Elateridae Research Articles

Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications.

Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)2) as reducer and stabilizing agents. Hybrid Au/F3O4 nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by l-cysteine (Cys). The four types of nanoparticles, PEG(SH)2AuNPs, PEG(SH)2Au/F3O4NPs, CysAuNPs, and CysAu/F3O4NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)2 was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes.Graphic abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s43630-021-00111-0.

Study of the possibility of using pheromones to control the number of polyphagous soybean (Glycine max) pests

The soybean is an economically important crop for Krasnodar krai. The biological control of polyphagous soybean pests is necessary for organic farming. The aim of our study was to study the possibility of using a synthetic analogs of sex pheromones to control the number of polyphagous soybean pests (Glycine max). The present investigation consisted in the fact that as a primary means of regulation of the number of click beetles (Coleoptera: Elateridae), such as Agriotes gurgistanus Heyden, A. tauricus (Faldermann) and A. sputator (L.) and the cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), methods of mass capture of males (elimination) and disruption of chemical communication (disorientation) were used on soybeans.. The studies were carried out in the soybean plot with an area of 8 hectares (GPS: 45.03° N 38.52° E; FSBSI FRCBZR, Krasnodar), and in the experimental plot “Zolotaya Niva” with an area of 10 hectares (GPS: 45.13°N 39.35°E Voronezhskaya village, Krasnodar Krai). Preparative forms of pheromones were made on specially designed technological equipment at FSCBPP. Glue traps for monitoring and mass catching of cotton bollworms (type “Attracon”) were also manufactured using FSCBPP technological equipment. Estrontype pheromone traps were purchased from the manufacturer. To determine the dynamics of flight and the number of pests, pheromone traps were placed on experimental plots at the rate of one trap per 1–3 hectares. In experiments on mass trapping of insects (elimination), traps were placed in the amount of 5–10 traps per hectare. The effectiveness of the method was determined by the fertilization of females in control and experiment. Females click beetles were captured using the lure method and LED traps.It was found that the effectiveness of the method of elimination of male click beetles (Agriotes gurgistanus, A. tauricus and A. sputator) was 89–95%, and the level of impaired chemical communication (disorientation) reached 91–99%. The disorientation effect for the cotton moth H. armigera was 92%, with the damage to plants and soybeans not exceeding 2.0%. The effectiveness of this method was 88.6–94.5%, during disorientation of three representatives of Elateridae family and cotton moth H. armigera (Lepidoptera: Noctuidae) it was found that the effectiveness of this method was 91.0–99.6% for Elateridae, and 92.0% for H. armigera. This work has presented a novel approach to the possibility of using pheromones as not only the main means of monitoring phytophages, but also a method for controlling polyphagous pests of soybeans, which is important in the development of protection for organic farming systems.

Three years of surveillance associates agro-environmental factors with wireworm infestations in Manitoba, Canada.

Wireworms, the soil-dwelling larvae of click beetles, are a major threat to global agricultural production. This is largely due to their generalist polyphagous feeding capabilities, extended and cryptic life cycles, and limited management options available. Although wireworms are well-documented as economically important pests in the Canadian Prairies, including Manitoba, there are gaps in knowledge on species distributions, subterranean behaviour and life cycles, feeding ecology and damage capacity, and economic thresholds for crop yield loss. We carried out 3 years (2018-2020) of intensive surveillance of larval populations across Manitoba. A total of 31 fields (24 in ≥ 2 consecutive years) were surveyed in early spring using standardized bait trapping approaches. Wireworms were present in 94% of surveyed sites, but the catch within fields varied year to year. While Hypnoidus bicolor predominated (94% of larvae), several other pest species were identified. We then explored the relationships between wireworm trap numbers and agro-environmental factors. The larval catch tended to decrease under conditions of low soil temperatures and increased clay content, coupled with high soil moisture and precipitation during the trapping period. Treatment and cultural methods appeared less influential; however, wheat production in either of the previous two growing seasons was associated with increased wireworm catch. Our models failed to predict a relationship between wireworm catch and crop yields, although infestations were rare in our region. Our findings better infer the risks posed by wireworms to crop production in the Canadian Prairies, and the agro-environmental factors that represent the greatest contributors to these risks. This information should be incorporated into future integrated pest management (IPM) strategies for wireworms. © 2021 Her Majesty the Queen in Right of Canada Pest Management Science © 2021 Society of Chemical Industry Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Preventive field application of Metarhizium brunneum in cover crops for wireworm control

