European Foulbrood will increase dramatically, while those of G. mellonella and V. mandarinia will decrease only slightly. Thus, climate change will produce an unfavorable situation for even maintaining some A. cerana populations in China in the future. Genetic structure analyses showed that the A. cerana population from Hainan Island had significant genetic differentiation from that of the mainland, and there was almost no gene flow between the 2, suggesting that urgent measures are needed to protect the unique genetic resources there. Through taking an integrated planning technique with the Marxan approach, we optimized conservation planning, and identified potential nature reserves (mainly in western Sichuan and southern Tibet) for conservation of A. cerana populations. Our results can provide insights into the potential impact of climate change on A. cerana, and will help to promote the conservation of the keystone honeybee in China and the long-term sustainability of its ecosystem services.

Nickel-Catalyzed Remote Asymmetric Hydroalkylation of Alkenyl Ethers to Access Ethers of Chiral Dialkyl Carbinols.

Site- and enantio-selective alkyl-alkyl bond formation is privileged in the retrosynthetic analysis due to the universality of sp3-hybridized carbon atoms in organic molecules. Herein, we report a nickel-catalyzed remote asymmetric hydroalkylation of alkenyl ethers via synchronous implementation of alkene isomerization and enantioselective C(sp3)-C(sp3) bond formation. Regression analysis of catalyst structure-activity relationships accelerates the rational ligand modification through modular regulation. This reaction has several advantages for synthesizing chiral dialkyl carbinols and their ether derivatives, including the broad substrate scope, good functional group tolerance, excellent regioselectivity (>20:1 regioisomeric ratio), and high enantioselectivity (up to 95% enantiomeric excess).

Photocatalytic Alkyl Radical Addition Tandem Oxidation of Alkenyl Borates.

Photocatalytic oxidation is a popular transformation way for organic synthesis and is widely applied in academia and industry. Herein, we report a blue light-induced alkylation-oxidation tandem reaction for the synthesis of diverse ketones by combining alkyl radical addition and oxidation of alkenyl borates. This reaction shows excellent functional group compatibility in acceptable yields, and diversity of radical precursors is applicable.

Ligand-Controlled Stereoselective Synthesis of 2-Deoxy-ß-C-glycosides by Cobalt Catalysis.

2-Deoxy-ß-C-glycosides represent an important class of carbohydrates that are present in many bioactive molecules. However, owing to the lack of substituents at the C2 position, the stereoselective synthesis of 2-deoxy-ß-C-glycosides is highly challenging. Herein, we report a ligand-controlled stereoselective C-alkyl glycosylation reaction to access 2-deoxy-ß-C-alkyl glycosides from readily available glycals and alkyl halides. This method exhibits broad substrate scope and excellent diastereoselectivity under very mild conditions. In addition, unprecedented stereodivergent synthesis of 2-deoxy-C-ribofuranosides is achieved using different chiral bisoxazoline ligands. Mechanistic studies suggest that hydrometallation of the glycal with the bisoxazoline-ligated Co-H species may be the turnover-limiting and stereodetermining step of this transformation.

Nucleophilicity Prediction Using Graph Neural Networks.

The quantitative description between chemical reaction rates and nucleophilicity parameters plays a crucial role in organic chemistry. In this regard, the formula proposed by Mayr et al. and the constructed reactivity database are important representatives. However, the determination of Mayr's nucleophilicity parameter N often requires time-consuming experiments with reference electrophiles in the solvent. Several machine learning (ML)-based models have been proposed to realize the data-driven prediction of N in recent years. However, in addition to DFT-calculated electronic descriptors, most of them also use a set of artificially predefined structural descriptors as input, which may result in a biased representation of the nucleophile's structural information depending on descriptors' definition preference. Compared with traditional ML algorithms, graph neural networks (GNNs) can naturally take the molecule's structural information into account by applying the message passing technique. We herein proposed a SchNet-based GNN model that only takes the molecular conformation and solvent type as input. The model achieves a comparable performance to the previous benchmark study on 10-fold cross-validation of 894 data points (R2 = 0.91, RMSE = 2.25). To enhance the model's ability to capture the molecule's electronic information, some DFT-calculated parameters are then incorporated into the model via graph global features, and substantial improvement is achieved in the prediction precision (R2 = 0.95, RMSE = 1.63). These results demonstrate that both structural and electronic information are important for the prediction of N, and GNN can integrate these two kinds of information more effectively.

