Insects

The black soldier fly, Hermetia illucens, is increasingly valued in applied entomology due to its remarkable capacity to upcycle organic waste and for high nutritional value of its larvae. As a result of global expansion and domestication, the species now displays substantial genetic diversity, yet performance differences between strains remain poorly documented. This study aimed to better understand the relationship between genotype and phenotype, as well as their interaction, to support the improvement of its domestication. Five distinct strains collected from the wild by artisanal farmers or obtained from industrial farms were genetically characterized using whole genome sequencing. These analyses revealed high genetic divergence based on mitochondrial genome and SNP nuclear genome phylogeny. To assess phenotypic performance, the strains were reared on three diets differing in nutritional value: poor (alfalfa meal), intermediate (wheat bran) and rich (chicken feed) and their growth rate was assessed. At harvest, we evaluated different life history traits including survival rate, average larval mass, feed conversion ratio, substrate reduction and bioconversion rate. Statistical analyses revealed strong effects of both diet and strain (p < 0.001), but the key result was the pronounced strain x diet interaction. Performance varied drastically depending on substrate quality: some strains showed high versatility across all diets, while others performed mainly on nutrient-rich substrates or excelled in substrate degradation. In contrast, other strains displayed more specialized profiles, with marked sensitivity to fibrous diets. These contrasted reaction norms highlight that diet performance cannot be interpreted independently of the strain genetics. Overall, these findings underscore the value of preserving diverse local genetic resources and the need for improved molecular tools to guide strain selection. ImplicationThis study shows that performance of the black soldier fly depends strongly on interactions between genetic background and diet, confirming the importance of genotype-environment relationships. While results are based on a limited number of strains and substrates, the consistent strain x diet interaction suggests broader relevance for rearing systems. These findings highlight the need to integrate genomic data into phenotypic assessments. Practically, they indicate that strain selection should be tailored to substrate type to optimize productivity and efficiency. This has direct economic benefits for insect farming and waste management industries because improved strain-diet matching can enhance organic waste bioconversion and support circular economy strategies. Overall, preserving genetic diversity and developing molecular tools for strain selection are key steps toward more sustainable and efficient insect production systems of this study have implications for the development and sustainable BSF systems production.

Black soldier fly larvae (BSFL) have quickly become one of the most farmed animals in the world. However, little is known about how to monitor stress and welfare in these animals. The difficulty of welfare assessment is compounded by the fact that BSFL live in their feed and prefer darkness. This behaviour makes it challenging to observe potential welfare indicators without inducing stress via disturbing the larvae or moving them into the light. However, acoustic devices may be able to pick up signatures of stress in the population even while they are out of sight, allowing for remote monitoring of animals in natural conditions (in the feed and/or in the dark). Acoustic monitoring of this type has been deployed for the detection of insects in stored grains, suggesting this method holds some promise for assessing insect behavioural signatures. In this study, we aimed to identify general, acoustic signatures of stress in BSFL by recording them during exposure to two stressors (light or shaking) or in a low-stress control condition. Our data suggest there are consistent differences in the acoustic recordings of the non-stressed and stressed conditions that may indicate the animals behaviours shift consistently in response to stress. Ultimately, the data suggest acoustic monitoring may hold promise for larval behaviour and/or welfare assessment and should be further explored in response to a variety of stressors across the larval life stage.

Life-history traits play an important role in insect population dynamics and ecological processes. The azuki bean beetle Callosobruchus chinensis is a common pest of stored legumes and is also widely used as a model species in ecological and evolutionary research. In this study, we tested whether machine learning models could be used to estimate several traits of C. chinensis, including elytral length, development time and adult lifespan. Experimental data were obtained from laboratory populations. The dataset included biological and environmental variables such as strain, treatment condition, developmental day, sex, temperature, and CO2. Six different machine learning models were tested, including linear regression, random forest, support vector machine (SVM), neural network, gradient boosting and AdaBoost. Model performance was evaluated using cross-validation. The coefficient of determination (R2) and root mean square error (RMSE) were used to measure prediction accuracy. Prediction accuracy differed among traits. Elytral length showed relatively higher predictability than the other traits, while development time was difficult to estimate in most models. Lifespan was easier to predict than the other traits, and the neural network produced one of the highest prediction accuracies among the tested models. Feature importance analysis also showed that factors such as sex and treatment condition contributed to variation in several traits. Machine learning models therefore helped reveal relationships among biological variables and life-history traits in C. chinensis. Combining ecological experiments with machine learning analysis may help improve our understanding of insect traits and may support future studies in insect ecology and pest management.

