Insects

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control. Full article

Nosema ceranae (N. ceranae) infection is prevalent globally, causing a decline in bee populations and significant economic losses to apiarists. Although several methods have been proposed for diagnosing Nosema infections, limitations in these methods have hindered their broad applications. Therefore, this current study aimed to develop a specialized method for diagnosing Nosema infections. To achieve this, a sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatography assay (ICG) were developed, and their effectiveness in screening and diagnosing Nosema infection was assessed. In sandwich ELISA, the combination of the monoclonal antibodies (mAb) 19B2 and biotinylated-19B2 exhibited stronger binding affinity to the antigen than did other combinations of mAbs that were tested. Furthermore, the antigen detection limit achieved with the sandwich ELISA surpassed that previously reported with Western blotting. The ICG was designed using the same antibody combination as that used in sandwich ELISA; however, the assay exhibited a lower diagnostic ability for Nosema infection than the ELISA. The diagnostic models developed in this study offer practical applications for conducting rapid nosemosis detection tests. These innovative techniques will help to improve the timely identification and management of nosemosis. Full article

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of “Candidatus Liberibacter asiaticus”, the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi “penetrance” and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12–34% and 18–39%, 5 days post-IAP on dsRNA at 10–500 and 100–500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance. Full article

The phylogenetic status of the family Nitidulidae and its sister group relationship remain controversial. Also, the status of the subfamily Meligethinae is not fully understood, and previous studies have been mainly based on morphology, molecular fragments, and biological habits, rather than the analysis of the complete mitochondrial genome. Up to now, there has been no complete mitochondrial genome report of Meligethinae. In this study, the complete mitochondrial genomes of Meligethinus tschungseni and Brassicogethes affinis (both from China) were provided, and they were compared with the existing complete mitochondrial genomes of Nitidulidae. The phylogenetic analysis among 20 species of Coleoptera was reconstructed via PhyloBayes analysis and Maximum likelihood (ML) analysis, respectively. The results showed that the full lengths of Meligethinus tschungseni and Brassicogethes affinis were 15,783 bp and 16,622 bp, and the AT contents were 77% and 76.7%, respectively. Each complete mitochondrial genome contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (A + T-rich region). All the PCGs begin with the standard start codon ATN (ATA, ATT, ATG, ATC). All the PCGs terminate with a complete terminal codon, TAA or TAG, except cox1, cox2, nad4, and nad5, which terminate with a single T. Furthermore, all the tRNAs have a typical clover-leaf secondary structure except trnS1, whose DHU arm is missing in both species. The two newly sequenced species have different numbers and lengths of tandem repeat regions in their control regions. Based on the genetic distance and Ka/Ks analysis, nad6 showed a higher variability and faster evolutionary rate. Based on the available complete mitochondrial genomes, the results showed that the four subfamilies (Nitidulinae, Meligethinae, Carpophilinae, Epuraeinae) of Nitidulidae formed a monophyletic group and further supported the sister group relationship of Nitidulidae + Kateretidae. In addition, the taxonomic status of Meligethinae and the sister group relationship between Meligethinae and Nitidulinae (the latter as currently circumscribed) were also preliminarily explored. Full article

The application of the sterile insect technique (SIT) requires the adaptation of insects to mass-rearing conditions. It is generally accepted that this adaptation may include a reduction in genetic diversity and an associated loss of desirable characteristics for the effective performance of sterile insects in the field. Here, we compare the genetic diversity of two mass-reared strains of the Mexican fruit fly, Anastrepha ludens, and a wild (WIL) population collected near Tapachula, Mexico, using seven DNA microsatellites as molecular genetic markers. The mass-reared strains were a bisexual laboratory strain (LAB) with approximately 130 generations under mass-rearing and a genetic sexing strain, Tapachula-7 (TA7), also under mass-rearing for 100 generations. Our results revealed an overall low level of genetic differentiation (approximately 15%) among the three strains, with the LAB and WIL populations being genetically most similar and TA7 most genetically differentiated. Although there were some differences in allele frequencies between strains, our results show that overall, the adaptation to mass-rearing conditions did not reduce genetic variability compared to the wild sample in terms of heterozygosity or allelic richness, nor did it appear to alter the level of inbreeding with respect to the wild populations. These results are contrary to the general idea that mass-rearing always results in a reduction in genetic diversity. Overall, our findings can contribute to a better understanding of the impact that adaptation to mass-rearing conditions may have on the genetic make-up of strains. Full article