Ligand-Controlled Cobalt-Catalyzed Regiodivergent Alkyne Hydroalkylation.

Regiodivergent alkyne hydroalkylation to generate different isomers of an alkene from the same alkyne starting material would be beneficial; however, it remains a challenge. Herein, we report a ligand-controlled cobalt-catalyzed regiodivergent alkyne hydroalkylation. The sensible selection of bisoxazoline (L1) and pyridine-oxazoline (L8) ligands led to reliable and predictable protocols that provided (E)-1,2-disubstituted and 1,1-disubstituted alkenes with high E/Z stereoselectivity and regioisomeric ratio starting from identical terminal alkyne and alkyl halide substrates and produced trisubstituted alkenes in the case of internal alkynes. This method exhibits a broad scope for terminal and internal alkynes with a wide range of activated and unactivated alkyl halides and shows excellent functional group compatibility.

Nickel-Catalyzed Switchable Site-Selective Alkene Hydroalkylation by Temperature Regulation.

Regiodivergent alkene functionalization that produces either regioisomer starting from the same raw materials is desirable. Herein, we report a nickel-catalyzed switchable site-selective alkene hydroalkylation. The selection of reaction temperatures leads to protocols that provide regiodivergent hydroalkylated products starting from a single alkene substrate. This protocol allows the convenient synthesis of α- and ß-branched protected amines, both of which are important to the fields of pharmaceutical chemistry and biochemistry. In addition, enantioenriched ß-branched alkylamines can be accessed in a catalytic asymmetric variant. Preliminary mechanistic studies indicate that the formation of a more stable nickelacycle provides the driving force of migration. The thermodynamic and kinetic properties of different reduction elimination intermediates are responsible for the switchable site-selectivity.

Increasing risk of aphids spreading plant viruses in maize fields on both sides of China's Heihe-Tengchong line under climate change.

BACKGROUND: In the coming decades, geographical distribution patterns of farmland organisms may undergo drastic changes due to climate change, with significant implications for global food security. In China, Rhopalosiphum maidis and its spread of sugarcane mosaic virus (ScMV) can become an increasingly serious threat to maize (Zea mays) production. We conducted ecological niche modeling for Z. mays, R. maidis, and ScMV under current and future (2041-2060 and 2081-2100) climate scenarios by using MaxEnt software to explore changes in this system. RESULTS: The Heihe-Tengchong line (an imaginary separation line of human population density) can divide China into main (east of the line) and secondary (west of the line) habitats for the three species. With climate change, rapid expansion in suitable areas is projected for ScMV and the aphid vector R. maidis. Taking species interactions into consideration, our overlaying analyses show that most areas east of the Heihe-Tengchong line (optimal for maize and suitable for R. maidis) will become increasingly highly suitable for ScMV, suggesting that the prevention and control of this plant virus and its aphid vector in China's main maize-growing areas (e.g. northeast) will become an increasing challenge in the future. CONCLUSION: Climate change will profoundly affect ScMV-vector-maize interactions, which may contribute favorably to invasion of this virus into new areas. Our comprehensive and in-depth analyses on shifts in this multi-species system under climate change provide useful and insightful information for devising strategies for the prevention and control of plant viruses and aphid vectors on maize in the future. © 2022 Society of Chemical Industry.

Rapid Changes in Composition and Contents of Cuticular Hydrocarbons in Sitobion avenae (Hemiptera: Aphididae) Clones Adapting to Desiccation Stress.