Wireworms, the larvae of click beetles (Coleoptera: Elateridae), can cause substantial losses in marketable yield of potatoes, yet control options are limited. The entomopathogenic fungus Metarhizium brunneum (Ascomycota; Hypocreales) isolate ART2825 is highly virulent against two of the most detrimental wireworm species, Agriotes obscurus L. and A. lineatus L., but field application of this isolate during potato cultivation has never succeeded. In this study, we integrated the fungus into the agricultural crop rotation prior to potato cultivation, with the aim of better adapting the application strategy to the fungus’ ecological and environmental requirements. Application preceded sowing of cover crops in late summer. We hypothesized that higher temperatures and undisturbed development for several months would support the establishment of the entomopathogen and enhance biocontrol efficiency in the following season. In two subsequent seasons, we quantified (1) fungal establishment in the soil, (2) efficiency of treated soils against wireworms in vitro, and (3) levels of wireworm damage in field potatoes. Spore concentration was enhanced in treated plots and we recovered the released Metarhizium isolate from all mycosed, field-collected wireworms. Treated soils increased wireworm mortality in the laboratory, but a statistically significant reduction of potato damage was only achieved in two out of ten field trials. The application strategy shows potential for fungal enhancement and opens new avenues for biological wireworm control.

Assessment of the Attraction Range of Sex Pheromone Traps to Agriotes (Coleoptera, Elateridae) Male Click Beetles in South-Eastern Europe.

Simple SummaryClick beetles are the adults of wireworms, soil-dwelling larvae that damage multiple arable crops. The attraction range of YATLORf pheromone traps to click beetles of four species of Agriotes wireworms was studied to provide additional information about the implementation of integrated pest management against these harmful pests in Europe. This should allow a significant reduction in insecticide use. Male click beetles were marked and released at different distances from a pheromone trap. Recapture rate, maximum sampling ranges, and effective sampling areas were calculated. The recapture rate was significantly affected by distance, species, and wind direction and decreased as distance increased. The majority of beetles were caught from short distances (up to 10 m) within the first five days. The estimated attraction range was low for all the considered Agriotes species, suggesting that pheromone traps were unsuitable for use as mass trapping instruments to disrupt mating. However, after results and previous research outputs were evaluated, it seems possible to use the traps not only as monitoring tools, but also as attract-and-kill strategies for most beetle populations.The attraction range of YATLORf pheromone traps to adults of four species of Agriotes (A. brevis, A. sordidus, A. litigiosus, and A. ustulatus) was studied to provide additional information about the most harmful Agriotes species in Europe. Male click beetles were marked and released at different distances from a pheromone trap. The recapture rate was calculated and analyzed using analysis of variance. The recapture rate was significantly affected by distance, species, and wind direction. The recapture rate decreased as distance increased. The majority of beetles were caught from short distances (up to 10 m) within the first five days. A. brevis, a mainly crawling species, showed the lowest recapture rate. The wind direction affected the recovery rate, with a significantly lower number of beetles moving downwind from the release points. Maximum sampling ranges and effective sampling areas were calculated. The obtained estimations were low (53 to 86 m and 509 to 2602 m2, respectively) for all the considered Agriotes species, suggesting that they were unsuitable for use as mass trapping instruments to disrupt mating. However, it seems possible to use the traps not only as monitoring tools, but also as attract-and-kill strategies for most beetle populations.

Limoniic Acid and Its Analog as Trap Lures for Pest Limonius Species (Coleoptera: Elateridae) in North America.

Four species of Limonius wireworms (Coleoptera: Elateridae), L. californicus, L. canus, L. infuscatus and L. agonus, are serious crop pests in North America. Limoniic acid, (E)-4-ethyloct-4-enoic acid, has been reported as a sex pheromone component of female L. californicus and L. canus, and a sex attractant for male L. infuscatus. In the same study, both limoniic acid and the analog (E)-5-ethyloct-4-enoic acid were highly attractive in field experiments. Moreover, six carboxylic acids in headspace volatiles of Limonius females elicited responses from male antennae but were not tested for behavioral activity. Here, we report trap catch data of Limonius spp. obtained in field experiments at 27 sites across North America. All four Limonius species were attracted to limoniic acid and to the analog but not to the carboxylic acids. Adding these carboxylic acids to limoniic acid, or to the analog, reduced its attractiveness. In dose-response studies, trap lures containing 0.4 mg or 4 mg of limoniic acid afforded large captures of L. californicus and L. infuscatus. Neither limoniic acid nor the analog were deterrent to other elaterid pest species. The broad attractiveness of limoniic acid to Limonius spp., and its non-deterrent effect on heterogeners, may facilitate the development of generic pheromone-based monitoring and management tools for multiple click beetle species.

