The potential of acoustic monitoring of aquatic insects for freshwater assessment.

Aquatic insects are a major indicator used to assess ecological condition in freshwater environments. However, current methods to collect and identify aquatic insects require advanced taxonomic expertise and rely on invasive techniques that lack spatio-temporal replication. Passive acoustic monitoring (PAM) is emerging as a non-invasive complementary sampling method allowing broad spatio-temporal and taxonomic coverage. The application of PAM in freshwater ecosystems has already proved useful, revealing unexpected acoustic diversity produced by fishes, amphibians, submerged aquatic plants, and aquatic insects. However, the identity of species producing sounds remains largely unknown. Among them, aquatic insects appear to be the major contributor to freshwater soundscapes. Here, we estimate the potential number of soniferous aquatic insects worldwide using data from the Global Biodiversity Information Facility. We found that four aquatic insect orders produce sounds totalling over 7000 species. This number is probably underestimated owing to poor knowledge of aquatic insects bioacoustics. We then assess the value of sound producing aquatic insects to evaluate ecological condition and find that they might be useful despite having similar responses in pristine and degraded environments in some cases. Both expert and automated identifications will be necessary to build international reference libraries and to conduct acoustic bioassessment in freshwaters. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Emerging technologies in citizen science and potential for insect monitoring.

Emerging technologies are increasingly employed in environmental citizen science projects. This integration offers benefits and opportunities for scientists and participants alike. Citizen science can support large-scale, long-term monitoring of species occurrences, behaviour and interactions. At the same time, technologies can foster participant engagement, regardless of pre-existing taxonomic expertise or experience, and permit new types of data to be collected. Yet, technologies may also create challenges by potentially increasing financial costs, necessitating technological expertise or demanding training of participants. Technology could also reduce people's direct involvement and engagement with nature. In this perspective, we discuss how current technologies have spurred an increase in citizen science projects and how the implementation of emerging technologies in citizen science may enhance scientific impact and public engagement. We show how technology can act as (i) a facilitator of current citizen science and monitoring efforts, (ii) an enabler of new research opportunities, and (iii) a transformer of science, policy and public participation, but could also become (iv) an inhibitor of participation, equity and scientific rigour. Technology is developing fast and promises to provide many exciting opportunities for citizen science and insect monitoring, but while we seize these opportunities, we must remain vigilant against potential risks. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Modeling insect growth regulators for pest management.

Insect growth regulators (IGRs) have been developed as effective control measures against harmful insect pests to disrupt their normal development. This study is to propose a mathematical model to evaluate the cost-effectiveness of IGRs for pest management. The key features of the model include the temperature-dependent growth of insects and realistic impulsive IGRs releasing strategies. The impulsive releases are carefully modeled by counting the number of implements during an insect's temperature-dependent development duration, which introduces a surviving probability determined by a product of terms corresponding to each release. Dynamical behavior of the model is illustrated through dynamical system analysis and a threshold-type result is established in terms of the net reproduction number. Further numerical simulations are performed to quantitatively evaluate the effectiveness of IGRs to control populations of harmful insect pests. It is interesting to observe that the time-changing environment plays an important role in determining an optimal pest control scheme with appropriate release frequencies and time instants.

Heatwaves inflict reproductive but not survival costs to male insects.

Climate change is having a dramatic effect on the environment, with rising global temperatures and more frequent extreme climatic events, such as heatwaves, that can hamper organisms' biological functions. Although it is clear that sudden and extreme temperatures can damage reproductive processes, there is limited understanding of the effects of heatwaves on male mating behaviour and reproductive success. We tested for the effects of heat stress induced by ecologically relevant heatwaves (33°C and 39°C for five consecutive days) on the mating behaviour, reproductive success, body mass and survival of male field crickets Gryllus bimaculatus, paired with untreated females. We predicted life-history and reproductive costs would increase with increasing heatwave intensity. Consistent with our expectations, males exposed to the highest heatwave temperature produced the fewest offspring, while having to increase courtship effort to successfully mate. Males also gained relatively more weight following heatwave exposure. Given that we found no difference in lifetime survival, our results suggest a potential trade-off in resource allocation between somatic maintenance and reproductive investment. Taken together, our findings indicate that sublethal effects of heatwaves could reduce the growth and persistence of animal populations by negatively impacting reproductive rates. These findings highlight the need for considering thermal ecologies, life history and behaviour to better understand the consequences of extreme climatic events on individuals and populations.