Biogeographically, Anatolia harbours a rich diversity of short-horned grasshoppers (Orthoptera, Caelifera). The number of species recorded from Anatolia so far stands at 300. They inhabit diverse habitats ranging from arid Eremial to Euro-Siberian-like montane meadows, aligning with the topographical and climatological heterogeneity of Anatolia. Alongside some swarming species, the pest potential of several pullulating species needs attention. This is especially important concerning global warming, a scenario expected to be more severe in the Northern Mediterranean Basin in general and Anatolia specifically. A faunal list of biogeographic Anatolia, the area extending from the Aegean Sea in the west to the intermountain basin of the Caucasus in the northeast, the lowlands of Lake Urmia in the east, and Mesopotamia in the southeast, was developed. The recorded species were classified according to the phytogeographical provinces of Anatolia. Distributions of the species with the potential for pullulating were modelled using ecological-niche-modelling approaches for the present and future. The results have the potential to lead to the development of a concept that merges biogeography and the pest potential of certain Anatolian grasshopper species. Our results reveal the following: (i) Acrididae and Pamphagidae are the most diverse families represented in Anatolia; (ii) roughly 40% of Caelifera and 71% of Pamphagidae are endemics, suggesting Anatolia is a biodiversity hotspot; (iii) according to Caelifera diversity, the phytogeographical provinces of Anatolia follow an order of Irano-Anatolia, Euro-Siberia, Mediterranean, and Mesopotamia; and (iv) based on ecological modelling and personal observations, Dociostaurus maroccanus, Locusta migratoria, Calliptamus italicus, Heteracris pterosticha, Notostaurus anatolicus, Oedipoda miniata, and O. schochii should be monitored regarding their pest potential. Full article

Toxicological studies have shown that the American cockroach Periplaneta americana (Linnaeus) is a classical model for studying the mode of action of commonly used insecticides. In a previous study, we demonstrated that thiamethoxam and clothianidin decreased locomotor activity in an open-field-like apparatus. Here, we tested the effect of the neonicotinoid acetamiprid when applied orally, topically, or injected into the haemolymph. We found that acetamiprid was also able to impair locomotor activity in the open-field-like apparatus. When treated with acetamiprid, a strong alteration in locomotor activity was observed 1 h, 24 h, and 48 h after haemolymph and topical applications. Oral application induced an impairment of locomotor activity at 24 h and 48 h. A comparison of the present data with our previously published results showed that neonicotinoids were more active when injected into the haemolymph compared to oral and topical applications. These findings increased our understanding of the effect of neonicotinoid insecticides on insect locomotor activity, and demonstrated that the cyano-substituted neonicotinoid, acetamiprid, was able to alter cockroach locomotor activity. Full article

Predator–prey coevolution, particularly chemo-ecological arms races, is challenging to study as it requires the integration of behavioral, chemical ecology, and phylogenetic studies in an amenable system. Moths of the genus Hemileuca (Saturniidae) are colorful, diurnal, and fast and often fly well above the vegetation canopy layer. However, several Hemileuca species have been reported as being captured in spider webs, specifically Argiope species (Araneidae). Female Hemileuca are known to produce mating pheromones and spiders of the Araneidae family are known to use pheromone lures to attract lepidopteran prey. We presented primarily female Argiope aurantia, which are attractive to male Anisota pellucida (Saturniidae), to different populations of Hemileuca species across the southern and western United States to categorize the homing response strength of different species of male Hemileuca. When we mapped these Argiope lure attraction strength categories onto the most recently published Hemileuca phylogeny, the behavioral patterns suggested a potential co-evolutionary arms race between predators and prey. Males of Hemileuca maia, H. grotei, and H. nevadensis (all in the same clade) appeared to have no attraction to A. aurantia, while H. magnifica and H. hera (within a different, separate clade) appeared to be strongly attracted to A. aurantia, but H. nuttalli (also within the H. hera and H. magnifica clade) displayed no attraction. Furthermore, Hemileuca eglanterina (yet a different clade) displayed strong, weak, and no attraction to A. aurantia, depending on the population. These apparent clade partitioning patterns of Argiope lure effectiveness and within-species variation in Hemileuca lure responses suggest a predator–prey coevolutionary history of measures and countermeasures. Full article