Evaluation of 13-Tetradecenyl Acetate Pheromone for Melanotus communis (Coleoptera: Elateridae) Detection in North Carolina Row Crop Agroecosystems.

Melanotus communis Gyllenhal (Coleoptera: Elateridae) larvae are a common soil-dwelling pest of many crops, including sweet potato, grains, and tobacco. Although many studies have focused on the larval stage of this pest, the seasonal activity and ecology of the adults (click beetles) are not well understood. The overarching goal of this study was to relate M. communis adult activity to host crops in the North Carolina row-crop agroecosystem. To do this, we conducted a two-year study documenting male M. communis activity, using a recently identified sex attractant pheromone, 13-tetradecenyl acetate. This project was divided into two parts: 1) a pheromone assessment study testing the efficacy and specificity of 13-tetradecenyl acetate, and two analogs, 13-tetradecenyl butyrate and 13-tetradecenyl hexanoate, and 2) a landscape survey using traps baited with 13-tetradecenyl acetate. Results of the efficacy study showed that 13-tetradecenyl acetate was the most effective M. communis lure when compared to non-baited control traps or traps baited with the two homologs. The landscape study documented a strong association between M. communis catch and the adjacent crop type. We found that adult M. communis abundance was greatest near corn, followed by sweet potato, and then cotton. Analysis of activity over time found that the peak activity occurred during July. Overall, this project demonstrates the usefulness of pheromone-baited traps in providing new information about M. communis activity.

Distribution of two European elaterids, Agriotes obscurus and A. lineatus in British Columbia: New records, and potential implications of their dispersal

• Two invasive click beetle pest species have established in southwestern British Columbia. • Surveys conducted in 2017–2019 reveal both species have now dispersed widely. • Crop damage, displacement of native species, and continued spread is expected. • Several native, non-pest species are attracted to the pheromone of these pests. Two European click beetle species, Agriotes obscurus L. (AO) and A. lineatus L. (AL) have become serious pests of agriculture in the Fraser Valley and Vancouver Island areas of British Columbia (BC), but prior to the start of this survey (2017) it was unknown if they had established elsewhere in the Province. Pitfall traps baited with sex pheromone of AO and AL were placed throughout BC in 2017–2019 and collected a total of 4,988 AO and 20,103 AL beetles in 150 trap pairs. Both species were found to be distributed throughout southern BC, including high numbers in Creston, Kelowna, Pemberton, and Salmon Arm. Traps placed in northern BC, northern Alberta, northern Idaho, or the Willamette valley in western Oregon did not collect AO or AL. Traps were also deployed for A. sputator L., a related European species that has become a serious pest of potato in eastern Canada, but this species was not collected. AO and AL traps placed in the Pemberton valley of BC also collected 474 A. ferrugineipennis (LeConte), and traps placed in the northern Okanagan valley collected 75 A. oregonensis Becker. Both are native Agriotes species not closely related to AO or AL; this is the first time A. oregonensis was collected in Canada. We discuss the potential implications of the spread of AO and AL to other agricultural areas in BC and beyond.

Efficacy of Naturally Occurring and Commercial Entomopathogenic Nematodes Against Sugar Beet Wireworm (Coleoptera: Elateridae).

Wireworms are the larval stage of click beetles (Coleoptera: Elateridae), and some of their species are serious pests of many crops. In the present study, we evaluated the efficacy of naturally occurring and commercial entomopathogenic nematode species against the sugar beet wireworm, Limonius californicus (Mannerheim), in the laboratory. First, efficacies of Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) collected from an irrigated (S. feltiae-SSK) and a dryland (S. feltiae-SSC) field and the two commercial entomopathogenic nematode species, S. carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), were examined. Efficacies of the two field-collected S. feltiae isolates were also compared against a commercial S. feltiae strain. In the first bioassay, S. feltiae-SSK caused 63.3% wireworm mortality, followed by 30% caused by S. carpocapsae, 23.3% by S. feltiae-SSC, and 6.7% by H.bacteriophora. In the second assay, S. feltiae-SSK killed 56.7% of the wireworms, ≈2.1- and ≈5.7-fold higher than S. feltiae-SSC and the commercial isolate, respectively.