Cuticular hydrocarbons (CHCs) are diverse in insects, and include variable classes of cuticular lipids, contributing to waterproofing for insects under desiccation environments. However, this waterproofing function of CHCs is still not well characterized in aphids. In this study, we compared CHC profiles for desiccation-resistant and nonresistant genotypes of the grain aphid, Sitobion avenae (Fabricius), in responses to desiccation. Our result showed that a total of 27 CHCs were detected in S. avenae, and linear alkanes (e.g., n-C29) were found to be the predominant components. Long-chain monomethyl alkanes were found to associate closely with water loss rates in S. avenae in most cases. Resistant genotypes of both wing morphs had higher contents of short-chain n-alkanes under control than nonresistant genotypes, showing the importance of short-chain n-alkanes in constitutive desiccation resistance. Among these, n-C25 might provide a CHC signature to distinguish between desiccation-resistant and nonresistant individuals. Compared with linear alkanes, methyl-branched CHCs appeared to display higher plasticity in rapid responses to desiccation, especially for 2-MeC26, implying that methyl-branched CHCs could be more sensitive to desiccation, and play more important roles in induced desiccation-resistance. Thus, both constitutive and induced CHCs (linear or methyl-branched) can contribute to adaptive responses of S. avenae populations under desiccation environments. Our results provide substantial evidence for adaptive changes of desiccation resistance and associated CHCs in S. avenae, and have significant implications for aphid evolution and management in the context of global climate change.

Structure and distribution of antennal sensilla in Pseudosymmachia flavescens (Brenske) (Coleoptera: Scarabaeidae: Melolonthinae).

Morphology, microstructure, and distribution of antennal sensilla were compared between female and male Pseudosymmachia flavescens (Brenske) (Coleoptera: Scarabaeidae: Melolonthinae). Lamellate antennae of P. flavescens were shown to have typical scape, pedicel, and flagellum segments. The flagellum consists of a four-segmented funicle and a three-segmented club. The lengths of their pedicel and funicle were found to be similar in females and males. Distinct sexual differences were observed in the length of lamellar segments. Nine types/subtypes of sensilla were identified on the antennae of both sexes, including Böhm sensilla, sensilla trichodea, sensilla basiconica (SB), and two subtypes for sensilla chaetica, sensilla coeloconica, and sensilla placodea each. Olfactory sensilla (e.g., SB and placodea) are mainly located on three lamellar segments of the antennal club. Variation was also seen in abundance of various types of antennal sensilla, with males possessing significantly more sensilla than females. Sensilla placodea were the most abundant, and their number in males was twice of that in females, showing a clear sexual dimorphism. The difference in the distribution of sensilla placodea might reflect their roles in sexual chemical communication.

Transition-metal-free decarboxylative thiolation of stable aliphatic carboxylates.

A transition-metal-free decarboxylative thiolation protocol is reported in which primary, secondary, tertiary (hetero)aryl acetates and α-CN substituted acetates undergo the decarboxylative thiolation smoothly, to deliver a variety of functionalized aryl alkyl sulfides in moderate to excellent yields. Aryl diselenides are also amenable substrates for construction of C-Se bonds under the simple and mild reaction conditions. Moreover, the protocol is successfully applied to the late-stage modification of pharmaceutical carboxylates with satisfactory chemoselectivity and functional-group compatibility.

Functional Characterization of Chemosensory Protein AmalCSP5 From Apple Buprestid Beetle, Agrilus mali (Coleoptera: Buprestidae).

