Leishmaniases are infectious diseases whose vectors are female sand flies that depend on the blood of vertebrates for egg maturation. In this study, we investigated the sand fly species and DNA from Leishmania spp. This entomological/epidemiological study was conducted in a rural area in Nova Andradina, Mato Grosso do Sul, Brazil. From March 2019 to February 2020, five modified Falcão light traps were installed at night once a month. The collected insects were separated by sex into pools of 10 specimens. A sample was used for species identification via microscopic analysis, and another sample was used to detect the DNA of Leishmania in females via polymerase chain reaction (PCR). A total of 42,933 insects were collected, 77.6% (n = 33,313) of which were female. A total of 4,993 (11.6%) insects were identified, most were Nyssomyia neivai (n = 4,839; 96.9%). For PCR, 4,130 (9.6%) insects were analyzed, and four pools were positive for Leishmania spp. (with a natural infection rate of 0.1%). When the number of sand flies collected and the climatic variables were correlated, a significant difference was detected when precipitation > 60 mm (p = 0.0314) and relative humidity ≤ 60% (p = 0.0201). The analyzed area is inhabited by one of the main vectors of cutaneous leishmaniasis in Brazil, and we also found that some specimens were infected with Leishmania DNA, which might favor the occurrence of the disease in the region. This information is essential for understanding the dissemination of leishmaniasis and planning actions to prevent in the municipality.
Bacterial symbionts play a significant role in the biology of tsetse flies (genus Glossina). However, their infection prevalence in Glossina spp. is not well documented. Hence, the systematic review followed PRISMA guidelines to determine bacterial infection prevalence among different Glossina spp. across sub-Saharan Africa. Six databases were used to search for studies published between 1980 and 2024. The search was conducted from February to March 2025. Meta-analysis was conducted using estimated pooled infection prevalence of bacterial symbionts in different Glossina species. Spearman's rank correlation coefficient was calculated to examine changes in bacterial infection prevalence between 2010 and 2024. A total of 30 studies from 19 countries met the inclusion criteria and were included in the final systematic review. Of the 30 studies, 173 records were extracted consisting of data from 14 Glossina species. Burkina Faso (n = 25) recorded highest number of studies and G. pallidipes (n = 27) was the most studied species. Significant associations were observed between bacterial infection prevalence and the tested variables (Glossina spp. and country). Temporal analysis results showed positive association in Spiroplasma infection prevalence over the years (rho = 0.418, p = 0.017) whilst Sodalis infections showed negative association (rho = -0.319. p = 0.008). Heterogeneity among different studies was substantially high (I2 > 75%) and evidence of publication bias was reported. The results indicated that bacterial infection prevalence was influenced by factors such as country, sample size and symbiont-specific species. However, heterogeneity remained high even after meta-regression analysis suggesting that other variables not included in the analysis or rather the combination of different variables are responsible for the high heterogeneity observed. Despite study limitations, the study provided the most comprehensive and complex results on the relationship between bacterial symbiont infections and Glossina species.
Many countries operate trapping programs to detect incursions of pestiferous fruit flies (Diptera: Tephritidae). Three male-specific attractants form the cornerstone of these surveillance networks: methyl eugenol for Bactrocera species, cue-lure for Bactrocera and Zeugodacus species, and trimedlure for Ceratitis species. Traditionally, these lures were applied as a liquid to a cotton wick, but polymeric plugs are being used more widely. Regulations require that any toxicant placed in a trap with a lure-bearing plug must be presented separately from the plug, which, most commonly, is held in a plastic, perforated basket inside a Jackson trap. The present study examined whether the presence or position of an insecticidal DDVP (dichlorvos) strip within a Jackson trap affects capture of Bactrocera dorsalis, Zeugodacus cucurbitae, and Ceratitis capitata males. Captures were compared among (i) traps without a DDVP strip, (ii) traps with a DDVP strip placed in a separate basket from the lure-bearing plug, and (iii) traps with a DDVP strip placed in the same basket as the plug. Results were similar for B. dorsalis and Z. cucurbitae: traps with the toxicant in the same basket as the male-lure plug captured significantly more males than traps with the toxicant in a separate basket. For B. dorsalis, traps with the toxicant in a separate basket from the lure-bearing plug had significantly higher captures than traps lacking a toxicant, while results for Z. cucurbitae were inconsistent between study sites. Neither the presence nor position of DDVP strips in Jackson traps influenced captures of C. capitata males.