Efficiency of pre-planting treatment of potato tubers against the background of high indicators of the presence of harmful organisms

We present the results of long-term studies on evaluating the effectiveness of pre-planting treatment of tubers with modern insectofungicides – Prestige, KS, Celest Top, KS and Emesto Quantum, KS – in protecting potatoes in the conditions of high levels of the presence of harmful organisms in seed material and soil in the North-West Russia. The infestation of planting tubers rhizoctonia disease varied from year to year within the range of 24,4–74,0 %, the intensity of the infestation — within the range of 6,0–16,1 %. The density of the larvae of click beetles in the experimental fields at the time of the spring cultivation ranged from 15 to 81 ind./m2 in different years. High efficiency (62,8–99,0 %) of these insectofungicides in reducing the infestation of new crop tubers rhizoctonia disease was established. The highest protective effect was achieved by using the Emesto Quantum, KS (98,5–99,0 %). The effect of the insecticides against the larvae of click beetles, with their high abundance in potato plantings, was weak (8,5–36,2 %), but significant (42,9–100 %) with respect to a decrease in the proportion of heavily damaged tubers. The overall economic effect of processing planting material with Prestige, KS was 28,4 center/ha (8,6 %), Celest Top, KS – 47,8 center/ha (15,3 %), Emesto Quantum, KS – 15,4 center/ha (5,3 %).

A survey on the click beetles (Coleoptera, Elateridae) in the province of Kurdistan (Iran), with three new records for the Iranian fauna

A survey on the click beetles (Coleoptera, Elateridae) in the province of Kurdistan (Iran), with three new records for the Iranian fauna

Molecular identification of Haemonchus contortus in goats

Haemonchus contortus commonly called the stomach worm or wire worm of ruminants inhabits the abomasum and is considered to be one of the economically important gastrointestinal strongyles in goats. In the present study, H. contortus was identified by PCR using the primers targeting partial 5.8S and partial internal transcribed spacer region 2 (ITS-2). Adult worms were identified morphologically and genomic DNA was extracted using DNeasy Blood and Tissue kit (QIAGEN, Germany). Gradient PCR protocol was standardised using the extracted genomic DNA. Ten-fold serial dilution of adult DNA was used to analyse the minimum detection limit and the products were amplified upto the tenth dilution. Cross reaction of primer sets was checked using the DNA extracted from predominant adult srongyles like Oesophagostomum columbianum and Trichostrongylus colubriformis and no cross reaction was seen at the optimum annealing temperature (60.7°C).

Evaluation of Pheromone Traps and Lures for Trapping Male Agriotes sputator (Coleoptera: Elateridae) Beetles in Eastern Canada

In North America, monitoring for Agriotes spp. click beetles typically has been done using Vernon beetle traps baited with bubble cap sex pheromone lures. This trap and lure are no longer produced commercially and a new trap, the Vernon pitfall trap, and lure design are used now for both invasive Agriotes and native pest species. Herein we compare the 2 trapping methods for Agriotes sputator (L.) (Coleoptera: Elateridae), and provide a calibration factor between them to allow comparison of survey results using the different methods. When deployed for the entire swarming season, Vernon pitfall traps fitted with the new capsule lures collect 0.7× as many A. sputator as Vernon beetle traps fitted with the bubble cap lures, and Vernon beetle traps fitted with capsule lures collect 0.5× as many beetles as Vernon beetle traps fitted with bubble cap lures. Unlike bubble cap lures, however, capsule-style lures need to be primed (maintained at room temperature for 3 wk) before deployment in the field, or else their initial attractiveness will be limited and trap catches will not be representative of populations present at the time. In addition, results from field studies indicate that these capsule lures deplete over the swarming season, and depending on the trapping objective (e.g., mass trapping) may need to be replaced after 5 to 6 wk of deployment. Increasing the lure load to 2× or 4× the regular 40 µL geranyl butanoate, or the capsule container size from 1.0 to 2.5 mL, did not significantly increase the number of A. sputator collected.

Alternative Strategies for Controlling Wireworms in Field Crops: A Review

Wireworms, the soil-dwelling larvae of click beetles (Coleoptera: Elateridae), comprise major pests of several crops worldwide, including maize and potatoes. The current trend towards the reduction in pesticides use has resulted in strong demand for alternative methods to control wireworm populations. This review provides a state-of-the-art of current theory and practice in order to develop new agroecological strategies. The first step should be to conduct a risk assessment based on the production context (e.g., crop, climate, soil characteristics, and landscape) and on adult and/or larval population monitoring. When damage risk appears significant, prophylactic practices can be applied to reduce wireworm abundance (e.g., low risk rotations, tilling, and irrigation). Additionally, curative methods based on natural enemies and on naturally derived insecticides are, respectively, under development or in practice in some countries. Alternatively, practices may target a reduction in crop damage instead of pest abundance through the adoption of selected cultural practices (e.g., resistant varieties, planting and harvesting time) or through the manipulation of wireworm behavior (e.g., companion plants). Practices can be combined in a global Integrated Pest Management (IPM) framework to provide the desired level of crop protection.

