Despite the potential ecological and economic impacts of invasive species, there is a dearth of data on the presence, impacts, and management implications of potentially invasive Orthoptera species. This lack of research and inconsistent data, including risk screenings and impact assessments, is especially evident in Europe. Consequently, assessing the status, distribution, and potential threats of nonnative Orthoptera in Europe remains challenging, impeding the development of effective management strategies. To address this gap, we call for increased efforts to collect and curate data on non-native and possibly invasive Orthoptera in Europe. Such efforts will improve our understanding of this order’s invasion dynamics, facilitate the identification of priority areas for conservation, and support the development of effective management policies and preventive measures.

Insects are the most diverse group of organisms in the animal kingdom, and some species exhibit complex social behaviors. Although research on insect object use is still in its early stages, insects have already been shown to display rich object-use behaviors. This review focuses on patterns and behavioral flexibility in insect object-use behavior, and the role of cultural evolution in the development of object-use behaviors. Object use in insects is not widespread but has been documented in a diverse set of taxa. Some insects can use objects flexibly and display various object-use patterns. Like mammals and birds, insects use objects in diverse activities, including foraging, predator defense, courtship, and play. Intelligence, pre-existing manipulative behaviors, and anatomical structure affect innovations in object use. In addition, learning and imitation are the main mechanisms underlying the spread of object-use behaviors within populations. Given that insects are one of the major animal groups engaging in object use, studies of insect object use could provide general insights into object use in the animal kingdom.

Although CRISPR/Cas9 has been widely used in insect gene editing, the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with Sogatella furcifera, the white-backed planthopper (WBPH), a significant pest of Oryza sativa (rice) that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)-CRISPR, in which Cas9 and single-guide RNAs (sgRNAs) for the WBPH eye pigment gene tryptophan 2, 3-dioxygenase were injected into the hemocoel of adult females. Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components. An evaluation of the offspring indicated that the highest G0 gene-edited efficacy (56.7%) occurred in females injected 2 d posteclosion, and that those mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.

MicroRNAs play critical roles in multiple developmental processes in insects. Our previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands, thereby improving the silk production capacity. In this study, we elucidate the molecular mechanism underlying of let-7 regulates growth. Identification of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication. let-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN. Dual-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7, with only 1 binding site on its 3’-untranslated region. RNA interference of BmCDK1 inhibited cell proliferation, but this effect was counteracted by co-transfection with let-7 antagomir. Moreover, let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues, and further induced endomitosis in the silk gland in vivo. Knock down of BmCDK1 resulted in abnormal formation of a new epidermis, and larval development was arrested at the 2nd or 3rd molt stage. Taken together, our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination, possessing potential for improving silk yield in silkworm.

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm,Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50 708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles.

Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.

Many aphid species exhibit both cyclical parthenogenesis (CP) and the obligate parthenogenesis (OP) life history, which are genetically determined. In CP aphid lineages, the parthenogenetic individuals can switch from asexual to sexual reproduction quickly in response to environmental factors such as changes in photoperiod and temperature. However, the OP aphid lineages do not undergo sexual reproduction under any conditions. So far, mechanisms underlying the reproduction switch in CP aphids have not been fully elucidated. Rhopalosiphum padi, a serious worldwide insect pest of wheat, has both CP and OP lineages. Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that participate in the metabolic detoxification of xenobiotics. Here, we identified 43 RpUGT genes from R. padi genome and transcriptome sequences, and found that: (1) the UGT content of the CP lineage was significantly higher than that in the OP lineage at the key time points when CP lineage mainly produce virginoparae, gynoparae, and males under inducing condition, while there were no significant difference under normal conditions; (2) RpUGT344J7 gene was highly expressed during the time points when CP lineages produce gynopara and males; (3) the critical time points for CP lineages to produce virginoparaee, gynoparae, and males were affected when the CP lineages were injected with dsRpUGT344J7; (4) the knockdown of RpUGT344J7 caused a significant reduction in the total number of virginoparae, gynoparae, and males in the offspring under inducing condition. The findings contribute to our understanding of the molecular mechanisms underlying the quick shift from asexual to sexual reproduction in aphid species.