The Mediterranean fruit fly (medfly) (Ceratitis capitata (Wiedemann, 1824) is a major agricultural pest, and egg desiccation is a critical constraint during handling and mass-rearing, as even short periods without moisture may compromise developmental success and downstream adult performance. The Wolbachia-medfly symbiosis is a relatively recently established artificial association, generated less than three decades ago using Rhagoletis cerasi as the Wolbachia donor. In this study, we evaluated the effects of egg-stage desiccation on developmental success and subsequent adult performance in three medfly lines differing in Wolbachia status: the uninfected Benakeion line, the wCer2-infected 88.6 line, and the wCer4-infected S10.3 line. Eggs were exposed to desiccation for 0-24 h at 4-h intervals before transfer to larval diet, and hatching, pupation, and adult emergence were recorded. We additionally assessed adult survival under stress for flies emerging from the 0, 8, and 10 h egg-desiccation treatments. Under control conditions, Benakeion showed the highest hatching and developmental recovery, S10.3 the lowest, and 88.6 intermediate performance. Across all strains, short desiccation exposures were comparatively well tolerated, whereas prolonged exposure sharply reduced hatching, pupation, and adult emergence, with the clearest decline at 20-24 h. Strain-dependent differences were expressed mainly at the hatching stage, while later developmental transitions were more similar among strains once larvae had hatched. In the adult follow-up, strain, rather than moderate egg-stage desiccation, was the main determinant of short-term survival and survival under extreme stress, with S10.3 again showing the weakest performance. These results indicate that Wolbachia-associated fitness costs in medfly are strain dependent and that egg-stage desiccation primarily acts at the embryonic bottleneck. Beyond providing insight into the Wolbachia-medfly artificial symbiosis, our findings are directly relevant to egg-handling and strain-evaluation protocols in medfly mass-rearing systems.

Introduced to Hawaii in 2016, Lema equestris has become a garden pest commonly reported on Solanum americanum, which is grown as a native and cultural plant in Hawaii and supports native vertebrates elsewhere across Oceania. Originally identified as L. solani, the species was later found to have been misidentified. Here, molecular and morphological evidence is used to discriminate Hawaiian specimens from L. solani and support the updated identification of L. equestris. As a new invasive species, it is important to confirm host associations and determine whether it will prey on important species, such as endemic or endangered plants, in its new range or any potential range to which it could spread. To this end, feeding assays were performed with adults, first-instar larvae, and newly hatched naive larvae on 11 potential hosts, comprising mostly Solanum species: Solanum americanum, potato, tomato, tomatillo, poha (gooseberry), chili pepper, eggplant, tobacco, tree tobacco, cabbage, and Brazilian nightshade. While feeding was attempted on cabbage, poha berry, and Brazilian nightshade, no host besides S. americanum supported survival. Rearing was used to further characterize the biology and life history of L. equestris, including instar length and distinctive morphological traits for identifying each life stage. While many basic biological traits are confirmed here, much remains to be studied to better understand this species and why it has begun to spread.