Mate choice plays a critical role in reproductive success and species survival. In Drosophila melanogaster, female mating preferences are shaped by various factors, including male pheromones and experience. Here, we investigated whether exposure to distinct fruit-derived volatiles could influence male attractiveness. We found that fruits released unique volatile profiles and that males exposed to these substrates acquired detectable amounts of substrate-specific volatiles. When presented with a choice, females consistently preferred males previously exposed to apple volatiles over those exposed to pear or parsnip, with parsnip-exposed males being least attractive. Analysis of similarities revealed that body odor profiles of males reflected their respective substrates. Importantly, male exposure to different substrates was not associated with detectable differences in courtship latency or in pheromone levels. Our results suggest that environmentally acquired odors may affect female mate choice, pointing to a possible role for fruit-derived volatiles in modulating reproductive behavior in Drosophila melanogaster.
The Pantanal biome harbors exceptional biodiversity but has been increasingly impacted by climate change and human activities. This region is considered a high-risk zone for zoonotic spillover, making viral studies in sylvatic mosquitoes and other invertebrates indispensable, as these vectors are involved in the transmission of pathogens of public health concern. This study aimed to describe viral genomes identified in Aedes spp., Ochlerotatus sp., Mansonia sp., Phlebotomus sp., Psorophora spp., and Anopheles spp. dipterans collected in March and June 2019, in Pirizal and Porto São Luiz, Alto Pantanal, Mato Grosso State, Brazil. Diptera specimens were pooled by genera, and nucleic acids were extracted, followed by library preparation and sequencing on the Illumina NextSeq 500/550 platform. A total of 39 putative viral sequences were recovered, including 23 potentially novel viruses. Coding-complete genomes were identified from Virgaviridae (n=1), Rhabdoviridae (n=1), and Metaviridae (n=1), as well as seven coding-complete segments from Partitiviridae (n=4) and Solemoviridae (n=3). Additionally, 29 partial genomes were recovered from Partitiviridae (n=7), Metaviridae (n=6), Chuviridae (n=2), Sedoreoviridae (n=1), Nodaviridae (n=3), Tombusviridae (n=2), Phasmaviridae (n=2), Flaviviridae (n=3), Virgaviridae (n=1), and Solemoviridae (n=2). Viral characterization in Diptera specimens has gained increasing importance with the advancement of metagenomic approaches, which contribute to global One Health initiatives by providing data that may support the prediction and prevention of future viral spillover events.
Reproductive microbiota is known to modulate host immunity, reproductive physiology, and longevity, yet their interactions with host genetics, sex, and mating status remain relatively underexplored in most insects. Some studies suggest that species (and even populations) can vary in the microbial communities associated with their reproductive tissues. Sexually transmitted microbes can also directly influence population growth and reproductive fitness, making them relevant even for applied insect management strategies. The economically relevant, Black Soldier Fly (BSF; Hermetia illucens), is known for its applications in organic waste management and sustainable food production. Despite several papers on the larval gut microbiota, there is no study (to date) that explores the reproductive microbiota of adults. This study characterizes the reproductive microbial communities of eight genetically distinct BSF populations using 16S rRNA gene sequencing. The results revealed striking, sex-specific microbial signatures within reproductive tissues, dynamically shaped by mating. Virgin females harboured diverse and functionally intriguing taxa, including Staphylococcus, Brevibacterium, and Corynebacterium, which likely supported ovarian maturation and reproductive readiness. Remarkably, mating triggered a dramatic shift, with unclassified Enterococcaceae emerging as the dominant taxon in mated females, suggesting selective microbial retention or transmission with potential implications for post-mating longevity. In contrast, although mated males showed enrichment of Rhodococcus_C_375578, their overall microbiota composition remained comparatively stable pre- and post-mating. Most notably, the genetic lineage significantly influenced the magnitude of sex- and mating-related bacterial shifts in reproductive microbiota. These novel findings advance the understanding of the complex dynamics of reproductive microbiota in insects and provide a foundation for optimizing breeding strategies and colony management in commercial BSF production systems.