Optimal allocation of resources among general and species-specific tools for plant pest biosecurity surveillance.

This paper proposes a surveillance model for plant pests that can optimally allocate resources among survey tools with varying properties. While some survey tools are highly specific for the detection of a single pest species, others are more generalized. There is considerable variation in the cost and sensitivity of these tools, but there are no guidelines or frameworks for identifying which tools are most cost-effective when used in surveillance programs that target the detection of newly invaded populations. To address this gap, we applied our model to design a trapping surveillance program in New Zealand for bark- and wood-boring insects, some of the most serious forest pests worldwide. Our findings show that exclusively utilizing generalized traps (GTs) proves to be highly cost-effective across a wide range of scenarios, particularly when they are capable of capturing all pest species. Implementing surveillance programs that only employ specialized traps (ST) is cost-effective only when these traps can detect highly damaging pests. However, even in such cases, they significantly lag in cost-effectiveness compared to GT-only programs due to their restricted coverage. When both GTs and STs are used in an integrated surveillance program, the total expected cost (TEC) generally diminishes when compared to programs relying on a single type of trap. However, this relative reduction in TEC is only marginally larger than that achieved with GT-only programs, as long as highly damaging species can be detected by GTs. The proportion of STs among the optimal required traps fluctuates based on several factors, including the relative pricing of GTs and STs, pest arrival rates, potential damage, and, more prominently, the coverage capacity of GTs. Our analysis suggests that deploying GTs extensively across landscapes appears to be more cost-effective in areas with either very high or very low levels of relative risk density, potential damage, and arrival rate. Finally, STs are less likely to be required when the pests that are detected by those tools have a higher likelihood of successful eradication because delaying detection becomes less costly for these species.

Abundance and risk assessment of microplastics in water, sediment, and aquatic insects of the Nile River.

Microplastics (MPs) are a serious threat in freshwater environments. The ecological risk and abundance level of MPs in abiotic and biotic compartments of the Nile River haven't been systematically reported. Thus, these issues were highlighted in the present study during different seasons of the sampling year. The results showed that MP concentrations in the river ranged from 2.24 ± 0.6 to 3.76 ± 1.1 particles/L, 298 ± 63 to 520 ± 80 particles/kg dry weight, and 0.081 ± 0.051 to 4.95 ± 2.6 particles/individual in surface water, sediment, and different species of aquatic insects, respectively. All the extracted MPs are colored blue, red, and black. Fiber-shaped polyesters (<500-1500 µm) were the most common MPs in all the river compartments. MPs' dominance was observed during the summer in comparison with that in the other seasons. Environmental risk indicators indicate the high ecological risk of MPs, which are widely distributed in the Nile River. In conclusion, MP consumption by aquatic insects may not only be related to levels of environmental contamination, since other variables, such as taxon size, weight, and particular feeding behavior, may also be significant. Additionally, the presence of MPs in insects (at lower trophic levels) creates the potential for predation-based inter-trophic level transmission. Thus, higher trophic-level investigations of various feeding groups should be carried out to identify any possible harm that MPs cause to various aquatic organisms.

Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

Since the discovery of the RNA interference (RNAi) mechanism, it has been widely used in the fields of gene function, biomedicine, and crop pest control. In the direction of agricultural application, this technology is highly expected, especially in the field of pest control, which is called "the third revolution in the history of pesticides". Currently, RNA biopesticides are developing rapidly all over the world. A genetically modified product (MON87411) has been approved for marketing, and a large number of agricultural companies are developing products based on direct spraying RNA biopesticides and submitting them for regulatory approval. The biggest problem that has emerged for spray RNA biopesticides is the technology for large-scale and low-cost production of dsRNA. At present, the bacterial fermentation production technology can realize large-scale dsRNA production with a yield of 4.23~182 mg/L bacterial solution. This article describes the experimental protocol for dsRNA production technology based on bacterial fermentation.

Economic costs of invasive non-native species in urban areas: An underexplored financial drain.

Urbanization is an important driver of global change associated with a set of environmental modifications that affect the introduction and distribution of invasive non-native species (species with populations transported by humans beyond their natural biogeographic range that established and are spreading in their introduced range; hereafter, invasive species). These species are recognized as a cause of large ecological and economic losses. Nevertheless, the economic impacts of these species in urban areas are still poorly understood. Here we present a synthesis of the reported economic costs of invasive species in urban areas using the global InvaCost database, and demonstrate that costs are likely underestimated. Sixty-one invasive species have been reported to cause a cumulative cost of US$ 326.7 billion in urban areas between 1965 and 2021 globally (average annual cost of US$ 5.7 billion). Class Insecta was responsible for >99 % of reported costs (US$ 324.4 billion), followed by Aves (US$ 1.4 billion), and Magnoliopsida (US$ 494 million). The reported costs were highly uneven with the sum of the five costliest species representing 80 % of reported costs. Most reported costs were a result of damage (77.3 %), principally impacting public and social welfare (77.9 %) and authorities-stakeholders (20.7 %), and were almost entirely in terrestrial environments (99.9 %). We found costs reported for 24 countries. Yet, there are 73 additional countries with no reported costs, but with occurrences of invasive species that have reported costs in other countries. Although covering a relatively small area of the Earth's surface, urban areas represent about 15 % of the total reported costs attributed to invasive species. These results highlight the conservative nature of the estimates and impacts, revealing important biases present in the evaluation and publication of reported data on costs. We emphasize the urgent need for more focused assessments of invasive species' economic impacts in urban areas.

Aquatic insects for monitoring the health status of riverine potholes: A case study in Chalakudy river basin, Kerala, India.

Potholes are microhabitats in riverine ecosystems that substantially contribute to the unique regional faunal diversity and community composition. Investigations on the ecological status of potholes in riverine habitats are very scanty. The present work is of utmost importance, particularly as a pioneering study to evaluate the health status of riverine potholes using aquatic insects as potential biological indicators. Samples of water and aquatic insects were collected and analyzed during the pre-monsoon season from February 2022 to May 2022 in the potholes at Athirappilly and the Ezhattumugham regions of the Chalakudy river basin. The present study identified 208 individuals represented by 15 families in the potholes at Athirappilly, whereas 94 individuals belong to 10 families at Ezhattumugham. The computation of water quality parameters and benthic metrics revealed a significant variation between the stations. Spearman correlation analysis revealed that the water temperature, electrical conductivity, dissolved oxygen concentration, and biochemical oxygen demand were influencing the distribution of aquatic insects in the potholes. Families such as Perlidae, Ephemeridae, Baetidae, Stenopsychidae, and Hydropsychidae (sensitive families) reported from the well-oxygenated potholes at Athirappilly, where the water temperature and biochemical oxygen demand were minimum. The significantly high percentage composition of Ephemeroptera, Plecoptera, and Trichoptera taxa and a low family biotic index value indicate good health condition of the potholes at Athirappilly. In contrast, the low percentage composition of Ephemeroptera, Plecoptera, and Trichoptera taxa and high family biotic index values, with the abundance of tolerant families (Micronectidae, Caenidae, and Chironomidae), reflect the impact of organic pollution at Ezhattumugham.

Gene regulation supporting sociality shared across lineages and variation in complexity.