The ovary generally undergoes tissue remodeling during larval to pupal transition, which includes membrane degeneration and ovariole growth. At the same time, the hormones produced by insects significantly change during metamorphosis. However, the regulatory mechanism for ovarian development and hormones is not fully understood in insects. Herein, we found that matrix metalloproteinase 2 (MMP2) was highly expressed in the ovarian capsules and ovarioles, and the development was abnormal after knocking out MMP2 in Bombyx mori. The process of abnormal degradation of collagen I due to MMP2 deletion, which resulted in abnormal development of ovarioles and eggs, was analyzed in detail. The proteomics of ovaries in the MMP2-knock out and wild type strains showed a critically significant difference in the expression of a protein, insulin-like peptide (ILP). Additional analysis revealed significant alteration of ILP during ovarian development, and abnormal expression of ILP significantly affected ovarian development in vivo and MMP2 expression in vitro and in vivo. These results showed that MMP2 regulation of ovarian tissue remodeling is closely related to ILP expression. Our study provides new insights into the regulatory mechanism of MMP2 and ovarian development in B. mori.

In insects, the odorant receptor (OR) multigene family evolves by the birth-and-death evolutionary model, according to which the OR repertoire of each species has undergone specific gene gains and losses depending on their chemical environment, resulting in taxon-specific OR lineage radiations with different sizes in the phylogenetic trees. Despite the general divergence in the gene family across different insect orders, the ORs in moths seem to be genetically conserved across species, clustered into 23 major clades containing multiple orthologous groups with single-copy gene from each species. We hypothesized that ORs in these orthologous groups are tuned to ecologically important compounds and functionally conserved. cis-Jasmone is one of the compounds that not only primes the plant defense of neighboring receiver plants, but also functions as a behavior regulator to various insects. To test our hypothesis, using Xenopus oocyte recordings, we functionally assayed the orthologues of BmorOR56, which has been characterized as a specific receptor for cis-jasmone. Our results showed highly conserved response specificity of the BmorOR56 orthologues, with all receptors within this group exclusively responding to cis-jasmone. This is supported by the dN/dS analysis, showing that strong purifying selection is acting on this group. Moreover, molecular docking showed that the ligand binding pockets of BmorOR56 orthologues to cis-jasmone are similar. Taken together, our results suggest the high conservation of OR for ecologically important compounds across Heterocera.

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp,Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A. calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host’s resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1,LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L. serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S. aureus. LsCole and LsDef2 exhibited antibiofilm activity against S. aureus. The 2 AMPs disrupted cell membranes and caused leakage of S. aureus cell contents. The results indicated that the 3 AMPs in L. serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect’s defensive AMP genes.

The brown planthopper (BPH,Nilaparvata lugens) is a notorious sap-sucking insect pest that damages rice (Oryza sativa) plants throughout Asia. During BPH feeding, saliva enters rice plant tissues, whereas during oviposition egg-associated secretions (EAS) are deposited in damaged plant tissue. Dynamic changes in rice to planthopper salivary effectors have been widely reported. However, the effects of EAS from planthopper on rice immunity remains largely unexplored. In this study, we found that both infestation of rice by gravid BPH female adults and treatment with the EAS elicited a strong and rapid accumulation of jasmonic acid (JA), JA-isoleucine, and hydrogen peroxide in rice. EAS enhanced plant defenses not only in rice but also in tobacco, and these impaired the performance of BPH on rice, as well as the performance of aphids and whiteflies on tobacco. High-throughput proteome sequencing of EAS led to 110 proteins being identified and 53 proteins with 2 or more unique peptides being detected. Some proteins from BPH EAS were also found in the salivary proteome from herbivores, suggesting potential evolutionary conservation of effector functions across feeding and oviposition; however, others were only identified in EAS, and these are likely specifically related to oviposition. These findings point to novel proteins affecting interactions between planthoppers and rice during oviposition, providing an additional source of information for effector studies.

Extracellular superoxide dismutase (EcSOD) protects tissues from oxidative stress, and thus is considered as a therapeutic agent for many diseases such as atherosclerosis, hypertension, and cancer. However, cost-effective production of bioactive recombinant human EcSOD (rhEcSOD) remains a challenge. Herein, we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms. rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48 ± 0.21 mg/g. Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50% and a yield of 3.5 ± 0.5 mg/g. Additionally, N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified. The purified rhEcSOD gained the potent enzymatic activity of 4 162 ± 293 U/mg after Cu/Zn ions incorporation. More importantly, rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway. These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.