In the sensitive and complex chemo-sensation system of insects, chemosensory proteins (CSPs) can facilitate the transfer of chemical information and play important roles for variable behaviors of insects. We cloned the chemosensory protein AmalCSP5 from antennae of the apple buprestid beetle (Agrilus mali Matsumura), a serious invasive pest of wild apple trees. Expression profiling showed that AmalCSP5 was expressed in various tissues, suggesting its significance in multiple physiological activities and behaviors of A. mali. AmalCSP5 was preferentially expressed in female antennae and male abdomens. AmalCSP5 was able to bind a variety of test volatiles, especially alcohols and esters. AmalCSP5 exhibited good binding affinity for all five test secondary compounds (i.e., procyanidin, phlorizin, kaemferol, chlorogenic acid, and rutin), suggesting its preferential binding abilities to nonvolatile host plant secondary metabolites and critical roles in gustatory perception of nonvolatiles. Tyr27 and Ser69 of AmalCSP5 could form hydrogen bonds with hexyl benzoate and hexyl hexanoate, respectively. Procyanidin, the best ligand among all test compounds, could form hydrogen bonds with three amino acid residues (i.e., Arg7, Leu8, and Lys41) of AmalCSP5. Thus, high ligand binding affinity for AmalCSP5 seemed to be dependent mainly on the formation of hydrogen bonds. The putative key amino acid residues of AmalCSP5 can be used as molecular targets for designing and screening new attractants and repellents for A. mali. Our results provide insights into binding interactions of AmalCSP5 with volatile and nonvolatile ligands, and a firm basis for developing eco-friendly management strategies of A. mali.

Analyses of structural dynamics revealed flexible binding mechanism for the Agrilus mali odorant binding protein 8 towards plant volatiles.

BACKGROUND: Volatiles from host plants are an important source of insect pest attractants and repellents. Insect odorant binding proteins (OBPs) have been widely characterized, but the molecular binding dynamics and underlying mechanisms are still not well understood. Thus, we characterized binding characteristics of AmalOBP8 from the apple buprestid beetle (Agrilus mali Matsumura), an unprecedented serious threat to rare apple germplasm resources and local ecosystems. RESULTS: Fluorescence studies demonstrated that the quenching mechanism was clearly static. AmalOBP8 was found to bind with both volatiles at single independent sites. Negative thermodynamic parameters suggested that binding interactions between AmalOBP8 and both volatiles could occur spontaneously. Hydrogen bonding was the key force in AmalOBP8's binding to geranyl formate, for which the amino acid residue Trp106 played a critical role in the binding pocket. Multiple Leu residues in AmalOBP8 created a strong hydrophobic environment, and formed the binding pocket for (Z)-3-hexenyl hexanoate. Compared to classic OBPs, in addition to lack of one disulfide bridge, AmalOBP8 had a small α-helix (α7) at the C-terminus, resulting in greater flexibility and adaptability for this protein to bind with different compound molecules. CONCLUSION: Key residues of AmalOBP8 in binding interactions with plant volatiles were clarified. AmalOPB8 had a large ligand binding spectrum and great flexibility in binding with plant volatiles, providing good molecular targets for screening insect attractants and repellents. Our results can promote understanding of insects' perception of various odorants, and establish a foundation for discovery of new pest control agents. © 2020 Society of Chemical Industry.

Divergence of Desiccation-Related Traits in Sitobion avenae from Northwestern China.

The impact of drought on insects has become increasingly evident in the context of global climate change, but the physiological mechanisms of aphids' responses to desiccating environments are still not well understood. We sampled the wheat aphid Sitobion avenae (Fabricius) (Hemiptera: Aphididae) from arid areas of northwestern China. Both desiccation-resistant and -nonresistant genotypes were identified, providing direct evidence of genetic divergence in desiccation resistance of S. avenae. Resistant genotypes of wingless S. avenae showed longer survival time and LT50 under the desiccation stress (i.e., 10% relative humidity) than nonresistant genotypes, and wingless individuals tended to have higher desiccation resistance than winged ones. Both absolute and relative water contents did not differ between the two kinds of genotypes. Resistant genotypes had lower water loss rates than nonresistant genotypes for both winged and wingless individuals, suggesting that modulation of water loss rates could be the primary strategy in resistance of this aphid against desiccation stress. Contents of cuticular hydrocarbons (CHC) (especially methyl-branched alkanes) showed significant increase for both resistant and nonresistant genotypes after exposure to the desiccation stress for 24 h. Under desiccation stress, survival time was positively correlated with contents of methyl-branched alkanes for resistant genotypes. Thus, the content of methyl-branched alkanes and their high plasticity could be closely linked to water loss rate and desiccation resistance in S. avenae. Our results provide insights into fundamental aspects and underlying mechanisms of desiccation resistance in aphids, and have significant implications for the evolution of aphid populations in the context of global warming.