The development of antidepressants has traditionally relied on vertebrate models. Drosophila melanogaster offers a promising alternative approach, yet evidence of antidepressant-like effects is fragmented. The aim of this systematic review was to analyse the literature on the effects of antidepressants on D. melanogaster behaviours. A comprehensive search of a number of bibliographic databases (i.e. PubMed, Web of Science, Scopus and EMBASE) was followed by a further screening of the retrieved articles (i.e. inclusion of studies on any D. melanogaster strain, sex, age, dose or outcome, tested in any behavioural assay; exclusion of reviews, studies on co-treatments and/or on non-prototypical antidepressants, and studies lacking control groups). This resulted in the final selection of 12 articles, comprising 146 studies. Most studies involved adult flies (k = 131), males (k = 56), fluoxetine treatment (k = 109) and courtship outcomes (k = 85). Some studies involved larvae (k = 15), females (k = 39), treatment with other antidepressants (citalopram, k = 12; desipramine, k = 6; amitriptyline, k = 2; imipramine, k = 2) and other outcomes (locomotion, k = 11; sucrose preference, k = 5; light preference, k = 2; aggression, k = 2; climbing, k = 1). Studies on larvae had, on average, larger sample sizes than studies on adult flies, resulting in more conclusive effect sizes. The effect sizes indicated that most studies presented inconclusive effects. However, sucrose preference, courtship and light preference were outcomes with conclusive findings, depending on the conditions (sex, treatment, stress), and thus these may represent more reliable indicators of antidepressant effects. However, the lack of consistency among studies precluded drawing any definitive conclusions on reliable indicators. In addition, incomplete reporting precluded an accurate risk-of-bias assessment. These findings highlight the need for standardisation and transparent reporting, in order to establish flies as a reproducible, ethically acceptable alternative to vertebrates in antidepressant research.
Fungal infections are an escalating global health challenge, exacerbated by increasing antifungal resistance and limited treatment options. Insects have emerged as a promising reservoir, possessing robust immune defences that enable them to thrive in microbe-rich environments. Their ability to fend off fungal pathogens through the production of antimicrobial peptides (AMPs), enzymes, and secondary metabolites provides a new avenue for antifungal drug development. Insect-derived antifungal compounds exhibit diverse mechanisms of action. AMPs such as defensins (from beetles and flies), cecropins (from moths, flies, and bees), and diptericin (from Drosophila melanogaster) disrupt fungal cell membranes, inhibit cell wall synthesis, and interfere with metabolic pathways. Honeybees, for instance, defend their colonies against fungal threats through immune responses and the production of propolis a resinous substance with potent antifungal properties. Additionally, compounds like thanatin (from soldier bugs), coprisin (from dung beetles), and metchnikowin (from fruit flies) have demonstrated strong antifungal activity, even against drug-resistant fungi. However, challenges remain, including large-scale production, regulatory hurdles, and ecological concerns. Addressing these limitations through interdisciplinary research could lead to innovative solutions for managing fungal infections, reinforcing the importance of insects as a largely untapped resource in the fight against antifungal resistance.
