Wei Liu

Olfaction is crucial for insects adapting to agroecosystems and becoming significant crop pests, but how the antenna, a multicellular olfactory organ, is systematically organized at the cellular and molecular levels is not well understood. This study focuses on tephritids, particularly Bactrocera dorsalis , due to its global invasiveness and severe agricultural impact. We conducted comparative single‐nucleus transcriptomics of the antennal olfactory organ in B. dorsalis and Drosophila melanogaster , revealing uneven transcriptomic divergence across cell types: structural cells showed higher cross‐species similarity, while sensory neuronal populations and receptor expression were more divergent. Notably, in B. dorsalis , ammonia detection involves an additional candidate neuronal population with BdorAmt/BdorOrco transcript co‐detection, contrasting with D. melanogaster , where ammonia detection is mediated by Amt ‐expressing neurons independently of Orco . This derived molecular sensing logic helps meet the ecological demands of B. dorsalis , in which females use ammonia as an important cue to locate bird droppings as a potential supplementary nutritional resource. Our results suggest that cell‐type‐dependent transcriptomic divergence may represent one important route through which insect olfactory systems adapt, with neuronal diversification contributing to molecular sensing pathways tailored to specific ecological demands.

Dipteran insects include numerous harmful species that cause significant agricultural damage. However, assembly of genomes for species in this order has been difficult due to their small body size, poor conservation of telomere and centromere structures, high levels of heterozygosity, and complex genetic backgrounds. In this study, we assemble a high-quality 596 Mb telomere-to-telomere genome for Bactrocera dorsalis, a fruit crop pest, using a strategy with a low-input HiFi CCS library from a male individual and an ONT sequence from pooled inbred individuals. The assembly includes complete structural organization information for centromeres and telomeres, providing insights into the evolution of chromosome structure in insects. Comparative genomic analysis reveals the polyphyletic origin of sex chromosomes across Diptera. Furthermore, we identify a homolog of ATPsynβ as a Y chromosome-specific gene that is highly expressed across multiple male tissues and may provide critical support for male-specific physiological activities. Additionally, we discover several tandem duplications of odorant receptor genes, including a triplet of the OR88a family, which was validated to be involved in the behavioral response to methyl eugenol. In summary, this complete reference genome provides a foundation for future genomic research in Diptera and offers genetic insights for the control of B. dorsalis.

Behavioural manipulation techniques, particularly attractants, have been widely adopted as environmentally friendly approaches to manage Bactrocera dorsalis . These approaches primarily target odorant receptors (ORs), key molecular components in the pest's chemotactic pathways, to guide pests towards traps or lethal stimuli, offering a sustainable and effective alternative to chemical pesticides. However, pest behaviour extends beyond chemotaxis. Crucial behaviours such as mating, oviposition and defence also directly influence population dynamics, underscoring the need to investigate molecular targets that regulate diverse behavioural processes. IR25a, a co‐receptor of ionotropic receptors (IRs), exhibits functional versatility by detecting diverse signals, including both volatile and non‐volatile compounds and environmental cues (e.g., temperature, humidity, acidity). These characteristics make IR25a a promising candidate target that influences various behavioural processes. In this study, CRISPR/Cas9 was employed to generate a BdorIR25a knockout homozygous strain ( BdorIR25a ^‐/‐ ). Its effects on various aspects of pest behaviour were systematically investigated to evaluate its potential as a molecular target for behavioural modification. Results showed that BdorIR25a was highly expressed in pupal and adult stages, particularly in antennae and mouthparts. Knockouts exhibited impaired responsiveness to specific chemicals, especially dimethylamine. Additionally, mutants displayed reduced courtship wing vibration, pheromone responsiveness and mating success. Oviposition rates and egg‐laying numbers were significantly reduced in females. Importantly, knockouts had no detectable effects on growth or survival, confirming BdorIR25a's primary role in behavioural modification. In conclusion, our findings identify BdorIR25a as a key molecular target with strong potential for manipulating diverse behavioural processes, providing a new perspective for pest control strategies. Future studies should focus on identifying IR25a ‐associated IRs and other molecular targets within its linked regions to clarify their respective roles in modulating critical behavioural traits. These studies could provide detailed insights into the molecular mechanisms of behaviour manipulation, advancing the development of innovative and targeted pest control strategies.