Transcriptome profiling revealed potentially important roles of defensive gene expression in the divergence of insect biotypes: a case study with the cereal aphid Sitobion avenae.

BACKGROUND: Many insects can develop differential biotypes on variable host plants, but the underlying molecular factors and mechanisms are not well understood. To address this issue, transcriptome profiling analyses were conducted for two biotypes of the cereal aphid, Sitobion avenae (Fabricius), on both original and alternative plants. RESULTS: Comparisons between both biotypes generated 4174 differentially expressed unigenes (DEGs). In their response to host plant shift, 39 DEGs were shared by both biotypes, whereas 126 and 861 DEGs occurred only in biotypes 1 and 3, respectively. MMC (modulated modularity clustering) analyses showed that specific DEGs of biotypes 1 and 3 clustered into five and nine transcriptional modules, respectively. Among these DEGs, defense-related genes underwent intensive expression restructuring in both biotypes. However, biotype 3 was found to have relatively lower gene transcriptional plasticity than biotype 1. Gene enrichment analyses of the abovementioned modules showed functional divergence in defensive DEGs for the two biotypes in response to host transfer. The expression plasticity for some defense related genes was showed to be directly related to fecundity of S. avenae biotypes on both original and alternative plants, suggesting that expression plasticity of key defensive genes could have significant impacts on the adaptive potential and differentiation of S. avenae biotypes on different plants. CONCLUSIONS: The divergence patterns of transcriptional plasticity in defense related genes may play important roles in the phenotypic evolution and differentiation of S. avenae biotypes. Our results can provide insights into the role of gene expression plasticity in the divergence of insect biotypes and adaptive evolution of insect populations.

Genetic Divergence of Two Sitobion avenae Biotypes on Barley and Wheat in China.

Host plant affinity and geographic distance can play critical roles in the genetic divergence of insect herbivores and evolution of insect biotypes, but their relative importance in the divergence of insect populations is still poorly understood. We used microsatellite markers to test the effects of host plant species and geographic distance on divergence of two biotypes of the English grain aphid, Sitobion avenae (Fabricius). We found that clones of S. avenae from western provinces (i.e., Xinjiang, Gansu, Qinghai and Shaanxi) had significantly higher genetic diversity than those from eastern provinces (i.e., Anhui, Henan, Hubei, Zhejiang and Jiangsu), suggesting their differentiation between both areas. Based on genetic diversity and distance estimates, biotype 1 clones of eastern provinces showed high genetic divergence from those of western provinces in many cases. Western clones of S. avenae also showed higher genetic divergence among themselves than eastern clones. The Mantel test identified a significant isolation-by-distance (IBD) effect among different geographic populations of S. avenae, providing additional evidence for a critical role of geography in the genetic structure of both S. avenae biotypes. Genetic differentiation (i.e., FST) between the two biotypes was low in all provinces except Shaanxi. Surprisingly, in our analyses of molecular variance, non-significant genetic differentiation between both biotypes or between barley and wheat clones of S. avenae was identified, showing little contribution of host-plant associated differentiation to the divergence of both biotypes in this aphid. Thus, it is highly likely that the divergence of the two S. avenae biotypes involved more geographic isolation and selection of some form than host plant affinity. Our study can provide insights into understanding of genetic structure of insect populations and the divergence of insect biotypes.

Transcriptome Profiling Revealed Potentially Critical Roles for Digestion and Defense-Related Genes in Insects' Use of Resistant Host Plants: A Case Study with Sitobion Avenae.