In strawberry production, western flower thrips (WFT) and two-spotted spider mites (TSSM) inflict feeding damage and reduce the yield. Biological control for these pests often includes phytoseiid predatory mites and entomopathogenic fungi. The hemipteran family Nabidae have been reported as prominent predators in open-field strawberry. Nabis americoferus Carayon is a new biocontrol agent developed in Canada. This study examined if this species was a good candidate for integration with biological control for greenhouse strawberry production. The laboratory trials showed that Phytoseiulus persimilis Athias-Henriot and Amblyseius swirskii Athias-Henriot were compatible with N. americoferus, especially when alternative food was available. In contrast, the nabid was not compatible with the Beauveria bassiana (Balsamo) GHA strain. A greenhouse cage study was conducted to determine if it was beneficial to add N. americoferus to the phytoseiid-mites-based biological control program for WFT and TSSM in greenhouse strawberry. The release of N. americoferus on a banker plant together with the placement of sachets of Neoseiulus cucumeris (Oudemans) and Neoseiulus californicus (McGregor) was beneficial, not only potentially reducing the number of sachet applications, but also providing better pest control than phytoseiid mites alone. Neither the phytoseiids nor the N. americoferus numbers were significantly affected by the presence of each other. Full article

Chironomids are found in all types of freshwater habitats; they are a ubiquitous and highly diverse group of aquatic insects. Plitvice Lakes National Park is the oldest and largest national park in Croatia and consists of numerous and diverse freshwater habitats, making the area an ideal location for long-term research into the chironomid emergence patterns and phenology. The main objectives of this study were to identify the composition of the chironomid community, determine the phenology of the identified species, and assess the main factors influencing their emergence in Plitvice Lakes. During 14 years of research, more than 13,000 chironomids belonging to more than 80 species were recorded. The most abundant species was found to be Parametriocnemus stylatus. The highest abundance of chironomids was recorded in lotic habitats with faster water current over substrates of moss and algae and pebbles. Water temperature and the availability of organic matter were found to be the main factors that drive chironomid emergence at the tufa barrier studied. In the last years of this study, a prolonged flight period was observed. Although this is not statistically significant (at this stage of the study), it could be due to a higher water temperature in winter. Full article

Monolepta is one of the diverse genera in the subfamily Galerucinae, including 708 species and 6 sub-species worldwide. To explore the information on the mitogenome characteristics and phylogeny of the section “Monoleptites”, especially the genus Monolepta, we obtained the newly completed mitochondrial genomes (mitogenomes) of four Monolepta species using high-throughput sequencing technology. The lengths of these four new mitochondrial genomes are 16,672 bp, 16,965 bp, 16,012 bp, and 15,866 bp in size, respectively. All four mitochondrial genomes include 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), and one control region, which is consistent with other Coleoptera. The results of the nonsynonymous with synonymous substitution rates showed that ND6 had the highest evolution rate, while COI displayed the lowest evolution rate. The substitution saturation of three datasets (13 PCGs_codon1, 13 PCGs_codon2, 13 PCGs_codon3) showed that there was no saturation across all datasets. Phylogenetic analyses based on three datasets (ND1, 15 genes of mitogenomes, and 13 PCGs_AA) were carried out using maximum likelihood (ML) and Bayesian inference (BI) methods. The results showed that mitogenomes had a greater capacity to resolve the main clades than the ND1 gene at the suprageneric and species levels. The section “Monoleptites” was proven to be a monophyletic group, while Monolepta was a non-monophyletic group. Based on ND1 data, the newly sequenced species whose antennal segment 2 was shorter than 3 were split into several clades, while, based on the mitogenomic dataset, the four newly sequenced species had close relationships with Paleosepharia. The species whose antennal segment 2 was as long as 3 were split into two clades, which indicated that the characteristic of “antennal segment 2 as long as 3” of the true “Monolepta” evolved multiple times in several subgroups. Therefore, to explore the relationships among the true Monolepta, the most important thing is to perform a thorough revision of Monolepta and related genera in the future. Full article