Hydrogen sulfide (H2S) is a vital gasotransmitter essential for maintaining redox homeostasis and modulating inflammatory responses. Herein, we examined a collection of Drosophila melanogaster knockout (KO) lines generated in our laboratory, lacking key genes involved in transsulfuration (cbs, cse) and sulfide metabolism (tst1), to elucidate the role of this adaptive system in immunity. Genetic ablation of these pathways results in profound H2S deficiency and hyperhomocysteinemia, leading to chronic oxidative stress. This leads to constitutive activation of major immune pathways-including IMD, Toll, and JAK-STAT-even in the absence of infection, a hallmark of chronic inflammation. Indeed, transcriptomic analysis and qRT-PCR studies revealed significant upregulation of these pathways in double (cbs; cse) and triple (cbs; cse; tst1) KO flies under control conditions. Following septic injury with Bacillus subtilis, double and triple KO flies exhibited increased expression of antimicrobial peptides (AMPs) and pattern recognition receptors compared to control and TST1-/- single KO lines. Notably, double and triple KO lines showed more prolonged immune activation. The triple KO flies exhibited the worst survival rate following septic injury. These results demonstrate that H2S deficiency causes chronic inflammation through the sustained activation of immune pathways, highlighting sulfur metabolism as a crucial regulator of homeostasis and innate immunity in Drosophila.
This study reports the first re-isolation of a wild-type Salehabad virus (SALV) since 1959, from a pool of male sand flies collected in Türkiye in August 2023. The isolate and the 1959 prototype strain shared 86-92% nucleotide identity across the four coding sequences but 95-99% amino acid identity; for the L protein, 12% nucleotide divergence corresponded to 1% amino acid divergence. Because male sand flies do not blood-feed, this isolation indicates transmission between sand flies by a route other than blood-feeding. These findings highlight the cryptic circulation of SALV and underscore the importance of monitoring historically overlooked phleboviruses.
Cornsilk flies are important pests of sweet corn in Florida due to destructive feeding by larvae on corn ears, which render them unmarketable. Pupation, primarily in the soil, presents a vulnerable stage for predation and parasitism. In this laboratory study, pupal parasitism of Euxesta eluta Loew (Diptera: Ulidiidae) by Spalangia cameroni (Perkins) (Hymentopera: Spalangiidae) was examined in both muck and sandy loam soils typical of Florida corn production areas. Pupae were either placed on the surface or buried 1 or 3 cm under the soil surface to examine effects on parasitoid searching and oviposition. Potential effects of soil moisture were examined by preparing soil that was either dry or at 25% or 50% of the field-holding water capacity, and soils with pupae were exposed to adult S. cameroni for 48 h. Overall percents of killed and successfully parasitized pupae were significantly higher in sandy loam than in muck soil. Soil depth and moisture affected both killed and parasitized pupae in both sandy loam and muck soil, with significant interactions. Pupae in sandy soils were most likely to be killed and parasitized when they were located 3 cm below the surface, especially in moist soil. In contrast, pupal mortality and parasitism in muck soils were mostly observed in pupae on the surface; no parasitism was observed in pupae placed at 3 cm. These findings demonstrate that soil type, depth and moisture heavily influence S. cameroni efficacy, providing critical insights for optimizing biological control strategies against cornsilk flies in diverse habitats.
Phenotypic development is regulated by multiple mechanisms that ensure tight control of gene expression. Post-transcriptional regulation, including the silencing or degradation of messenger RNAs by microRNAs (miRNAs), is an important component of this process. Here, we use gain-of-function and loss-of-function screens to examine the effects of miRNAs on cuticular pigmentation in adult Drosophila melanogaster. We found that 48 of 166 miRNAs ectopically expressed in a stripe along the dorsal side of developing flies were each sufficient to affect pigmentation. We also found that 22 of 41 miRNAs competitively inhibited in the same tissue visibly altered pigmentation, showing that they were necessary for adult pigmentation development. For each of the 15 miRNAs with opposing effects in the gain- and loss-of-function screens, computational tools identified possible targets among 93 genes previously reported to affect adult pigmentation. Using cell culture, we found that one of these miRNAs (miR-8) was able to regulate gene expression through 3' UTR sequences from at least three pigmentation genes: ebony, bric-a-brac 1, and bric-a-brac 2. All three of these genes reduce development of black pigments, suggesting that miR-8 coordinately regulates expression of multiple genes with similar effects on pigmentation. These data show that miRNAs are developmental regulators of body pigmentation, which could also allow them to contribute to pigmentation divergence, as has been shown for miR-193 in butterflies.