Culex tarsalis Coquillett (Diptera: Culicidae) mosquitoes are capable of vectoring numerous pathogens affecting public and animal health. Unfortunately, the probing behaviors of mosquitoes are poorly understood because they occur in opaque tissues. Electropenetrography (EPG) has the potential to elucidate these behaviors by recording the electrical signals generated during probing. We used an AC–DC EPG with variable input resistors (Ri levels) to construct a waveform library for Cx. tarsalis feeding on human hands. Biological events associated with mosquito probing were used to characterize waveforms at four Ri levels and with two electrical current types. The optimal settings for EPG recordings of Cx. tarsalis probing on human hands was an Ri level of 10⁷ Ohms using an applied signal of 150 millivolts alternating current. Waveforms for Cx. tarsalis included those previously observed and associated with probing behaviors in Aedes aegypti L. (Diptera: Culicidae): waveform families J (surface salivation), K (stylet penetration through the skin), L (types 1 and 2, search for a blood vessel/ingestion site), M (types 1 and 2, ingestion), N (type 1, an unknown behavior which may be a resting and digestion phase), and W (withdrawal). However, we also observed variations in the waveforms not described in Ae. aegypti, which we named types L3, M3, M4, and N2. This investigation enhances our understanding of mosquito probing behaviors. It also provides a new tool for the automated calculation of peak frequency. This work will facilitate future pathogen acquisition and transmission studies and help identify new pest and disease management targets.

Our understanding of the nervous tissues that affect the wing flapping of insects mainly focuses on the brain, but wing flapping is a rhythmic movement related to the central pattern generator in the ventral nerve cord. To verify whether the neural activity of the abdominal ganglion of the honeybee (Apis mellifera) affects the flapping-wing flight, we profiled the response characteristics of indirect flight muscles to abdominal ganglion excitation. Strikingly, a change in the neural activity of ganglion 3 or ganglion 4 has a stronger effect on the electrophysiological activity of indirect flight muscles than that of ganglion 5. The electrophysiological activity of vertical indirect flight muscles is affected more by the change in neural activity of the abdominal ganglion than that of lateral indirect flight muscles. Moreover, the change in neural activity of the abdominal ganglion mainly causes the change in the muscular activity of indirect wing muscles, but the activity patterns change relatively little and there is little change in the complicated details. This work improves our understanding of the neuroregulatory mechanisms associated with the flapping-wing flight of honeybees.

Sex pheromones are considered to play critical roles in partner communication of most parasitic Hymenoptera. However, the identification of sex pheromone components remains limited to a few families of parasitoid wasps. In this study, we functionally characterized a candidate sex pheromone component in Microplitis mediator (Hymenoptera: Braconidae), a solitary parasitoid of Noctuidae insects. We found that the body surface extract from female wasps could significantly stimulate courtship behavior of males. Gas chromatography-electroantennographic detection (GC-EAD) analysis revealed that a candidate semiochemical from extract triggered significant electrophysiological response of antennae of males. By performing gas chromatography-mass spectrometer (GC-MS) measurement, GC-EAD active compound was identified as n-octyl acrylate, a candidate sex pheromone component in female M. mediator. In electroantennogram (EAG) tests, antennae of male wasps showed significantly higher electrophysiological responses to n-octyl acrylate than those of females. Y-tube olfactometer assays indicated that male wasps significantly chose n-octyl acrylate compared with the control. Furthermore, male wasps showed a remarkable preference for n-octyl acrylate in a simulated field condition behavioral trial; simultaneously,n-octyl acrylate standard could also trigger significant courtship behavior in males. We propose that n-octyl acrylate, as a candidate vital sex pheromone component, could be utilized to design behavioral regulators of M. mediator to implement the protection and utilization of natural enemies.

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid,Diaphorina citri Kuwayama, which serves as vector of phytopathogens causing citrus greening. However, development of resistance to neonicotinoids among populations of D. citri has coincided with occasional control failures in the field. The objectives of this research were to (1) survey current levels of imidacloprid resistance in Florida citrus; (2) compare feeding behavior between imidacloprid-resistant and susceptible D. citri using electrical penetration graph recordings, and (3) investigate the possible amplification of insecticide hormoligosis associated with resistance. Field surveys confirmed that the susceptibility of D. citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population. Following 12 generations of selection, resistance to imidacloprid increased by 438 fold compared with the susceptible strain. Imidacloprid-susceptible D. citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway (C), phloem penetration (D), phloem salivation (E1), and nonprobing (Np) activities than imidacloprid-resistant counterparts. However, there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D. citri. There was no statistical difference in fecundity between resistant and susceptible strains. However, the fecundity of imidacloprid-susceptible female D. citri treated with a sublethal concentration of imidacloprid (LC₂₅) increased significantly compared with controls, while such hormoligosis was less pronounced among imidacloprid-resistant psyllids. Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide, indicative of a behavioral resistance mechanism.