Across evolutionary lineages, insects vary in social complexity, from those that exhibit extended parental care to those with elaborate divisions of labor. Here, we synthesize the sociogenomic resources from hundreds of species to describe common gene regulatory mechanisms in insects that regulate social organization across phylogeny and levels of social complexity. Different social phenotypes expressed by insects can be linked to the organization of co-expressing gene networks and features of the epigenetic landscape. Insect sociality also stems from processes like the emergence of parental care and the decoupling of ancestral genetic programs. One underexplored avenue is how variation in a group's social environment affects the gene expression of individuals. Additionally, an experimental reduction of gene expression would demonstrate how the activity of specific genes contributes to insect social phenotypes. While tissue specificity provides greater localization of the gene expression underlying social complexity, emerging transcriptomic analysis of insect brains at the cellular level provides even greater resolution to understand the molecular basis of social insect evolution.

RNA m6 A Methylation Suppresses Insect Juvenile Hormone Degradation to Minimize Fitness Costs in Response to A Pathogenic Attack.

Bioinsecticides and transgenic crops based on the bacterial pathogen Bacillus thuringiensis (Bt) can effectively control diverse agricultural insect pests, nevertheless, the evolution of resistance without obvious fitness costs has seriously eroded the sustainable use of these Bt products. Recently, it has been discovered that an increased titer of juvenile hormone (JH) favors an insect host (Plutella xylostella) to enhance fitness whilst resisting the Bt pathogen, however, the underlying regulatory mechanisms of the increased JH titer are obscure. Here, the involvement of N6 -methyladenosine (m6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m6 A methyltransferase subunit genes, PxMettl3 and PxMettl14, repress the expression of a key JH-degrading enzyme JH esterase (JHE) to induce an increased JH titer, mitigating the fitness costs associated with a robust defense against the Bt pathogen. This study identifies an as-yet uncharacterized m6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m6 A methylation atlas in insects, which further expands the functional landscape of m6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions.

Allelopathy and allelobiosis: efficient and economical alternatives in agroecosystems.

Chemical interactions in plants often involve plant allelopathy and allelobiosis. Allelopathy is an ecological phenomenon leading to interference among organisms, while allelobiosis is the transmission of information among organisms. Crop failures and low yields caused by inappropriate management can be related to both allelopathy and allelobiosis. Therefore, research on these two phenomena and the role of chemical substances in both processes will help us to understand and upgrade agroecosystems. In this review, substances involved in allelopathy and allelobiosis in plants are summarized. The influence of environmental factors on the generation and spread of these substances is discussed, and relationships between allelopathy and allelobiosis in interspecific, intraspecific, plant-micro-organism, plant-insect, and mechanisms, are summarized. Furthermore, recent results on allelopathy and allelobiosis in agroecosystem are summarized and will provide a reference for the future application of allelopathy and allelobiosis in agroecosystem.

Intraspecific scaling and early life history determine the cost of free-flight in a large beetle (Batocera rufomaculata).

The scaling of the energetic cost of locomotion with body mass is well documented at the interspecific level. However, methodological restrictions limit our understanding of the scaling of flight metabolic rate (MR) in free-flying insects. This is particularly true at the intraspecific level, where variation in body mass and flight energetics may have direct consequences for the fitness of an individual. We applied a 13C stable isotope method to investigate the scaling of MR with body mass during free-flight in the beetle Batocera rufomaculata. This species exhibits large intraspecific variation in adult body mass as a consequence of the environmental conditions during larval growth. We show that the flight-MR scales with body mass to the power of 0.57, with smaller conspecifics possessing up to 2.3 fold higher mass-specific flight MR than larger ones. Whereas the scaling exponent of free-flight MR was found to be like that determined for tethered-flight, the energy expenditure during free-flight was more than 2.7 fold higher than for tethered-flight. The metabolic cost of flight should therefore be studied under free-flight conditions, a requirement now enabled by the 13C technique described herein for insect flight.