Understanding how fruit fly species partition resources along environmental gradients is important for predicting pest pressure under changing climatic conditions. The population ecology of Dacus bivittatus (Bigot) and Dacus punctatifrons (Karsch) (Diptera: Tephritidae) was examined across six sites spanning 526-1,650 m above sea level in the Uluguru Mountains, Tanzania, over eight years (2004-2012). A total of 2,200 weekly trap records were aggregated into 292 site-month observations and standardised as flies per trap per day (FTD). Dacus bivittatus showed strong seasonal structuring (H = 43.03, p < 0.001), with abundance peaking during the cool dry season (June-August), whereas D. punctatifrons showed no clear seasonal pattern. Both species declined significantly with increasing altitude ({rho} = -0.308 and -0.769, respectively; p < 0.001), but the decline was steeper for D. punctatifrons. Species dominance shifted along the gradient: D. punctatifrons dominated warm lowland conditions (>24 {degrees}C), whereas D. bivittatus prevailed at elevations above approximately 569 m. Seasonal niche overlap declined markedly with altitude, indicating increasing temporal segregation between the species in cooler environments. These findings demonstrate that altitude structures ecological divergence between two closely related fruit fly pests and provide a basis for site-specific monitoring and climate-sensitive pest forecasting in tropical mountain agroecosystems.

Gastropods are a highly diverse and often overlooked taxonomic group of significant ecological and economic importance. Some terrestrial gastropods are critical pests of commercial agriculture and home gardens worldwide. Malacopathogenic nematodes offer an effective biological control method of managing pest slugs and snails as a natural enemy. Pellioditis (syn. Phasmarhabditis) hermaphrodita and Pellioditis (syn. Phasmarhabditis) californica are two species of biocontrol nematodes that have been commercialized, sold as Nemaslug(R) and Nemaslug(R) 2.0 respectively on three continents. Although there is interest in bringing Nemaslug(R) products to the US, they are currently not permitted due to limited knowledge on their North American distribution and effects on non-target and native species. In this study, we investigated the impact of P. hermaphrodita and P. californica on Ariolimax columbianus across two slug-host life stages, in laboratory infectivity assays. The objectives were to 1. determine whether P. hermaphrodita and P. californica nematodes impact survival of A. columbianus, and 2. evaluate whether there are differential effects on survival in juvenile and adult life stages of A. columbianus, in laboratory infectivity trials. We found that P. hermaphrodita caused significant mortality in A. columbianus with 100% mortality observed in both juvenile and adult slug hosts. The P. californica treatment had significant effects on the juvenile A. columbianus group only, with 80% mortality. By contrast, only 16% of unexposed control juveniles and 4% of control adult slugs died during the experiment. These results indicate that P. hermaphrodita and P. californica are lethal to the native, non-target Pacific banana slug (A. columbianus) under laboratory conditions, with mortality differing between juvenile and adult host life stages. Given the ecological importance of A. columbianus, these findings raise concerns for potential non-target effects of P. californica and P. hermaphrodita on terrestrial gastropod communities and emphasize the need for testing biocontrol agents against multiple life stages.

Nocturnal migration is a remarkable phenomenon observed in many insect species, including moths. Migratory moths are capable of maintaining precise directional orientation during migration, as demonstrated in both laboratory and field studies, suggesting that they use multiple environmental cues for orientation and navigation. Recent studies on Australian Bogong moths revealed that these animals can use stellar cues and likely the geomagnetic field (in conjunction with local visual cues) to select and maintain population-specific migratory direction. However, the underlying orientation mechanisms used by most other migratory moths are still largely unresolved. Further, it remains unclear whether migratory moths can adjust their orientation using Earths magnetic field parameters for determining their position relative to the goal (i.e. location or map information) - an ability clearly shown in some migratory birds which respond to virtual magnetic displacements by correcting their orientation (experiments when animals are exposed to magnetic cues corresponding to other geographic locations). Here, we present results from virtual magnetic displacement experiments conducted on red underwings (Catocala nupta). In addition, we tested their orientation under simulated overcast conditions and in a vertical magnetic field to get indications whether this species relies on geomagnetic or celestial cues to maintain its population-specific migratory direction. Our results show that (1) red underwings did not compensate for virtual magnetic displacement, indicating the absence of a magnetic map; (2) they remained significantly oriented in the absence of geomagnetic information, suggesting the use of a stellar compass; and (3) there was no evidence of magnetic compass orientation in absence of any visual cues.

