The fall webworm, Hyphantria cunea, is a destructive invasive pest whose seasonal outbreaks depend largely on successful pupal diapause, but how photoperiod coordinates larval development and fat body transcription during pre-diapause preparation remains incompletely understood. Here, we characterized photoperiod-associated developmental and transcriptional remodeling before diapause and assessed candidate genes linked to diapause-related traits. Under short photoperiod, diapause-destined (DP) larvae developed an additional seventh instar. Relative to non-diapause (NDP) larvae under long photoperiod, they exhibited enlarged fat bodies, larger pupae, and greater terminal-stage body mass, consistent with enhanced storage capacity before pupation. Fat body transcriptomics across matched fourth- to sixth-instar comparisons yielded 4154 differential-expression calls, and functional profiling highlighted stage-specific metabolism- and hormone-related processes. Independent RT-qPCR analysis confirmed the RNA-sequencing expression patterns. Hc_CYP314A1 and Hc_Wat showed their strongest DP-biased expression at the sixth instar. Under diapause-inducing conditions, feeding-based RNA interference linked eight candidate genes to gene- and sex-dependent changes in developmental timing, survival, pupal weight, pharate-adult development, and eclosion timing. The dsDP_RFP control eclosed only after prolonged diapause, whereas Hc_Diap1-4 knockdown produced earlier eclosion in subsets of pupae at 0-30 or 90-110 d. Together, these findings reveal coordinated photoperiod-associated developmental and fat body transcriptional remodeling during pre-diapause preparation and identify candidate genes associated with diapause-related traits in H. cunea.
Despite its importance in the circular economy and the large amount of research published each year on this insect, the chemical ecology of adult Black Soldier Fly (BSF), Hermetia illucens, remains poorly understood. Only a few studies have described antennal olfactory sensilla and the ability of both sexes to detect chemicals associated with organic decomposition. In this context, the present study aims to deepen our understanding of the sensitivity of male and female BSF antennal sensilla to olfactory cues from decaying organic matter, its plasticity throughout the adult lifespan, and its potential role in reproduction. This is achieved by: (i) single sensillum recordings (SSR) targeting olfactory sensory neurons (OSNs) housed in different olfactory sensilla stimulated with volatiles from decaying substrates; and (ii) Y-tube olfactometer bioassays, to assess the attractiveness of these cues to males and females differing in age and reproductive status. Different antennal sensilla respond similarly in both sexes to VOCs emitted by decaying substrates. These cues elicit sex- and stage-specific taxis: males are always repelled, whereas females are repelled after emergence but become attracted upon reaching sexual maturity, when they need to oviposit. These findings suggest that the tested olfactory cues could drive sex- and stage-specific movements of H. illucens adults, relative to their emergence, mating and oviposition sites. This knowledge advances our understanding of BSF chemical ecology and should be considered for the optimisation of rearing systems to improve industrial productivity.
Predation risk can shape prey behavior as strongly as direct killing, yet the non-lethal effects of predators on termites remain largely unknown. Current study investigated how chemical, contact, and soil-derived cues from fire ant Solenopsis invicta affect the behavior and physiology of termite Reticulitermes chinensis. Termites exposed to ant-derived cues elicited significantly elevated allogrooming, trophallaxis, vibration, and avoidance behaviors by nestmates. Burial and cannibalism responses toward cadavers bearing predator cues were also significantly enhanced. Locomotion assays revealed reduced distance traveled and velocity, coupled with increased turn angle and angular velocity, indicating disrupted orientation under predator-associated stress. Predator cues further inhibited foraging by decreasing visits to the food zone and increasing time spent in inner zones. Biochemically, termites exposed to S. invicta cues exhibited significant upregulation of catalase (CAT) and hydrogen peroxide (H₂O₂), suggesting a potential activation of oxidative stress-related responses. These findings show that R. chinensis can detect predator presence through non-contact cues and respond with coordinated behavioral and physiological responses. The study highlights the ecological significance of non-consumptive predator effects in termites and provides new insight into how social insect colonies regulate risk through integrated social and oxidative stress responses.