Assessment of the impacts of GABA and AChE targeting pesticides on freshwater invertebrate family richness in English Rivers.

Globally, riverine system biodiversity is threatened by a range of stressors, spanning pollution, sedimentation, alterations to water flow, and climate change. Pesticides have been associated with population level impacts on freshwater invertebrates for acute high-level exposures, but far less is known about the chronic impact of episodic exposure to specific classes of pesticides or their mixtures. Here we employed the use of the UK Environment Agency's monitoring datasets over 40 years (covering years 1980 to 2019) to assess the impacts of AChE (acetylcholinesterase) and GABA (gamma-aminobutyric acid) receptor targeting pesticides on invertebrate family richness at English river sites. Concentrations of AChE and GABA pesticides toxic to freshwater invertebrates occurred (measured) across 18 of the 66 river sites assessed. For one of the three river sites (all found in the Midlands region of England) where data recorded over the past 40 years were sufficient for robust modelling studies, both AChE and GABA pesticides associated with invertebrate family richness. Here, where AChE total pesticide concentrations were classified as high, 46 of 64 invertebrate families were absent, and where GABA total pesticide concentration were classified as high, 16 of 64 invertebrate families were absent. Using a combination of field evidence and laboratory toxicity thresholds for population relevant endpoints we identify families of invertebrates most at risk in the selected English rivers to AChE and GABA pesticides. We, furthermore, provide strong evidence that the absence of the invertebrate family Polycentropodidae (caddisfly) from one field site is due to exposure effects to AChE pesticides.

Economic impact disharmony in global biological invasions.

A dominant syndrome of the Anthropocene is the rapid worldwide spread of invasive species with devastating environmental and socio-economic impacts. However, the dynamics underlying the impacts of biological invasions remain contested. A hypothesis posits that the richness of impactful invasive species increases proportionally with the richness of non-native species more generally. A competing hypothesis suggests that certain species features disproportionately enhance the chances of non-native species becoming impactful, causing invasive species to arise disproportionately relative to the numbers of non-native species. We test whether invasive species with reported monetary costs reflect global numbers of established non-native species among phyla, classes, and families. Our results reveal that numbers of invasive species with economic costs largely reflect non-native species richness among taxa (i.e., in 96 % of families). However, a few costly taxa were over- and under-represented, and their composition differed among environments and regions. Chordates, nematodes, and pathogenic groups tended to be the most over-represented phyla with reported monetary costs, with mammals, insects, fungi, roundworms, and medically-important microorganisms being over-represented classes. Numbers of costly invasive species increased significantly with non-native richness per taxon, while monetary cost magnitudes at the family level were also significantly related to costly invasive species richness. Costs were biased towards a few 'hyper-costly' taxa (such as termites, mosquitoes, cats, weevils, rodents, ants, and asters). Ordination analysis revealed significant dissimilarity between non-native and costly invasive taxon assemblages. These results highlight taxonomic groups which harbour disproportionately high numbers of costly invasive species and monetary cost magnitudes. Collectively, our findings support prevention of arrival and containment of spread of non-native species as a whole through effective strategies for mitigation of the rapidly amplifying impacts of invasive species. Yet, the hyper- costly taxa identified here should receive greater focus from managers to reduce impacts of current invasive species.

Insect pollinators can unlock an annual monetary value of more than US $100 million from crop production in Rwanda.