Electroantennography (EAG) is a valuable approach for monitoring the sensory responses of insects to insecticidal and repellent molecules and an effective tool for early screening of compounds aimed at controlling and protecting against medically important insect vectors. However, its predictive potential for repellent efficacy in triatomine vectors remains poorly explored. The objective of this study was to evaluate the EAG responses to different xenobiotics as a preliminary selection strategy for compounds with potential repellent action against triatomines. For this purpose, the antennae of adult triatomines subjected to prolonged fasting ([&ge;]30 days) were exposed to repellent molecules. In parallel, repellency bioassays were conducted using a live bait (Gallus gallus) and a newly designed laboratory device to validate the electroantennographic results. EAG recordings showed a significant reduction in olfactory capacity of> 60% in response to the chemical compounds IR3535 and carvone, consistent with the protection times observed in the repellency tests (135.6 {+/-} 43.29 min and 108 {+/-} 26.33 min, respectively). In conclusion, the compounds with the highest repellent activity were clearly discriminated by the insects olfactory system, a finding corroborated by the decrease in electrical signals recorded in the EAG bioassays.

Holometabolous polyphagous insects undergo complete metamorphosis and exploit multiple host plants, exposing them to highly variable ecological conditions across both life stages and host plants. Whether these species, like specialist ones, harbour a stable core microbiota, or whether life stages or host plants act as the primary drivers of microbiota assembly remain open questions. Here, we characterized the fungal and bacterial communities associated with Drosophila suzukii across life stages and host fruits using 16S and ITS metabarcoding. We tested the relative influence of life stage and host fruit on microbiota composition, using community and network-based analyses. We first identified that host fruit significantly structured fungal communities, but not bacterial ones. Yeast communities were rather fruit-specific: Hanseniaspora and Pichia mostly associated with cherries and strawberries, contrary to Metschnikowia with blackberries. In contrast, bacteria and filamentous fungi were shared across fruits, constituting for fruits a core microbiota dominated by Gluconobacter cerinus, Tatumella and Cladosporium. Second, we found that both bacterial and fungal D. suzukii communities were structured by life stage, and that fungal, but not bacterial communities, were also structured by host fruits. D. suzukii individuals harboured a core bacteria composed of G. cerinus and a niche-specific microbiota composed of yeasts: Hanseniaspora typical in individuals related to cherry and strawberry, and Metschnikowia to blackberry. Components of both core and niche-specific microbiota were most likely horizontally acquired by D. suzukii from host fruits. Taken together our results underline the importance of meta-community approaches to investigate tripartite interactions among insects, host plants and microbiota. IMPORTANCEThe role of gut microbiota in mediating interactions between phytophagous insects and their host plants has been well illustrated in specialist species. However, it has been less comprehensively studied in polyphagous species, which infest multiple host plants, and across life stages for holometabolous species experiencing separate ecological niches through development. We tested the existence of a core, a niche-specific and a stage-specific microbiota in a polyphagous holometabolous species, D. suzukii. We examined both fungal and bacterial communities in larvae, pupae and emerging flies infesting three host fruits. Our results showed first that the assembly of bacteria, filamentous fungi and yeasts on fruits is driven by different ecological processes. Second, that D. suzukii harbours a core bacterial microbiota, a niche-specific microbiota constituted by yeasts and no stage-specific microbiota. Our study emphasizes the importance of considering jointly the assembly of host plant and polyphagous insect microbial communities to better understand the ecology and evolution of insect-microbe interactions.

RNA viruses are common in tephritid fruit flies including the Queensland fruit fly, Australias most significant horticultural pest. For many their transmission, tissue tropism and load across host development remain unexplored. Yet these factors are important for host biology, ecology and pest management. We investigated Bactrocera tryoni orbivirus (OV), Bactrocera tryoni xinmovirus (XV), Bactrocera tryoni toti-like virus (TLV) and Bactrocera tryoni iflavirus species 2 (IVsp.2) that commonly coinfect B. tryoni laboratory populations. OV and XV transmission was vertical within and on eggs, while TLV transmission was vertical within eggs. IVsp.2 was not detected in eggs but was present in adults; however, IVsp.2 was horizontally transmitted, with viral load increasing with cohabitation time with infected flies. Horizontal transmission was not observed for the other viruses. OV had a similar load across all tissues, while XV was consistently more abundant in ovaries. TLV had a high viral load in the brain whereas IVsp.2 was abundant in the thorax, foregut and midgut. Besides differences in eggs, the viruses were detected in all other developmental stages, but viral load patterns differed: viral load remained constant for TLV, fluctuated for OV and XV, and was low in pre-adult stages and high in adults for IVsp.2. Our findings demonstrate distinct transmission strategies and tissue tropism among the viruses, providing new insights into their epidemiology and role in host biology. Furthermore, contrary to prevailing views that viruses are generally horizontally transmitted, most known RNA viruses of B. tryoni are vertically transmitted affecting the evolution of host-virus interactions.