Mitochondria play a central role in energy metabolism and cellular survival, yet their seasonal adaptations in insects enduring prolonged dormancy remain poorly understood. We investigated whether winter-like lab acclimation alters mitochondrial density/abundance and respiratory capacity in the flight muscles of three Drosophila species from subarctic climatic zone and D. melanogaster. Flies were acclimated to either warm (20 °C and long day) or cold (3 °C and short day) conditions, and mitochondrial traits were assessed using microCT, microscopy, molecular proxies of mtDNA abundance, and high-resolution respirometry. Contrary to expectations, cold acclimation induced little to no change in flight muscle structure, mitochondrial density and abundance across all species. Similarly, mitochondrial oxygen consumption capacity measured at 20 °C remained largely unchanged between warm- and cold-acclimated phenotypes, despite pronounced interspecific differences. The only consistent physiological trend was a modest increase in the relative contribution of glycerol-3-phosphate pathway to respiration in cold-acclimated flies. These findings suggest that subarctic Drosophila species enter seasonal dormancy with mitochondria that retain the capacity for normal rates of oxygen consumption. Our study provides a foundation for future work examining mitochondrial performance under ecologically relevant winter (low-temperature) conditions and their resistance to severe cold-induced stress.
Chemical insecticides remain a cornerstone of contemporary integrated pest management (IPM). However, the toxicological profile of Streltzoviella insularis, an economically important wood-boring pest, remains insufficiently characterized. In this study, a laboratory rearing system for S. insularis was established using a customized artificial diet, providing a reliable platform for toxicological assays. Bioassays indicated that chlorantraniliprole (CAP) exhibited significantly higher insecticidal activity against fifth-instar larvae than the three other insecticides tested. Focusing on disruption of gut bacterial symbiont homeostasis as a mechanistic entry point, we investigated whether sublethal CAP exposure imposes fitness costs on S. insularis through perturbation of gut symbiotic communities. 16S rRNA amplicon sequencing revealed substantial restructuring of the larval bacterial community under sublethal CAP stress. The abundance of the core genus Enterococcus was significantly reduced, whereas Carnimonas and Levilactobacillus were significantly enriched. These three taxa came to dominate the reshaped gut microbiota, suggesting a potential compensatory functional shift following depletion of core symbionts. Activities of key digestive enzymes, including α-amylase, lipase, and trypsin, were significantly reduced. Substantial fitness costs were observed, including reduced larval and pupal weights, prolonged larval development time, and decreased pupation success, adult emergence, egg hatching, and female fecundity. Collectively, these findings suggest that sublethal CAP exposure is closely associated with gut symbiont dysbiosis in S. insularis, accompanied by impaired digestive function and delayed larval growth and development.
Dietary supplementation with probiotics has been demonstrated to enhance nutritional efficiency and economic traits in the silkworms (Bombyx mori), with mechanistic insights indicating that these benefits are associated with gut microbiota activity and mucosal immune modulation through the regulation of enzymatic functions. This study investigated the effects of probiotic Bacillus species on intestinal microbiome diversity in fifth-instar silkworms. Two native isolates, Bacillus subtilis Y11 and Bacillus velezensis Z55, were administered either individually or in combination to experimental groups (CK, Y11, Z55, and Y11/Z55) on the first day of the fifth instar. Intestinal contents were collected at days 1, 3, and 5 after treatment for 16S rRNA gene sequencing, and cellulose content in silkworm excrement was measured on a daily basis. Experimental groups exhibited significantly elevated body weight, digestive capacity, and cocoon weight compared to controls. The cellulose content in silkworm excrement decreased by 24.7% and 31.8% on days 2 and 3, respectively, compared to the control (CK) group at the corresponding time points. Tax4Fun2 analysis further predicted a rapid increase in the potential abundance of cellulose degradation-related genes within the gut microbiome, an inference based on taxonomic composition. These findings support the hypothesis that probiotic treatment may enhance the net efficiency of cellulose degradation in silkworms by modulating the gut microbial community and structure, thereby offering new insights into improving their growth performance.