BACKGROUND: Temperature and humidity are critical environmental factors that shape insect growth, development and survival. The larval stage is particularly vulnerable, making the ability to detect and respond to adverse environmental conditions vital for avoiding harm. The oriental fruit fly, Bactrocera dorsalis, is a major global pest inflicting damage on >150 fruit crops. However, the behavioral responses of nonmodel insect larvae to temperature and humidity remain poorly characterized.RESULTS: We found that temperature and humidity significantly influence key larval behaviors. To uncover the molecular mechanisms driving these behaviors, we identified B. dorsalis painless (Bdorpainless) homologs and confirmed their expression during larval behavioral stages. Genetic disruption of Bdorpainless notably impaired the larvae's temperature‐escape response. Although humidity sensing remained unaffected in Bdorpainless‐mutant larvae, their pupation rate was significantly reduced.CONCLUSIONS: Our findings provide new insights into the role of environmental factors in B. dorsalis behavior and highlight Bdorpainless as a potential molecular target for pest control. For example, RNA‐based biopesticides targeting Bdorpainless could impair larvae's environmental adaptability, offering a novel pest management approach. © 2025 Society of Chemical Industry.

Numerous insects are attracted to low levels of ammonia, utilizing it as a cue to locate food sources. The Ammonium Transporter (Amt), a highly conserved, atypical olfactory receptor, has been shown to mediate the detection of ammonia in insects. While the attraction of Tephritidae to ammonia is well established, knowledge about the Amt in this family is limited. The species Bactrocera dorsalis (Hendel 1912), Bactrocera cucurbitae (Coquillett 1899), Bactrocera correcta Bezzi 1916 and Bactrocera tau (Walker 1849), which are common agricultural pests within Tephritidae, exhibit numerous ecological similarities, offering a solid foundation for studying Amt characteristics in this family. In this study, we elucidated the sequences, evolutionary relationships, and expression patterns of Amt in these four species. The results indicated that these Amts share the same open reading frame, containing 1770 bp that encode a protein of 589 amino acid residues. These Amt proteins exhibit the typical structural characteristics of Amts, including an 11-transmembrane domain with an extracellular N-terminus and an intracellular C-terminus. They also have the ability to form trimers in the membrane. Additionally, they contain three conserved amino acid residues essential for ammonia transport: A189, H195, and H352. Phylogenetic and expression pattern analyses showed that they are highly conserved in Diptera and are significantly expressed in antennae. This study is the first report characterizing the Amt gene in four Tephritidae species. These findings provide a foundation for further exploration into the roles of these genes in their particular biological contexts.

Alternative splicing is an essential post-transcriptional regulatory mechanism that diversifies gene function by generating multiple protein isoforms from a single gene and act as a crucial role in insect environmental adaptation. Olfaction, a key sense for insect adaptation, relies heavily on the antennae, which are the primary olfactory organs expressing most of the olfactory genes. Despite the extensive annotation of olfactory genes within insect antennal tissues facilitated by high-throughput sequencing technology advancements, systematic analyses of alternative splicing are still relatively less. In this study, we focused on the oriental fruit fly (Bactrocera dorsalis), a significant pest of fruit crops. We performed a detailed analysis of alternative splicing in its antennae by utilizing the full-length transcriptome of its antennal tissue and the insect’s genome. The results revealed 8600 non-redundant full-length transcripts identified in the oriental fruit fly antennal full-length transcriptome, spanning 4,145 gene loci. Over 40% of these loci exhibited multiple isoforms. Among these, 161 genes showed sex-biased isoform switching, involving seven different types of alternative splicing. Notably, events involving alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS) were the most common. Of all the genes undergoing ATSS and ATTS alternative splicing between male and female, 32 genes were alternatively spliced in protein coding regions, potentially affecting protein function. These genes were categorized based on the length of the sex-biased isoforms, with the highest difference in isoform fraction (dIF) associated with the ATSS type, including genes such as BdorABCA13, BdorCAT2, and BdorTSN3. Additionally, transcription factor binding sites for doublesex were identified upstream of both BdorABCA13 and BdorCAT2. Besides being expressed in the antennal tissues, BdorABCA13 and BdorCAT2 are also expressed in the mouthparts, legs, and genitalia of both female and male adults, suggesting their functional diversity. This study reveals alternative splicing events in the antennae of Bactrophora dorsalis from two aspects: odorant receptor genes and other types of genes expressed in the antennae. This study not only provides a research foundation for understanding the regulation of gene function by alternative splicing in the oriental fruit fly but also offers new insights for utilizing olfaction-based behavioral manipulation techniques to manage this pest.