We investigated the diversity and occurrence of wood wasps in Lithuania and determined communities of associated fungi. Trapping of wood wasps resulted in three different species, including Sirex juvencus, Urocerus gigas, and Tremex fuscicornis. Fungal culturing from adult females of T. fuscicornis mainly resulted in fungi from the genera Penicillium and Trichoderma. High-throughput sequencing of ITS2 rDNA resulted in 59,797 high-quality fungal sequences, representing 127 fungal OTUs. There were 93 fungal OTUs detected in U. gigas, 66 in S. juvencus, and 10 in T. fuscicornis. The most common fungi were Fusarium sporotrichioides (63.1% of all fungal sequences), Amylostereum chailletii (14.9%), Penicillium crustosum (7.8%), Microascus sp. 2261_4 (5.0%), and Pithoascus ater (2.1%). Among these, only A. chailletii was found in all three insect species with the highest relative abundance in U. gigas (15.2%), followed by S. juvencus (7.7%), and the lowest in T. fuscicornis (0.3%) (p < 0.0003). Correspondence analysis of fungal communities showed a distant placement of different species of wood wasps, indicating that fungal communities in each of these were largely different. In conclusion, the study showed that the economically important tree pathogen A. chailletii was among the most common fungal OTUs vectored by siricid wood wasps. Full article

In 2020, the invasive Thrips parvispinus (Karny) was first detected in Florida, United States. In response to the implemented regulatory restrictions, we conducted laboratory experiments under containment conditions. Thrips larvae and adults were exposed to 32 products (conventional and biorational insecticides) either directly or indirectly. Direct exposure was performed using a Spray Potter Tower, while indirect exposure was conducted by evaluating residue toxicity against the thrips. Water served as a control. We assessed mortality and leaf-feeding damage 48 h post-treatment. Among the conventional insecticides, chlorfenapyr, sulfoxaflor-spinetoram, and spinosad caused high mortality across all stages in both direct and residue toxicity assays. Pyridalyl, acetamiprid, tolfenpyrad, cyclaniliprole-flonicamid, acephate, novaluron, abamectin, cyantraniliprole, imidacloprid, cyclaniliprole, spirotetramat, and carbaryl displayed moderate toxicity, affecting at least two stages in either exposure route. Additionally, chlorfenapyr, spinosad, sulfoxaflor-spinetoram, pyridalyl, acetamiprid, cyclaniliprole, cyclaniliprole-flonicamid, abamectin, and acephate inhibited larvae and adult’s leaf-feeding damage in both direct and residue toxicity assays. Regarding biorational insecticides, mineral oil (3%) and sesame oil caused the highest mortality and lowest leaf-feeding damage. Greenhouse evaluations of spinosad, chlorfenapyr, sulfoxaflor-spinetoram, and pyridalyl are recommended. Also, a rotation program incorporating these products, while considering different modes of action, is advised for ornamental growers to avoid resistance and to comply with regulations. Full article

We evaluated a novel push–pull control strategy for protecting highbush blueberry, Vaccinium corymbosum, against spotted-wing drosophila (SWD), Drosophila suzukii. Methyl benzoate (MB) was used as the pushing agent and a previously tested SWD attractive blend of lure-scents was used as the pulling agent. MB dispensers (push) were hung in the canopy and lure-scent dispensers (pull) were hung in yellow jacket traps filled with soapy water around the blueberry bushes. Blueberries were sampled weekly, and any infestation was inspected by examining the breathing tubes of SWD eggs which protrude through the skin of infested fruit. The frequency of infestation, i.e., the proportion of berries infested with at least one egg, and the extent of infestation, i.e., the mean number of eggs in infested berries, were significantly reduced in treatments receiving MB dispensers as a pushing agent when infestation rates were very high. However, the mass trapping devices as a pulling agent did not provide comparable protection on their own and did not produce additive protection when used in combination with the MB dispensers in push–pull trials. We conclude that MB has the potential to be implemented as a spatial repellent/oviposition deterrent to reduce SWD damage in blueberry under field conditions and does not require the SWD attractant as a pulling agent to achieve crop protection. Full article

The morphological diversity of insect mouthparts is closely related to changes in food sources and diets. Research into the structures of insect mouthparts may help to establish a fundamental basis for a better understanding of insect feeding mechanisms. In this study, we examined the fine morphology of the mouthparts of Illeis chinensis using scanning electron microscopy. We paid particular attention to the types, quantities, and distribution of sensilla on the mouthparts. Our results showed that the basic components of the mouthparts of I. chinensis are the same as those in other lady beetles, i.e., the labrum, mandible, maxillae, labium, and hypopharynx. We also found structural specialization indicating adaptation to fungal feeding. On the mouthparts, there are eight kinds of sensilla and two kinds of glandular structures, including sensilla chaetica, sensilla basiconica, sensilla styloconica, sensilla coeloconica, sensilla campaniformia, sensilla placodea, sensilla digitiformia, Böhm bristles, perforated plates, and cuticular pores. This is the first time that sensilla digitiformia has been reported in ladybirds. Finally, variations in mouthparts among ladybirds with differing diets, as well as the putative functions of each of the mouthparts and sensilla, were discussed. This research can provide a reference for understanding the functions of the mouthparts in ladybird feeding behavior and thereby contribute to the development of precise insect behavior regulation and management strategies. Full article