Body-colour polymorphism is closely associated with fitness variation and environmental adaptation in insects, yet the genetic and physiological significance of rare colour morphs remains poorly characterized. During field surveys, we discovered a wild parthenogenetic yellow morph of the pea aphid, Acyrthosiphon pisum, and examined its inheritance pattern, fitness performance, and heat-stress physiology. Controlled crosses among yellow, deep-red, and green morphs showed that yellow coloration could not be fully explained by the classical single-locus red/green dominance model. Segregation patterns in selfed F₁ lineages were consistent with a two-locus recessive epistasis model, in which the inferred Y locus permits red/green pigmentation and homozygosity for the recessive y allele results in the yellow phenotype. Under control conditions, the yellow parental clone showed reduced fecundity, suggesting a potential fitness cost under non-stressful conditions. However, under heat stress, yellow-associated lineages consistently showed relatively low mortality. Notably, yellow F₂ segregants retained low heat-stress-induced mortality while exhibiting higher fecundity than the yellow parental clone, suggesting that the tolerance-fecundity trade-off may be partially decoupled through hybridization and recombination. When parental, selfed, and cross-derived yellow morphs were pooled and compared with additional wild-derived red and green reference lineages, the yellow group still showed the lowest mortality and the highest HSP70 and HSP83 expression under heat stress. These results suggest that the yellow phenotype may be associated with enhanced survival under heat stress and elevated heat-shock protein expression. Our findings reveal a previously underappreciated component of pea aphid colour polymorphism and provide new insights into the ecological significance of rare colour variants under thermal stress.
Understanding how morphological and behavioral traits diverge and develop independently is essential for explaining phenotypic diversification in insects. Ostrinia furnacalis (Guenée) exhibits stable forewing pattern polymorphism, providing a useful system for examining whether geometric shape variation in wing morphology is associated with differences in acoustic courtship signaling. In this study, we combined geometric morphometric analysis of wing venation with bioacoustic characterization of male courtship songs across four laboratory-maintained morphotypes. Wing shape was quantified using homologous venation landmarks after accounting for size-related variation, while courtship signals were recorded from adult males and analyzed for temporal and spectral characteristics. Distinct morphological differences in wing venation were detected among morphotypes, indicating shape divergence beyond superficial wing pattern variation. Male courtship songs also varied among morphotypes in several temporal and spectral features, suggesting differentiation in acoustic signaling. However, variation in wing venation was not significantly associated with differences in courtship song characteristics. These findings indicate that geometric shape variation in wing morphology does not impose detectable constraints on the acoustic properties of courtship signals in O. furnacalis. Instead, wing morphology and acoustic communication appear to vary as relatively independent phenotypic components. This decoupling suggests that forewing polymorphism in O. furnacalis reflects multidimensional phenotypic diversification, in which morphological shape and behavioral traits can diverge independently, highlighting the potential role of phenotypic modularity in the evolutionary diversification of insect morphology and communication systems.