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors. Author summaryRhodnius prolixus is a blood-feeding insect and a major vector of Chagas disease, a parasitosis endemic in Latin America and affecting millions of people worldwide. In the absence of effective drugs and vaccines, the control of the insect population represents a promising strategy to reduce the diffusion of the disease. Yet, genetic and functional studies in Rhodnius are extremely challenging due to its feeding habit and life cycle. To overcome these limitations, researchers have previously developed two stable cell lines derived from Rhodnius embryos. In this study, we provide the first characterization of the genes expressed in these cell lines. We found that, while the two cell lines share many expressed genes, each of them also has distinct gene expression patterns pointing to two different cell types with specialized functions. These differences likely affect the way they respond to stress and regulate biological processes. Our findings provide an important resource for researchers studying Rhodnius prolixus and other insect vectors, helping advance our understanding of the genetic and molecular mechanisms that control the insect development and mediate the interactions between insect vectors and the parasites they transmit

Over the past decade, the global rise in invasive species has accelerated at an unprecedented rate, intensifying threats to ecosystems, human health, and economies worldwide. Newly invasive taxa, such as Tropilaelaps mites, are of particular concern for apiculture and agroecosystems. Despite growing concern about the spread of Tropilaelaps mites and other arthropods, limited resources are available to assess their invasive potential. We characterized 118 invasive arthropod species using available literature to identify key biological and ecological traits associated with invasive potential. We developed predictive generalized linear mixed models (GLMMs) to determine the traits most important for predicting invasive potential (number of invaded regions), and the top-performing models were subsequently applied to Tropilaelaps mercedesae. Several traits were identified as significant predictors of invasiveness, including the degree of human association, resilience at small population sizes, diet breadth, maximum annual number of generations, altitude range, and the interaction between human association and temperature range. Notably, T. mercedesae was predicted to be capable of invading 160 regions, ranking it within the top 10% most invasive species among those evaluated (12th out of 119), ranked just below the cosmopolitan Varroa destructor mite. These findings position T. mercedesae as a high-risk, yet under-recognized, invasive threat. Collectively, this demonstrates the power of predictive trait-based modeling to inform invasion risk prior to widespread establishment and underscores the urgency of reallocating resources toward surveillance, research, and proactive management strategies rather than relying on costly, often ineffective post-establishment eradication.

Triatomines are the vectors of Chagas disease, one of the main endemic diseases from South to North America, now expanding to other continents. These hemimetabolous insects have been considered poorly visual animals. However, recent findings challenge this idea. Here, we used Rhodnius prolixus as a model species to comprehensively characterize triatomine compound eyes. We found that in the adult stage, eye size significantly exceeds the dimensions predicted by the nymphal eye growth rate. Moreover, while the compound eye grows symmetrically in its dorsal and ventral directions throughout the nymphal instars, in the adult, the eye undergoes greater ventral growth, resulting in a dorsoventrally asymmetrical eye. By studying a bright pseudopupil induced by fluorescence in natural mutant animals, we observed no major differences in sampling resolution between the last nymphal instar and the adult stage. However, the adult eye possesses significantly larger ommatidia, particularly in its ventral region, shifting the area of highest sensitivity from the equatorial region in the nymphal instars to the ventral region in the adult. A similar eye growth pattern was observed in Triatoma infestans and Panstrongylus megistus. The analysis of photographic records from 39 species across 10 genera indicates that an asymmetrical eye is the predominant eye pattern in adult triatomines. Notable exceptions in wingless adults of Mepraia spinolai, reveal a tight association between possessing a large asymmetrical eye and the presence of wings. This suggests that vision might support triatomine dispersal flights among other visual behaviors. Significance StatementKissing bugs are hematophagous insects known for being the vectors of Chagas disease, one of the main endemic diseases in the Americas. Vision was not considered a relevant sensory system in these insects. Here, we show that their eyes increase in size beyond expected by ontogeny and become asymmetrical when transitioning from the last nymphal instar to the adult stage. The eyes undergo a ventral expansion that shifts the region of greatest light sensitivity from the equatorial zone in nymphs to the ventral region in adults. We found this asymmetrical eye only in winged kissing bugs, suggesting that vision supports flight. This is relevant in ecological and epidemiological terms since kissing bugs disperse by flight for habitat colonization and host-seeking.