Wireworms in Florida Sugarcane

Wireworms, the larval stage of a click beetle, often cause severe damage to numerous crops in Florida. At least twelve species of wireworms have been found in southern Florida, but only the corn wireworm, Melanotus communis, is considered to cause significant economic damage to sugarcane. Since M. communis is the important wireworm species, the rest of this document will pertain to this species. Generally, wireworms are a pest of newly planted sugarcane and only rarely a pest in ratoon sugarcane. More studies are needed, but the current information suggests substantial percentages (e.g. 40% or more) of wireworms could be killed by flooding but, in general, the flood duration had to be at least 4 to 5 weeks at water temperatures above 24 °C. Soil insecticides are generally used in newly planted sugarcane for wireworm control. Insecticides are not used for wireworm control in ratoon sugarcane. Pheromone traps are untested in Florida sugarcane for click beetles but have an important function in for both mass trapping and monitoring in other agricultural systems.

The status of Elateridae (Coleoptera) in India

Beetles are a group of insects that form the order Coleoptera, in the superorder Endopterygota. It is estimated that a quarter of all described species in the world belongs to this order. The family Elateridae or click beetles is the ninth-largest family under this order. Wireworms are the larval stage of this family and they play a role in the ecosystem as they are omnivorous and act as pests. There are over 800 species of elaterids recorded from the Indian Subcontinent under eight subfamilies. India is rich in floral and faunal diversity and insect diversity in particular constitute about 6% of all identified insects in the world with a large number being endemic species. But in contrast to its extreme species richness, there is comparatively very little knowledge about this family of beetles when compared to other families. And as such, there is still a lot of potential to study its ecology and diversity. It is also because of this same reason that review of the status of the elaterid family is being done as a prequel to the actual field study.

Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae)

<p>Wireworms are larvae of click beetles (Coleoptera: Elateridae) and are well known pests of economic importance. Knowing the species diversity in a particular area is important, as the ecological requirements and consequent management strategies of individual species vary. Morphological identification of wireworms is challenging; separating between individual species is often almost impossible. Molecular methods therefore complement morphological methods. Mitochondrial gene for cytochrome oxidase I is most commonly used for molecular identification of wireworms. In this study we list molecular, morphological and behavioural methods for wireworm identification and also highlight the advantages and disadvantages of these methods. At the end of the article, a simplified morphological identification key for determining economically important wireworm species of the genus <em>Agriotes</em> in Slovenian language is attached.</p>

Уточнение видовой принадлежности некоторых видов щелкунов (Coleoptera: Elateridae) на основе молекулярно-генетического анализа

The article is devoted to data on the approbation of modern methods and the refinement of species affiliation of soil-inhabiting larvae of click beetles (wireworm) based on the molecular genetic analysis (DNAbarcoding). For the first time, data obtained on the complete identity of certain DNA sequences of a number of species of click beetles living in the Kostanay Region (Kazakhstan) — dangerous pests of agricultural crops. At the same time, the World genetic bank (GenBank) did not find identical DNA sequences of decoded DNA nucleotide sequences for a number of studied specimens. The basis for this study was the materials collected in 2018 in the subzone of ordinary black earth on sandy loam soils (Mendykarinsky district). The selection of larvae was carried out by the method of standard soil-zoological samples. The fixation and storage of the selected click beetles larvae was carried out according to the method of preparing samples for molecular genetic analysis with fixation in 96 % alcohol. After the classic identification of the taxonomic position of the collected specimens, the species were identified by genetic analysis on the nucleotide sequence of the cytochrome C oxidase I subunit gene (COI). The assembly and decoding of the DNA nucleotide sequences of the studied samples were carried out using the programs «Codon Code Aligner» and «MEGA-X». As a result of the work carried out in the DNA laboratory of the Museum of Natural History (University of Oslo, Norway), it was possible to identify the complete identity of the DNA sequences of several mass species of click beetles, while a number of decoded DNA sequences of model specimens were absent in the genetic bank, which requires replenishment in it with new data.

Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter—membrane-anchored Cypridina luciferase (maCLuc)—paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.