Sawflies (Symphyta: Hymenoptera) use specialized ovipositors to cut into soft plant tissues for egg deposition. These ovipositors employ a reciprocating sawing motion and exhibit intricate morphological and material adaptations that enable selective tissue cutting with minimal damage to surrounding structures and preservation of the mechanical integrity of the ovipositors. While wood-boring ovipositors have been widely researched in biomimetics, the structural and compositional basis of soft-tissue-cutting ovipositors remains largely unexplored. This study characterizes the hierarchical material organization of sawfly ovipositors in relation to a recently identified cutting mechanism. Using a combination of scanning electron microscopy (SEM), micro-computed tomography (μCT), confocal laser scanning microscopy (CLSM) and polarized light microscopy (PLM), we investigate the composition, microstructure, and density gradients of materials present in the ovipositor of Rhogogaster scalaris. Our results reveal multi-scale structural heterogeneity, including spatially resolved density gradients, chitin fibre-bundle arrangements, and protein composition variations suggestive of mechanical optimization. These findings suggest that cutting efficiency is supported primarily by hierarchical structure and compositional gradients, rather than requiring prominent metal-based hardening, although trace or localized metal enrichment remains to be assessed through quantitative elemental mapping. Preliminary dehydration observations support the functional significance of hydration in maintaining mechanical integrity. This study provides the first detailed compositional and structural analysis of soft-tissue-cutting ovipositors and offers a structural and compositional basis that may inform future bioinspired designs of cutting tools aiming to enhance tissue selectivity and structural preservation.
The reproductive division of labour is the defining feature of eusociality, in which reproduction is monopolised by one, or a small subset, of individuals within a colony. In the Western Honeybee Apis mellifera, workers are reproductively constrained by a suite of mechanisms, but primarily by queen mandibular pheromone (QMP). The adult developmental limits of QMP's ability to repress worker reproduction and its ability to suppress reproduction once ovaries have begun to activate remain unresolved. Here, we investigated the limits of plasticity of QMP mediated reproductive repression and compared it to Drosophila melanogaster. By manipulating the timing of QMP exposure and withdrawal in caged honeybee workers, we show that QMP inhibits ovary activation across early adult development, even when exposure is delayed for several days post-eclosion, and that this inhibition is reversible upon removal of QMP. We also show that QMP alone is unable to bring about repression of honeybee worker ovaries once they have activated, indicating that there is an intrinsic limit to QMP-mediated reproductive plasticity in honeybee workers. This contrasts with D. melanogaster where QMP exposure significantly reduced oogenesis even in active ovaries. These findings indicate that there are species differences in reproductive plasticity; and that in honeybees, QMP acts primarily as an inhibitor of adult reproductive development rather than as a reversible regulator of adult reproduction, with reproductive repression likely requiring other additional colony cues.
The insect larval fat body is the primary organ for detoxification of environmental pesticides and metabolic waste. During lepidopteran metamorphosis, the larval fat bodies undergo degradation and reprogramming for development, leaving the process of detoxification largely unknown at this stage. Using integrative approaches in the silkworm, Bombyx mori, we demonstrate that the pupal rectal sac, a greatly enlarged hindgut structure, serves as a central detoxification hub during the non-feeding metamorphic period. We show that it actively sequesters metabolic waste (uric acid) and, remarkably, clears exogenous toxins directly from the hemolymph. Proteomic and functional analyses reveal that the rectal fluid is enriched with a suite of active enzymes, including prophenoloxidase, laccase, and esterase, which create a stage-specific detoxification microenvironment. This organ thereby functions analogously to a vertebrate liver, providing remote clearance of toxins when feeding and excretion cease. Our findings uncover a key adaptive strategy in Lepidoptera and open new avenues for developing stage-specific pest management strategies by targeting this essential detoxification center.
Insects inhabiting temperate regions use photoperiodic cues to predict seasonal changes and regulate diapause. In some species, maternal photoperiodic experience determines offspring diapause fate. However, the physiological mechanisms underlying transgenerational transmission of seasonal information remain poorly understood. In the parasitoid jewel wasp Nasonia vitripennis, females exposed to long-day conditions produce offspring that develop continuously, whereas those exposed to short-day conditions produce offspring destined for diapause. Previous studies have shown that maternal juvenile hormone (JH) levels increase under long-day conditions and promote non-diapause development in offspring, suggesting that JH may participate in the transfer of maternal photoperiodic information. Here, we investigated whether this effect is mediated through canonical maternal JH signalling pathways or through changes in egg endocrine state. RNA interference-mediated silencing of methoprene-tolerant, taiman, and krüppel-homologue 1 did not alter offspring diapause fate. In contrast, eggs from long-day females contained significantly higher JH III levels than those from short-day females, and topical application of JH III to eggs from short-day females promoted non-diapause development. These findings suggest that photoperiod-dependent variation in egg JH levels contributes to offspring diapause determination and support a role for egg endocrine state as an intermediary between maternal environmental perception and offspring developmental fate.