Mosquito-borne diseases (MBDs) are important emerging diseases that affect humans and animals. Zoological parks can work as early warning systems for the occurrence of MBDs. In this study, we characterized the mosquito fauna captured inside Lisbon Zoo from May 2018 to November 2019. An average of 2.4 mosquitos per trap/night were captured. Five mosquito species potentially causing MBDs, including Culex pipiens biotypes, were found in the zoo. The sympatric occurrence of Culex pipiens biotypes represents a risk factor for the epizootic transmission of West Nile virus and Usutu virus. The mosquito occurrence followed the expected seasonality, with the maximum densities during summer months. However, mosquito activity was detected in winter months in low numbers. The minimum temperature and the relative humidity (RH) on the day of capture showed a positive effect on Culex pipiens abundance. Contrary, the RH the week before capture and the average precipitation the week of capture had a negative effect. No invasive species were identified, nor have flaviviruses been detected in the mosquitoes. The implementation of biosecurity measures regarding the hygiene of the premises and the strict control of all the animals entering the zoo can justify the low prevalence of mosquitoes and the absence of flavivirus-infected mosquitoes. Full article

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy. Full article

The endemic and endangered Chinese firefly Pyrocoelia pectoralis is a sexually dimorphic, nocturnal species. A previous attempt by this team to assemble a draft genome of P. pectoralis using PacBio and Illumina HiSeq X Ten platforms was limited in its usefulness by high redundancy and contamination. This prompted us to conduct an improved chromosome-level genome assembly of P. pectoralis. Ten chromosomes were further assembled based on Hi-C data to a 532.25 Mb final size with a 52.87 Mb scaffold N50. The total repeat lengths in the genome of P. pectoralis amount to 227.69 Mb; 42.78%. In total, 12,789 genes could be functionally annotated using at least one public database. Phylogenetic inference indicated that P. pectoralis and P. pyralis diverged ~51.41 million years ago. Gene family expansion and contraction analysis of 12 species were performed, and 546 expanded and 2660 contracted gene families were identified in P. pectoralis. We generated a high-quality draft of the P. pectoralis genome. This genome assembly should help promote research on the species’ sexual dimorphism and its unique courtship behavior, which involves a combination of pheromonal and bioluminescent signals. It also can serve as a resource for accelerating genome-assisted improvements in the conservation of this species. Full article

Solenopsis invicta, often known as the red imported fire ants (RIFAs), is a well-known global invasive ant species that can be found in agricultural, urban, and natural environments worldwide. Simultaneously, it also inhabits the soil. Biochar is generated by the pyrolysis of organic matter under high-temperature anoxic environments and widely used in agricultural ecosystems and soil amendment. However, to date, it remains unknown as to whether soil application of biochar has a negative effect on RIFAs. In our study, we investigated the toxicity and irritability effects of different amounts of biochar (0%, 1%, 2%, 5%, 10%, and 20%) introduced into the soil on red fire ants; upon comparison with the control soil (0% biochar), the application of 1%, 2%, and 5% biochar did not result in significantly different results. But the utilization of biochar at a concentration over 10% effectively repelled the RIFAs, resulting in their departure from the treated soils. High doses of biochar were able to cause death of red fire ants; the mortality rate of red fire ants reached 55.56% after 11 days of 20% biochar treatment. We also evaluated the effects of biochar on four behaviors of red fire ants, namely aggregation, walking, grasping, and attacking; 20% of the biochar treatment group reduced aggregation by 64.22% and this value was 55.22%, 68.44%, and 62.36% for walking, grasping, and attacking. Finally, we measured the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activity and malondialdehyde (MDA) content in red fire ants; the results showed that the activities of the three enzymes increased with the increase in biochar addition, which indicated that a high dose of biochar induced oxidative stress in red fire ants. Our results indicate that biochar has the potential to cause toxicity and repel red imported fire ants (RIFAs) in a manner that is dependent on the concentration. We propose that biochar could be utilized in the control and manufacturing of baits for red fire ant management. This work establishes a foundation for the prevention and management of red fire ants and the logical utilization of biochar. Full article