Using host plant resistance (HPR) in management of insect pests is often environmentally friendly and suitable for sustainable development of agricultural industries. However, this strategy can be limited by rapid evolution of insect populations that overcome HPR, for which the underlying molecular factors and mechanisms are not well understood. To address this issue, we analyzed transcriptomes of two distinct biotypes of the grain aphid, Sitobion avenae (Fabricius), on wheat and barley. This analysis revealed a large number of differentially expressed genes (DEGs) between biotypes 1 and 3 on wheat and barley. The majority of them were common DEGs occurring on both wheat and barley. GO and KEGG enrichment analyses for these common DEGs demonstrated significant expression divergence between both biotypes in genes associated with digestion and defense. Top defense-related common DEGs with the most significant expression changes included three peroxidases, two UGTs (UDP-glycosyltransferase), two cuticle proteins, one glutathione S-transferases (GST), one superoxide dismutase, and one esterase, suggesting their potentially critical roles in the divergence of S. avenae biotypes. A relatively high number of specific DEGs on wheat were identified for peroxidases (9) and P450s (8), indicating that phenolic compounds and hydroxamic acids may play key roles in resistance of wheat against S. avenae. Enrichment of specific DEGs on barley for P450s and ABC transporters suggested their key roles in this aphid's detoxification against secondary metabolites (e.g., alkaloids) in barley. Our results can provide insights into the molecular factors and functions that explain biotype adaptation in insects and their use of resistant plants. This study also has significant implications for developing new resistant cultivars, developing strategies that limit rapid development of insect biotypes, and extending resistant crop cultivars' durability and sustainability in integrated management programs.

Identification and Genetic Differentiation of Sitobion avenae (Hemiptera: Aphididae) Biotypes in China.

The development of biotypes of the cereal aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae), was initially found only on wheat, but barley can also be critical in the process. To address this issue, S. avenae clones were collected on barley and wheat, genotyped with six microsatellite markers, and tested with 58 wheat/barley varieties. Based on the virulence response profiles on different resistant wheat/barley varieties and three susceptible controls, six biotypes of S. avenae were identified. We developed a new system to distinguish between S. avenae biotypes by using only five barley/wheat varieties (i.e., barley: Dulihuang, Zaoshu No.3, Xiyin No.2; wheat: Zhong 4 wumang, 186-TM12-34). The unique virulence profiles of different S. avenae biotypes were further verified by testing their life-history traits (i.e., 10-d fecundity and total developmental time of nymphs) on the abovementioned five barley/wheat varieties. Among all the identified biotypes, biotype 1 was predominant, occupying over 82% of the total in each province. Biotype 5 was found only in Xinjiang, whereas biotype 6 occurred only in Zhejiang. The principal coordinate analysis with microsatellite data suggested apparently low genetic differentiation between biotypes 1 and 2. In most cases, extents of genetic divergence between different S. avenae biotypes could reflect differences in virulence response profiles of these biotypes, implying a genetic component for evolutionary relationships among these biotypes. Our study provides insights into the development and evolution of aphid biotypes, and a firm basis for clarifying the underlying genetic and evolutionary mechanisms.

Electrophysiological and Alarm Responses of Solenopsis invicta Buren (Hymenoptera: Formicidae) to 2-Ethyl-3,5-dimethylpyrazine (Short Title: EAG and Behavioral Responses of Fire Ants to Pyrazine).

2-Ethyl-3,5-dimethylpyrazine is an isomer of 2-ethyl-3,6-dimethylpyrazine, the alarm pheromone component of the red imported fire ant, Solenopsis invicta Buren. The pyrazine was synthesized and its alarm activity was investigated under laboratory conditions. It elicited significant electroantennogram (EAG) activities, and released characteristic alarm behaviors in fire ant workers. The EAG and alarm responses were both dose-dependent. Two doses of the pyrazine, 1 and 100 ng, were further subjected to bait discovery bioassays. Fire ant workers excited by the pyrazine were attracted to food baits, and their numbers increased over time. Ants displayed very similar response patterns to both low and high doses of the pyrazine. The pyrazine impregnated onto filter paper disc attracted significantly more fire ant workers than the hexane control for all observation time intervals at the low dose, and in the first 15 min period at the high dose. The pyrazine loaded onto food bait directly tended to attract more fire ant workers than the hexane control. These results support the potential use of 2-ethyl-3,5-dimethylpyrazine to enhance bait attractiveness for the control of S. invicta in invaded regions.