Western honeybees (Apis mellifera, L.) play a crucial role in the pollination of numerous plants, including many economically important crops, and are essential for food production. In this study, we first developed and optimized a protocol for the extraction and characterization of proteins from the intestinal tissue of worker honeybees, which allowed the identification of 1787 distinct components. By feeding honeybees with distinct diets in a caged bee assay and using a Tandem Mass Tag (TMT)-based proteomic approach, we also investigated how the proteome of A. mellifera intestine varies its composition depending on different nutritional inputs. Quantitative proteomics revealed a strong diet-dependent modulation primarily affecting proteins involved in carbohydrate metabolism, protein synthesis and turnover, proteostasis, and cytoskeletal remodeling, reflecting adaptive intestinal responses to different nutritional regimes. On this basis, this study allowed to elucidate the molecular mechanisms underlying intestine adaptation in honeybees, highlighting the dynamic nature of gut physiology in response to nutritional variations.
The plum psyllid, Cacopsylla pruni (Hemiptera: Psyllidae), is the primary vector of 'Candidatus Phytoplasma prunorum', the causal agent of European Stone Fruit Yellows (ESFY). Its seasonal host alternation - from conifers in winter to Prunus species in spring - requires precise host recognition, yet the underlying sensory mechanisms remain poorly understood. We investigated the visual and olfactory responses of overwintered C. pruni during remigration. Electroretinographic recordings revealed a visual system comprising UV-, blue-, and green-sensitive photoreceptors, potentially allowing trichromatic colour vision. Colour vision modelling indicated that white Prunus flowers and white sticky traps provide nearly identical chromatic and achromatic signals, likely accounting for the pronounced attraction of remigrants to white traps in early spring. Complementary electroantennographic analyses demonstrated robust antennal responses to key Prunus floral volatiles, including benzaldehyde, benzyl alcohol, phenylacetaldehyde, linalool, 2-phenylethanol, 4-oxoisophorone, and methyl salicylate. Together, our results provide the first electrophysiological evidence that C. pruni has the potential to integrate both visual and olfactory cues during host location. These findings are consistent with the broader understanding of psyllid host finding, in which long-distance orientation is primarily mediated by visual cues, whereas olfactory inputs likely facilitate short-range host assessment. This multimodal recognition strategy likely enhances foraging efficiency during seasonal migration and has important implications for monitoring and management of this economically significant vector.
Symbiotic microbiota of insects play crucial roles in host development, metabolism, and immunity, but the molecular mechanisms underlying these interactions remain poorly understood, particularly in non-model lepidopteran pests. Traditional germ-free (GF) insect models are primarily generated using antibiotics, which may introduce confounding effects and fail to completely eliminate microbiota. Here, we present an antibiotic-free method to generate GF Spodoptera frugiperda larvae by rearing them on axenically cultured maize. The 3rd to 6th instar GF larvae exhibited significantly reduced weight and length compared to the conventionally reared (CR) larvae, and the developmental period was prolonged. Transcriptomic analysis of 3rd instar larvae revealed significant differences in gene expression between the two groups, especially in pathways related to total carbohydrate, protein, triglycerides metabolism, as well as juvenile hormone (JH) signaling pathway. In addition, three nutritional content and JH titer were tested between GF and CR larvae. Furthermore, GF groups showed lower pupation rate and eclosion rate, reduced pupal weight, and prolonged developmental period, while pupal length was not affected compare to CR groups. Additionally, the ovarian and testes sizes of GF adults were smaller than those of CR adults. Consistently, GF females laid fewer eggs with significantly lower hatching rate compared to the CR females. These findings demonstrate that microbiota profoundly influence egg, larval, pupal development and adult reproduction in S. frugiperda. This study provides a robust framework for microbiota-function research in agricultural pests and expands our understanding of lepidopteran insects and microbiota interactions.