Sand fly midgut microbiota plays a critical role in shaping Leishmania development and vector competence, yet functional evidence from natural vector populations remains limited. In this study, sand flies were collected between 2020 and 2022 in Cukurova region, Türkiye to characterize the gut bacterial composition of Phlebotomus tobbi and evaluate the anti-leishmanial potential of cultivable isolates. A total of 1739 sand flies were captured (878 females, 861 males), of which Ph. tobbi was the predominant species (n = 1312). 16S rRNA amplicon sequencing (V4-V6) showed that the gut microbiota was dominated by Proteobacteria, with Erwinia aphidicola/persicina representing the most abundant species across all analyzed groups. Fourteen cultivable bacterial species were identified by MALDI-TOF MS, including Serratia liquefaciens, Pantoea agglomerans, and Micrococcus luteus. Functional XTT assays against Leishmania infantum promastigotes demonstrated variable inhibitory activity among isolates. The strongest leishmanicidal effects were observed with S. liquefaciens (32.3%) and M. luteus (28.8%). Morphological examination confirmed promastigote rounding and cell death in isolates showing >25% activity. These findings define the gut bacterial landscape of Ph. tobbi in an endemic region and identify bacterial taxa with in vitro anti-leishmanial activity, highlighting their potential for future microbiota-based or paratransgenic control strategies.
Nursery pollination systems face an inherent conflict: pollinator larvae consume seeds or floral tissues. Known conflict-resolution mechanisms, including host sanctions, selective abortion, chemical defences, and third-party suppression, operate in systems where larvae feed on seeds. How mutualisms are stabilised when larvae feed on vegetative tissues remains unclear. We investigated a novel nursery pollination involving the dioecious aquatic plant Ottelia jingxiensis, combining field observations, exclusion experiments, floral manipulation, and chemical and colour analyses. We tested whether spatial decoupling of oviposition and larval feeding eliminates the conflict (conflict avoidance), or whether herbivory tolerance is required (conflict resolution). Shore flies (Hydrellia sp.) are primary pollinators; rewardless female flowers attract them via imperfect Bakerian mimicry. Shore flies oviposit on persistent female calyces, but larvae migrate to submerged leaves. Foliar herbivory is substantial (c. 62% of leaves with > 30% area consumed) yet uncorrelated with seed set (R2 < 0.001), consistent with substantial tolerance. A parasitoid wasp (Chaenusa naiadum) parasitises 67.5-80.1% of pupae and provides supplementary pollination but does not reduce within-generation herbivory or seed predation. Spatial decoupling and herbivory tolerance jointly resolve the pollination-herbivory conflict without requiring host sanctions or biotic regulation, representing a previously unrecognised stabilisation mechanism in nursery pollination.
To investigate an outbreak of canine visceral leishmaniasis and the circulation of Leishmania in phlebotomine species in a peri-urban area in the municipality of São Paulo. Twelve dogs were assessed for Leishmania infection and sandflies were collected using Shannon and CDC light traps. The presence of the parasite in the captured female sandflies was analyzed by molecular method. Of the dogs analyzed, four were seropositive both in the rapid serological test and by ELISA for Le. infantum. Among the 8,354 phlebotomines captured, Nyssomyia whitmani predominated in the area (98.2%) and DNA from Le. infantum and Le. braziliensis was detected in females of this species, indicating potential participation in the transmission cycle of the visceral leishmaniasis agent. The findings of this study demonstrate the occurrence of an outbreak of canine visceral leishmaniasis in the Perus district, in the municipality of São Paulo, characterized by the presence of dogs infected with Le. infantum and potential transmission associated with the phlebotomine Ny. whitmani. It also reinforces the importance of entomological surveillance of susceptible vectors and the integration of information in a one health approach, with a view to the early detection of potential transmission foci. Investigar um foco de leishmaniose visceral canina e a circulação de Leishmania nas espécies de flebotomíneos em área periurbana do município de São Paulo. Doze cães foram avaliados quanto à infecção por Leishmania e foram realizadas coletas de flebotomíneos com armadilhas de Shannon e luminosas do tipo CDC. A presença do parasito nas fêmeas de flebotomíneos capturados foi analisada por método molecular. Dos cães analisados, quatro foram soropositivos tanto no teste sorológico rápido quanto por ELISA para Le. infantum. Entre 8.354 flebotomíneos capturados, Nyssomyia whitmani predominou na área (98,2%) e foi detectado DNA de Le. infantum e Le. braziliensis em fêmeas desta espécie, indicando a potencial participação no ciclo de transmissão do agente da leishmaniose visceral. Os achados do presente estudo demonstram a ocorrência de um foco de leishmaniose visceral canina no distrito de Perus, município de São Paulo, caracterizado pela presença de cães infectados por Le. infantum e potencial transmissão associada ao flebotomíneo Ny. whitmani. Também reforça a importância da vigilância entomológica de vetores suscetíveis e da integração de informações em um enfoque de uma só saúde, visando a detecção precoce de potenciais focos de transmissão.