The oriental fruit fly, Bactrocera dorsalis (Hendel), poses a significant threat to the global fruit industry, causing damage to diverse fruits like citrus, mango, and guava. Chemical pesticides have limited effectiveness, and pesticide residues and pesticide resistance are pressing issues. Therefore, it is essential to develop environmentally friendly pest control methods to address this problem. Behavior-modifying chemicals, including male attractants and intersex protein baits, play a critical role in the control of B. dorsalis. The mature host fruit serves as both an oviposition site and food source under natural conditions, making it a potential attraction source for oriental fruit flies. Orange, Citrus sinensis, is a main host of B. dorsalis, and commercial orange juice is a common attractant for the egg laying of B. dorsalis. Although it can both attract and elicit oviposition behaviors in B. dorsalis adults, its active components are still unclear. This study utilized analytical chemistry, behavioral tests, and electrophysiology to identify the active components of commercial orange juice that attract B. dorsalis, with the aim of providing a reference for the development of behavior-modifying chemical-based techniques to control B. dorsalis. Five compounds with a high abundance were identified via a GC-MS, including D-Limonene, butanoic acid ethyl ester, β-myrcene, linalool, and α-terpineol. Behavioral and electrophysiological experiments uncovered that D-Limonene was the active substance that was the main attractant in the mixture of these five substances, evoking a strong electrophysiological response in adult B. dorsalis. D-Limonene strongly attracts adult B. dorsalis only when they are sexually mature, and the attraction is not rhythmic. Olfaction plays a leading role in the attraction of D-Limonene to adult B. dorsalis, and Orco^−/− mediates the perception of D-Limonene by B. dorsalis. Overall, D-Limonene is one of the key attractant compounds for B. dorsalis in the volatile compounds of commercial orange juice, offering possible support for the development of behavior-modifying chemical-based technology to control B. dorsalis in the future.

The Oriental fruit fly, Bactrocera dorsalis, is a significant pest that damages a variety of fruit crops. The effectiveness of chemical pesticides against such pests is limited, raising concerns about pesticide residues and resistance. Proteins naturally attract B. dorsalis and have led to the development of a management strategy known as protein bait attractant technology (BAT). Although the attraction of protein sources to B. dorsalis is well-documented, the biologically active components within these sources are not fully understood. This study employed analytical chemistry, behavioral tests, and electrophysiological techniques to investigate the behaviorally active components of beer yeast protein powder (BYPD), aiming to provide a basis for improving and developing protein baits. An olfactory trap assay confirmed the attractiveness of BYPD, and five components with high abundance were identified from its headspace volatiles using GC–MS. These components included ethanol, isoamyl alcohol, ethyl decanoate, benzaldehyde, and phenylethyl alcohol. Mixtures of these five components demonstrated significant attraction to B. dorsalis adults, with benzaldehyde identified as a potential key component. The attractiveness of benzaldehyde required a relatively large dose, and it was most attractive to adults that had been starved from dusk until the following morning. Attraction of adult flies to benzaldehyde appeared mainly mediated by inputs from olfactory receptors. While EAG data supports that ionotropic receptors could influence the detection of benzaldehyde in female adults, they did not affect female behavior towards benzaldehyde. These findings indicate that benzaldehyde is an important behaviorally active component in BYPD and offer insights for developing novel protein lures to control B. dorsalis in an environmentally friendly manner.

The ability to recognize a host plant is crucial for insects to meet their nutritional needs and locate suitable sites for laying eggs. Bactrocera dorsalis is a highly destructive pest in fruit crops. Benzothiazole has been found to induce oviposition behavior in the gravid B. dorsalis. However, the ecological roles and the olfactory receptor responsible for benzothiazole are not yet fully understood. In this study, we found that adults were attracted to benzothiazole, which was an effective oviposition stimulant. In vitro experiments showed that BdorOR49b was narrowly tuned to benzothiazole. The electroantennogram results showed that knocking out BdorOR49b significantly reduced the antennal electrophysiological response to benzothiazole. Compared with wild-type flies, the attractiveness of benzothiazole to BdorOR49b knockout adult was significantly attenuated, and mutant females exhibited a severe decrease in oviposition behavior. Altogether, our work provides valuable insights into chemical communications and potential strategies for the control of this pest.

Developing behavioral modifying chemicals through molecular targets is a promising way to improve semiochemical-based technology for pest management. Identifying molecular targets that affect insect behavior largely relies on functional genetic techniques such as deletions, insertions, and substitutions. Selectable markers have thus been developed to increase the efficiency of screening for successful editing events. However, the effect of selectable markers on relevant phenotypic traits needs to be considered. In this study, we cloned the wp gene ofBactrocera dorsalis. Knocking out Bdorwp causes white pupae phenotypes. Reproductive behaviors in both males and females were strongly regulated by Bdorwp. Remarkably, Bdorwp did not affect the antennal electrophysiology response to 63 chemical components with various structures. It is recommended to indirectly apply Bdorwp as a selectable marker in functional gene research on behavioral modifying chemicals. Moreover, Bdorwp could also be a potential molecular target for developing new insecticides for tephritid species control.