Social insects rely on multimodal communication systems to coordinate both individual and colony-level responses, yet the sensory foundations underlying the perception of combined signal components remain poorly understood. In ants, alarm communication often integrates chemical and vibroacoustic cues, but how these modalities are detected and processed by individual workers is still largely unexplored. In this study, we investigated the peripheral detection of multimodal alarm signals in Myrmica scabrinodis, examining how workers detect concurrent alarm pheromones and stridulations. Our electrophysiological recordings revealed that chemical cues are primarily detected through the antennae, which showed robust responses to pheromones but no detectable sensitivity to vibroacoustic stimuli. Leg recordings provided no conclusive evidence of chemical or vibroacoustic detection, although inconsistent responses to vibroacoustic stimuli in the left hindlegs suggest a potential mechanosensory role that requires further investigation. Chemical and vibroacoustic stimuli are probably detected independently at the peripheral level, likely converging only within central processing pathways in the brain. Moreover, we found evidence of lateralization in sensory detection: the right antenna exhibited greater sensitivity to alarm pheromones than the left, suggesting a left-right bias associated with population-level lateralization previously described in other social insects. Such asymmetries may contribute to enhanced group coordination by imposing consistent physiological biases on signal detection. Taken together, our results provide new insights into the specialization and lateralization of sensory structures involved in ant alarm communication, offering a foundation for further investigation into the neural mechanisms underlying multimodal signal integration in social insects.
Understanding how multiple stressors interact is essential for predicting insect performance under global change. Although pesticide toxicity is well-established to be temperature-dependent, the physiological mechanisms underlying these interactions remain poorly understood. Insects often display stage- and sex-specific sensitivities to environmental pressures, suggesting complex energetic trade-offs across their life cycles. Copper-based fungicides, such as Bordeaux mixture, are widely used to control fungal diseases but can stress non-target insects. Here, we experimentally examined how Bordeaux mixture exposure interacts with projected late 21st-century climatic conditions by measuring growth rate, energy reserves, standard metabolic rate (SMR), and Hsp70 concentrations across larval instars (3rd and 5th) and adult sexes in the insect pest Lobesia botrana. Our results show that larvae underwent substantial physiological adjustments to fungicide exposure (increased Hsp70 concentrations, reduced SMR and glycogen), suggesting reallocation of energy from maintenance to stress protection. Future climatic conditions strongly modulated larval responses to the fungicide, shifting the timing of metabolic adjustments across development and interacting with Hsp70 production. Adults displayed sex-specific sensitivity to fungicide exposure, without major interaction with climatic conditions. Females exposed to the fungicide emerged with depleted reserves but unchanged SMR, consistent with energy allocation to reproduction. For males, Hsp70 concentrations were increased by fungicide exposure without major energy reserve loss, suggesting preservation of flight and mate-searching performance. Thus, stress responses are not simply additive but mediated through shared physiological pathways linking Hsp70 concentrations, metabolic regulation, and energy reserve mobilization. This study provides a mechanistic framework for understanding how interacting stressors reshape insect life-history strategies.