Radiation-induced intestinal injury (RIII) significantly limits the efficacy of abdominal and pelvic radiotherapy while also impairing patient quality of life. This condition is primarily driven by excessive reactive oxygen species (ROS) and dysregulation of Caspase-dependent apoptosis. Quercetin (QUE), a natural antioxidant flavonoid, exhibits radioprotective potential; however, the key signaling pathways it employs to regulate radiation-induced apoptosis remain to be elucidated. In vitro studies, IEC-6 cells (1-10 µg/mL QUE pretreatment followed by 0-8 Gy X-ray exposure) were conducted to analyze proliferation, clonogenic survival, ROS levels, apoptosis, and expression of RIII-related proteins and genes. Network pharmacology identified 47 overlapping targets associated with QUE and RIII, with AKT1 and CASP3 identified as hub targets, and the PI3K-AKT pathway recognized as a key regulatory pathway. A Caspase-3/7 inhibitor (HY-103346, H10) and AutoDock-Vina docking analysis were used to explore the involvement of Caspase-3-related apoptotic signaling. In vivo experiments using Drosophila melanogaster (W1118) involved groups subjected to control, 50 Gy irradiation alone, or 50 Gy combined with 1/5/10/50 µg/mL QUE. Lifespan, locomotor capacity, and intestinal ROS levels were assessed, including validation with DCP-1RNAi transgenic flies (DCP-1: Drosophila Caspase-3 homolog). In vitro findings revealed that QUE enhanced the viability of irradiated IEC-6 cells, reduced ROS and apoptosis, upregulated anti-apoptotic markers (p-AKT, p-PI3K and p-mTOR), and downregulated pro-apoptotic markers (cleaved-Caspase-3 and Cytochrome C), while H10 inhibited the effects of QUE. Molecular docking suggested a potential interaction between QUE and Caspase-3 through hydrogen bonds and hydrophobic interactions to inhibit its activation. In vivo, pre-irradiation gavage of 10 µg/mL QUE mitigated RIII in Drosophila, an effect that was abolished in DCP-1 knockdown flies. In summary, QUE protects against RIII by scavenging ROS and modulating apoptosis-related signaling, with evidence supporting the involvement of the PI3K-AKT/Caspase-3 axis, with the PI3K-AKT/Caspase-3 axis identified as central to these protective effects. This study underscores the clinical potential of QUE for RIII and offers insights into the targeting of apoptosis for radioprotection.