With climate change, the spotted-wing Drosophila (SWD,Drosophila suzukii) invades a great number of fruit production regions worldwide. A plethora of insecticides are being applied for management of this pest. As expected, SWD develops resistance against some potent insecticides or is rather insensitive to some others. Therefore, there is an urgent need to identify and characterize alternative insecticides to control SWD populations. Here, we have studied the effects of the orally applied inhibitor of the fatty acid synthesis pathway spirotetramat on SWD fitness with respect to fecundity and surface barrier function at different stages. In our experiments, we applied spirotetramat mixed with baker’s yeast and showed that females and males were not repelled by effective concentrations of this insecticide. We found that spirotetramat, by trend, lowers egg numbers laid by fed females. These eggs rapidly desiccate, and only a few larvae hatch. Spirotetramat is lethal to larvae and reduces survival of adult flies under low-humidity conditions. Taken together, based on our data, we propose to use yeast supplemented with spirotetramat and additional SWD-specific attractants in traps in non-crop areas in order to eradicate SWD populations before they infest crop production sites.

The black soldier fly,Hermetia illucens, comes with big promises for industrial purposes since its larvae feed polyphagously on a broad spectrum of organic substrates. However, research focusing on adult flies is scarce, which is inconsistent with their reproductive relevance within the rearing cycle. In particular, directed oviposition is a challenge in artificial systems. Currently, decomposing organic matter is commonly used as oviposition substrate, which has extensive potential for improvement in view of the lack of standardization and the risk of microbial contamination. Here, we identified three fatty acids and one fatty acid methyl ester derived from the surface of old oviposition sites and targeted to elucidate their effect on preference behavior and oviposition site selection using Y-olfactometry and prepared oviposition sites, respectively. Exposure to tetradecanoic acid attracted gravid females and stimulated oviposition most strongly, while decanoic acid demonstrated a repulsive effect. Females kept in mixed-sex populations were attracted by tetradecanoic acid, resulting in a higher egg mass found in the compound box (3.0–11.4 fold), a ≥ 2.3 fold reduction of nonspecifically deposited eggs, and the highest total egg mass. Conversely, decanoic and dodecanoic acid caused females to lay a greater proportion of eggs nonspecifically outside both boxes. Our data suggest that fatty acids, especially tetradecanoic acid, are important cues for oviposition site selection in black soldier flies. In order to achieve a directed oviposition behavior, the role of further short- and long-chain fatty acids as attractants should be examined.

Phenylacetaldehyde (PAH), an aromatic compound, is present in a diverse range of fruits including overripe bananas and prickly pear cactus, the two major host fruits for Drosophila melanogaster. PAH acts as a potent ligand for the ionotropic receptor 84a (IR84a) in the adult fruit fly and it is detected by the IR84a/IR8a heterotetrameric complex. Its role in the male courtship behavior through IR84a as an environmental aphrodisiac is of additional importance. In D. melanogaster, two distinct kinds of olfactory receptors, that is, odorant receptors (ORs) and ionotropic receptors (IRs), perceive the odorant stimuli. They display unique structural, molecular, and functional characteristics in addition to having different evolutionary origins. Traditionally, olfactory cues detected by the ORs such as ethyl acetate, 1-butanol, isoamyl acetate, 1-octanol, 4-methylcyclohexanol, etc. classified as aliphatic esters and alcohols have been employed in olfactory classical conditioning using fruit flies. This underlines the participation of OR-activated olfactory pathways in learning and memory formation. Our study elucidates that likewise ethyl acetate (EA) (an OR-responsive odorant), PAH (an IR-responsive aromatic compound) too can form learning and memory when associated with an appetitive gustatory reinforcer. The association of PAH with sucrose (PAH/SUC) led to learning and formation of the long-term memory (LTM). Additionally, the Orco¹,Ir84a^MI00501, and Ir8a¹ mutant flies were used to confirm the exclusive participation of the IR84a/IR8a complex in PAH/SUC olfactory associative conditioning. These results highlight the involvement of IRs via an IR-activated pathway in facilitating robust olfactory behavior.