The insect cuticle, an important protective barrier covering the body surface, is mainly composed of cuticular proteins and chitin fibers, which together form the insect exoskeleton system. Resilin, an arthropod elastic protein, has attracted attention due to its unique mechanical properties. In this study, we identified BmCPR151, a short resilin-like cuticular protein from Bombyx mori (B. mori). The gene contains a 699-bp CDS encoding a 233-amino-acid full-length protein with shortened repetitive domains, distinguishing it from Drosophila resilin and the previously reported silkworm BmCPR140. Using the CRISPR/Cas9 gene-editing tool, we successfully constructed a systemic BmCPR151 knockout homozygous mutant, and the moths of this mutant exhibited smaller wing area, a significantly thinned wing membrane, and thinner wing veins. Furthermore, based on the previously obtained BmCPR140 knockout mutant, a Double-KO (BmCPR151-KO & BmCPR140-KO) homozygous mutant was generated. Comparative analysis of the three mutants (BmCPR140-KO, BmCPR151-KO, and Double-KO) and the wild type showed that all mutants had decreased wing area and wing vein width. The wing rigidity of female moths decreased by 36.91%, 43.65%, and 45.78%, while that of male moths decreased by 26.54%, 27.08%, and 32.25%, respectively. Moreover, wing patterns were markedly faded in the Double-KO mutant. Transcriptomic analysis revealed that a large number of cuticular proteins were significantly differentially expressed in the wings of Double-KO silkworms, among which 98 cuticular proteins were down-regulated. Collectively, these results indicate that insect cuticular proteins have a relative expression balance, and the loss of resilin-like proteins disrupts the balance, thereby affecting wing development. This study provides a reference for further exploring the functions of insect resilin-like proteins.
Bombykol, (E,Z)-10,12-hexadecadien-1-ol, is a sole sex pheromone component essential for mating in the silkmoth Bombyx mori. The bombykol isomer EE-kol is consistently present across all silkmoth strains tested, but its role in sex pheromone communication remains unclear. Previous studies have indicated that BmOR1 is involved in EE-kol detection. Here, we reevaluated the function using the Xenopus oocyte expression system and two-electrode voltage clamp recording method. EE-kol elicited inward currents in BmOR1-expressing oocytes only at very high concentrations and with a markedly smaller response than to bombykol. EE-kol co-application reduced the bombykol-induced currents in a dose-dependent manner at higher bombykol concentrations, whereas plant-derived alcohols did not cause significant suppression. At the antennal level, EE-kol evoked a weaker response than bombykol, and the bombykol-EE-kol mixture shifted the dose-response curve rightward, with little change in the maximal response. These results support the conclusion that EE-kol acts as a partial agonist of BmOR1 and competitively suppresses the bombykol-induced response. These findings raise the possibility that a well-conserved pheromone isomer may modulate sex pheromone receptor sensitivity in the silkmoth.
The oriental fruit fly, Bactrocera dorsalis (Hendel), depends on peripheral gustation for feeding decisions and host assessment, yet the physiological coupling between early sensory transduction and downstream behavioral output remains poorly resolved. Here, we investigated the physiological responses of mid-tarsal gustatory sensilla in mated females using non-invasive micro-test technology (NMT) to quantify real-time Ca2+ flux, single-sensillum recording (SSR) to measure neuronal activity, and proboscis extension response (PER) assays to evaluate feeding behavior. Upon stimulation with ecologically relevant phagostimulants (fructose and sucrose) and deterrents (L-nicotine and tannic acid), we found that all tastants induced measurable Ca2+ influx, but their downstream coupling among ionic, electrophysiological, and behavioral layers differed substantially. Fructose stimulation generally elicited close correspondence among Ca2+ influx, spike activity, and feeding responses, whereas sucrose exhibited partial dissociation at elevated concentrations. In contrast, deterrent stimuli produced pronounced functional uncoupling: sustained Ca2+ influx persisted even when spike output was strongly suppressed under high-intensity stimulation. The PER of B. dorsalis under deterrent stimulation was more closely associated with persistent ionic signaling than with spike frequency alone. These findings indicate that the tarsal sensilla of B. dorsalis function not simply as passive detectors, but as early peripheral sensory checkpoints in which tastant-specific coupling and uncoupling among ionic, electrophysiological, and behavioral layers shape early feeding decisions.