Insect pollinators provide a natural ecosystem service to more than 80% of known flowering plants, many of which are part of our diet. However, their importance in Africa and an agriculture-dependent country like Rwanda has yet to receive attention. This encumbers policy formulation and investments in insect pollinators as a strategic agronomic input. Meanwhile, Rwanda cultivates crops that can benefit significantly from insect pollinators for superior agronomic outputs. To uncover this, we characterized the dependence of the crop production subsector on insect pollinators. Using the bioeconomic approach, we assessed the total economic value and the value due to insect pollinators of crops cultivated in Rwanda. We also evaluated the crop's production value per ton and whether production would meet consumption demands in the complete absence of insect pollinators. Using 71 representative crops currently grown in Rwanda, we found a direct dependency of 62% on insect pollinators. Of 32 representative crops used for economic valuation in two years (2014 and 2020), their total monetary value is estimated at $2.551 billion to $2.788 billion. Direct insect pollinator-dependent crops accounted for 20% (2014) to 18% (2020) of this value, with the share attributed to insect pollinators above $100 million. The sector's vulnerability to insect pollinators decreased from 7.3% in 2014 to 4.3% in 2020. The mean production value per ton of the direct insect pollinator-dependent crops was found to be higher in 2014 before declining in 2020. Using 21 representative crops from 2014 to 2020, we found that many direct insect pollinator-dependent crops will struggle to meet consumption demands in the complete absence of all suitable insect pollinators. Finally, we propose interventions and future research that could be undertaken. These insights are a critical first step to propel the government to act on insect pollination to support its food security agenda.

Assessment of Contact Toxicity and Repellent Effects of Essential Oils from Piper Plants Piper yunnanense and Piper boehmeriifolium against Three Stored-Product Insects.

Storage is a crucial part during grain production for the massive spoilage caused by stored product insects. Essential oils (EOs) of plant origin have been highly recommended to combating insects which are biodegradable and safe mode of action. Hence, to make the fullest use of natural resources, essential oils of different parts from Piper yunnanense (the whole part, PYW; fruits, PYF; leaves, PYL) and Piper boehmeriifolium (leaves, PBL) were extracted by steam distillation method in the present study. Gas chromatography-mass spectrometry (GC-MS) characterization revealed bicyclogermacrene (PYW), γ-muurolene (PYF), δ-cadinene (PYL) and methyl 4,7,10,13,16,19-docosahexaenoate (PBL) as the principal compound of each essential oil. Sesquiterpene hydrocarbons were also recognized as the richest class accounting for 56.3 %-94.9 % of the total oil. Three storage pests, Tribolium castaneum, Lasioderma serricorne and Liposceis bostrychophila, were exposed to different concentrations of EOs to determine their insecticidal effects. All tested samples performed modest contact toxicity in contrast to a bioactive ingredient pyrethrin, among which the most substantial effects were observed in PYF EOs against T. castaneum (35.84 µg/adult), PBL EOs against L. serricorne (15.76 µg/adult) and PYW EOs against L. bostrychophila (57.70 µg/cm2 ). In terms of repellency tests, essential oils of PYF at 78.63 nL/cm2 demonstrated to have a remarkable repellence against T. castaneum at 2h and 4h post-exposure. The investigations indicate diverse variations in the chemical profiles and insecticidal efficacies of P. yunnanense and P. boehmeriifolium EOs, providing more experimental evidence for the use of the Piper plants.

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits. Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

A new flexible model for maintenance and feeding expenses that improves description of individual growth in insects.

Metabolic theories in ecology interpret ecological patterns at different levels through the lens of metabolism, typically applying allometric scaling to describe energy use. This requires a sound theory for individual metabolism. Common mechanistic growth models, such as 'von Bertalanffy', 'dynamic energy budgets' and the 'ontogenetic growth model' lack some potentially important aspects, especially regarding regulation of somatic maintenance. We develop a model for ontogenetic growth of animals, applicable to ad libitum and food limited conditions, based on an energy balance that expresses growth as the net result of assimilation and metabolic costs for maintenance, feeding and food processing. The most important contribution is the division of maintenance into a 'non-negotiable' and a 'negotiable' part, potentially resulting in hyperallometric scaling of maintenance and downregulated maintenance under food restriction. The model can also account for effects of body composition and type of growth at the cellular level. Common mechanistic growth models often fail to fully capture growth of insects. However, our model was able to capture empirical growth patterns observed in house crickets.

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.).

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.