Leishmaniasis, a neglected tropical disease endemic to 98 countries, affects more than 300 million people worldwide. The disease is transmitted through the bite of infected female phlebotomine sand flies harboring Leishmania parasites. Although infection initiates at the cutaneous inoculation site, Leishmania species exhibit distinct tissue tropisms, resulting in three primary clinical manifestations: cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). The initial stage of infection involves the engulfment of metacyclic promastigotes (MPs) by host phagocytes, a process mediated by early interactions between the MP flagellum and the host cell surface. This study investigates how species-specific MP-flagellar proteomic profiles dictate unique interactions with host cell populations, thereby driving divergent pathogenic outcomes. To address this, we conducted a comparative quantitative proteomic analysis of flagella from Leishmania species representing each clinical form. Our analysis revealed distinct flagellar proteomic signatures that differentiate the VL-causing species, L. infantum, from others. Notably, we identified five virulence factor families that were differentially expressed: amastins, cysteine peptidases, heat shock proteins, promastigote surface antigens, and leishmanolysins. These findings link flagellar surface composition to species-specific pathogenicity, providing molecular insights into early infection dynamics and identifying potential antigenic targets for developing species-specific vaccines and therapeutics.
The widely-used acaricide pyridaben is classified as a mitochondrial complex I inhibitor (IRAC Group 21). However, a persistent disconnect exists, as it induces acute neuro-excitatory symptoms including ataxia, tremors, leg paralysis and wing buzzing that are phenotypically inconsistent with a purely metabolic mode-of-action but hallmark cholinergic poisoning. This mechanistic ambiguity complicates resistance prediction and obscures the true sublethal hazard to nontarget arthropods. We therefore tested the hypothesis that its documented sublethal neurotoxicity is driven by direct, off-target inhibition of acetylcholinesterase (AChE) and characterized the mode of this inhibition. Sublethal pyridaben exposure (LC20 = 5.2 μg mL-1; LC30 = 8.2 μg mL-1) in Drosophila melanogaster caused significant neurobehavioral impairment, including 58% reduced locomotion and abolished olfactory attraction with response index declining from +0.50 to -0.20. In vitro, pyridaben acted as a competitive AChE inhibitor (mean inhibitory concentration IC50 = 9.8 ± 1.2 μm; inhibitory Kᵢ = 7.2 ± 0.8 μm; n = 6 replicates). Critically, ex vivo analysis of exposed flies confirmed dose-dependent AChE inhibition (29% reduction at LC20; 46% reduction at LC30), concurrent with behavioral deficits, demonstrating in vivo target engagement. This mechanism was conserved in the agriculturally significant pest Drosophila suzukii. Competitive AChE inhibition is a novel secondary mechanism contributing to pyridaben's sublethal neurotoxicity. These findings resolve the toxicological paradox of its symptomology and provide a mechanistic basis for understanding behavioral impairment in exposed insects, with implications for refining diagnostic resistance monitoring approaches and informing mechanism-based insecticide rotation strategies for sustainable pest management. © 2026 Society of Chemical Industry.
Background/Objectives: The study sought to investigate the neurotoxicity of NBOHs, which are a class of new psychoactive substances (NPSs) that act as agonists of serotonin 5-HT2A and 5-HT2C receptors, resulting in hallucinogenic effects similar to those of LSD, often found in paper blotters. Methods: Differentiated SH-SY5Y cells were incubated for 24 h with 0.5, 1.0, 3.5, 5 and 10 nM of the derivatives 25I-NBOH, 25E-NBOH and 25B-NBOH. Cell viability was assessed by MTT reduction and neutral red uptake assays. Mitochondrial membrane potential and production of free radicals were also evaluated. In addition, Drosophila melanogaster flies were exposed to 5, 10 and 50 nM of each NBOH derivative for 4 h. Negative geotaxis, CAT, and AChE enzymatic activities were evaluated. Results: Both 25E-NBOH and 25B-NBOH decreased cell viability. Furthermore, mitochondrial hyperpolarization of cells and an increase in oxidative species were observed in response to higher concentrations of 25E-NBOH and 25B-NBOH. In D. melanogaster, 25B-NBOH increased climbing time and both 25E-NBOH and 25B-NBOH increased CAT and AChE activities at 50 nM. Conclusions: These findings indicate greater toxicity of the 25E- and 25B-NBOH compared to 25I-NBOH, suggesting metabolic activity or mitochondrial signaling, oxidative stress, and cholinergic dysregulation as a potential mechanism related to NBOH neurotoxicity.