Ultra-low volume (ULV) insecticide spraying with deltamethrin as the active ingredient is widely used in mosquito control programs, yet its effectiveness against target mosquitoes and its ecological side effects remain poorly quantified under field conditions in Central Europe. Here, we experimentally evaluated the short-term impact of ground ULV spraying on both mosquito populations and non-target flying insects in Hungary using a paired before-after-control-impact (BACI) design. Mosquitoes were sampled with BG Sentinel traps, while non-target insects were collected using malaise traps. ULV treatment resulted in a significant reduction in mosquito abundance at treated sites, with an average decline of approximately 45%. Native and invasive mosquito species, including Aedes albopictus and Aedes koreicus, showed similar proportional decreases. However, treatment effectiveness varied substantially among sites and was influenced by initial mosquito abundance and wind conditions. In parallel, malaise trap samples revealed a marked decline in non-target flying insects, with reductions exceeding 40% across multiple taxonomic groups, particularly among small- and medium-sized insects, and also when considering pollinator taxa together. Our results indicate that while ULV spraying can temporarily reduce mosquito abundance, it also imposes considerable short-term impacts on non-target insect communities, highlighting trade-offs between vector control and insect conservation within mosquito management programs.

The increasing use of microbial biopesticides in sustainable agriculture requires a deeper understanding of their potential impact on non-target pollinators. Although biocontrol agents are generally considered safer than synthetic pesticides, they may still cause subtle but ecologically relevant adverse effects on non-target organisms, especially when exposed to multiple stressors that are often overlooked in current risk assessment frameworks. Among these, nutritional stress, caused by habitat loss, fragmentation and reduced floral diversity, is becoming increasingly widespread. In this study, we investigated the lethal and sublethal effects of the bacterial biopesticide Bacillus velezensis (formerly B. amyloliquefaciens) strain QST713 at field-relevant concentrations on two key pollinators: Apis mellifera and Bombus terrestris. For the first time for a biopesticide, oral toxicity was assessed under environmental stress represented by diets with varying sugar concentrations (optimal and suboptimal) to identify potential synergistic effects on bee health. Sublethal effects were examined by studying learning performance and memory retention through a conditioning experiment under laboratory conditions. The results showed marked species-specific differences. While B. velezensis did not impact bee survival under realistic nutritional conditions, we observed a synergistic lethal effect in B. terrestris when biopesticide exposure was coupled with extreme nutritional stress (sugar deprivation). Similar species-specific differences emerged at the behavioral level: unlike A. mellifera, B. terrestris showed impaired visual learning and early long-term memory recall. Taken together, these results show that sublethal cognitive endpoints and multi-stressor contexts may reveal vulnerabilities not immediately evident through mortality-based assessments alone. Our findings also highlight the importance of including multiple pollinator species in risk assessment, as sensitivity to biopesticides might greatly vary among species.