Genes on the Y chromosome play important roles in male sex determination and development. The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development, but also offers genetic control targets for agricultural pests. However, Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization, especially in the oriental fruit fly. In this study, 1 011 Y-chromosome-specific candidate sequences were screened from 2 to 4 h Bactrocera dorsalis embryo datasets with the chromosome quotient method, 6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction, including typo-gyf, a 19 126-bp DNA sequence containing a 575-amino acid open reading frame. Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos. After typo-gyf knockout with clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 in the embryonic stage, the sex ratio of the emergent adults was unbalanced, with far more females than males. A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal. Typo-gyf knockout led to the death of XY individuals in the embryonic stage. We conclude that typo-gyf is an essential gene for male survival, and is also involved in testicular development and spermatogenesis. The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra-primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses.

Spodoptera frugiperda, also known as fall armyworm (FAW), is an invasive crop pest that can feed on a variety of host plants, posing a serious threat to food security. There are two sympatric strains of FAW that are morphologically identical but described with different food preferences: the “rice strain” (SfR) and the “corn strain” (SfC). A few genetic loci exist to identify these two strains. Mitochondrial and Z-chromosome-linked haplotypes are the most used, but the biggest part of the genome displays little polymorphism between strains that could explain their adaptation to different plants. We have previously observed consistent transcription differences between the strains in both laboratory and natural populations. Therefore, we wonder if there are effects from host-strain-associated loci, maternally or paternally inherited, on FAW performance that could explain the divergence between the two FAW strains. To test this hypothesis, we first produced two F₁ hybrid generations (SfR ♀ × SfC ♂, SfC ♀ × SfR ♂). These reciprocal hybrids should be heterozygous for all chromosomes except for the maternally inherited mitochondrial and sexual W chromosomes. To evaluate whether plant preference is determined by these genetic loci, we cultivated the two hybrids and the two parental strains in triplicate on an artificial diet and recorded several phenotypic traits such as weight over time, survival rate, emerging rate, developmental time, and sex ratio. Then, the same performance experiment was carried out on corn plants. Surprisingly, on the artificial diet, the two hybrid genotypes were both more performant than the two parental strains in terms of survival rate, pupal emerging rate, and developmental time, whereas they were intermediate to the inbred parental strains in pupal weight. On the corn plant diet, both hybrid genotypes outperformed the two parental strains in larval weight. Although these asymmetrical results revealed that mitochondrial or sex-linked haplotypes alone cannot explain the performance differences, they suggested a heterosis effect in FAW. A reduction of the female number for the CR genotype and the decreased F₁ offspring reproduction in both hybrids suggested the possibility of Haldane’s rule, which might be explained by the dominance model.

Interspecific competition is an important factor in the population dynamics and geographical distribution of insect populations. Fall armyworm (FAW),Spodoptera frugiperda, an invasive species, and the Asian corn borer (ACB),Ostrinia furnacalis, a native pest species are major pests on maize in China, posing a threat to maize yield and grain quality. A series of laboratory, greenhouse, and field experiments were conducted to elucidate the competitive interactions between FAW and ACB. In the laboratory experiments, FAW exhibited aggressive behaviors more frequently when compared with ACB, while the latter species exhibited defense behaviors more frequently. Higher intraspecific competition was recorded in the FAW conspecific rather than in ACB, particularly in the 6th larval stage. FAW had a higher interspecific competitive advantage through intraguild predation over ACB, resulting in partial or complete displacement ACB when initially the ratio of the 2 species was 1:1. The interspecific competition also had significantly influenced on the population parameters, defensive enzymes, and nutrient of these 2 species. Competitive interaction proved that the response of superoxide, catalase, and soluble protein in FAW were significantly increased, whereas the total sugar content in both species was substantially decreased. Survival rate, and the plant damage that co-infested by both species varied significantly among the sequential combinations under greenhouse and field conditions. FAW consistently exhibited stronger intraspecific aggression than ACB under laboratory and field conditions when co-existing on the same feeding guild. These findings contribute to efforts toward the improvement of integrated pest management programs for FAW, in decision making for invasive and native pests’ management strategies to reduce the high risks of FAW and ACB outbreaks.