Methods for estimating and modeling the long-term and short-term adult flight dynamics of the conifer silk moth Dendrolimus superans (Lepidoptera: Lasiocampidae) are examined. The analysis uses light trap adult catch data collected over 21 years, from 2005 to 2025. Three models of adult flight are considered: a flight-initiation model driven by weather factors, an autoregressive model of long-term catch dynamics, and a binary model of seasonal catch. For the flight-initiation model, we propose estimating the accumulated temperature sum ST from the date when the first derivative of the remote sensing vegetation index NDVI becomes positive until the date of the first adult capture of the season. ST is shown to be sufficiently stable across all years of observation, with flight each year beginning after this temperature sum is reached. The second model demonstrates that the long-term light trap catch time series is well described by a second-order autoregressive model AR(2), in which the catch of the current year depends on catches from the two preceding years. This long-term series is compared with a previously studied larval population density series of the Siberian silk moth; both are shown to be AR(2) series with similar coefficient values, which suggesting that adult catch data may serve as a proxy for absolute larval population density. In the third model, we describe the transition from absolute-scale seasonal catch dynamics (number of adults per day) to a binary scale (0, 1), where 0 denotes days on which no adults were attracted to the trap, and 1 denotes days on which at least one individual was captured. The seasonal absolute catch series is thereby transformed into a binary series of zeros and ones, and relationships between adjacent values in such a binary series are examined. A linear relationship between the absolute and binary seasonal dynamics series is demonstrated, making it possible to estimate absolute catches from binary catch values and to analyze seasonal flight in sparse pest populations. This potentially opens new avenues for understanding how outbreak populations function at chronically low density. Author summaryForest pests can cause catastrophic damage, yet predicting their outbreaks remains challenging. During periods of low population density, standard monitoring methods become labor-intensive and uninformative, while the transition to an outbreak often occurs unexpectedly. Using a 21-year dataset of adult Siberian silk moth (Dendrolimus superans) captures from light traps, we developed an approach combining three complementary models. First, we showed that moth flight begins upon reaching a specific temperature sum, with the starting point determined by NDVI vegetation index dynamics rather than a calendar date--making the forecast more ecologically relevant. Second, long-term adult population dynamics follow a second-order autoregressive model AR(2), matching the dynamics previously observed for larval populations. This establishes light trap data as a reliable proxy for absolute population density when ground surveys are impractical. Third, we introduced a method to analyze seasonal flight using binary data (presence/absence of moths per day), which we showed is linearly related to absolute abundance. This enables studying population dynamics during periods of extremely low density, when traditional methods fail. Our approach opens new possibilities for early warning systems to detect when a population risks transitioning from a latent state to an outbreak phase.

Accurate species identification is crucial to assess the medical and veterinary relevance of a mosquito specimen, but it requires high experience of the observers and well-equipped laboratories. This study aimed to evaluate whether low-cost imaging in combination with geometric wing morphometrics can provide accurate identification of invasive, morphologically similar Aedes species. The right wings of 670 female specimens covering 184 Ae. aegypti, 156 Ae. albopictus, 166 Ae. j. japonicus and 164 Ae. koreicus, were removed, mounted and photographed with a professional stereomicroscope (Olympus SZ61, Olympus, Tokyo, Japan) and a macro lens (Apexel-24XMH, Apexel, Shenzhen, China) attached to a smartphone. The coordinates of 18 landmarks on the vein crosses were digitalized by a single observer for each image. In addition, the landmarks of 20 specimens per species and imaging device were digitalized by six different observers to assess the degree of the observer error. The superimposed shape variables were used to compare the species classification accuracy of linear discriminant analysis (LDA), support vector machine (SVM), Random Forest (RF), and XGBoost. In the single-observer landmark data, the LDA achieved the best classification results with a mean accuracy of 95 % for landmarks from microscope images and 92 % for those obtained from smartphone images. In the multi-observer landmark data, LDA consistently performed worse than the other three classifiers, and the reduction in the accuracy was more pronounced for smartphone images than for microscope images. This pattern was associated with a higher degree of observer error for smartphone images, as confirmed by a landmark-wise comparison across all landmarks. Geometric wing morphometrics provides a reliable method to distinguish the most common invasive Aedes species in Europe. Thereby, the image quality obtained by smartphones equipped with a macro lens is sufficient and represents a cost-effective alternative to professional microscopes. However, due to the greater degree of observer variation for smartphone images, landmark coordinates for such images should ideally be collected by a single observer.