Melolontha Larvae Research Articles

Back to the 'roots' of research: a hypothesis-driven approach provides predictive understanding of plant-herbivore-microbe interactions.

This article is a Commentary on Böttner et al. (2023), 239: 1475–1489.

Investigation of the species composition and harmfulness of dominant phytophages of onions in the Right-Bank Forest-Steppe of Ukraine

The research is aimed at studying the species composition, dynamics of the number and harmfulness of dominant pests, which is extremely important for the development and improvement of elements of the onion protection system. After all, the analysis of literature sources showed insufficient information about the distribution and harmfulness of the main phytophages under modern conditions of growing onions in the Right Bank Forest-Steppe of Ukraine.
 We conducted the monitoring of the phytosanitary condition of onion crops during 2017–2020 and determined the species composition of dominant phytophages. We studied the complex of soil pests: larvae of Melolontha sp., the population density of which over the years of research was 0.8–2.5 specimens/m2, larvae of Elateridae sp., or click beetles (wireworms) – 0.9–2.2 specimens/m2 and Gryllotalpa gryllotalpa L. – 2.5 specimens/m2. Dominant species of phytophages in onion crops included onion fly (Delia Antigua Mg.), onion thrips (Trips tabaci Lind.), and Ceuthorrhynchus jakovlevi Schultze.
 The four most vulnerable phases of growth and development of onions before colonization and damage by major phytophages were identified:
 1) phase of leaf development (a primary shoot) (ВВСН 1–19);
 2) phase of the beginning of thickening of leaf base – bulb formation (ВВСН 41–43);
 3) phase of bulb formation – beginning of leaf splitting (ВВСН 45–47);
 4) phase of the completion of growth and development (state of physiological rest) (ВВСН 48–49)

Stability ofMetarhizium anisopliae(Hypocreales: Clavicipitaceae) isolates during repeatedin vitrosubculture and evaluation of an oil-in-water mycoinsecticide

AbstractNineMetarhizium anisopliae(Hypocreales: Clavicipitaceae) isolates were evaluated for efficacy againstMelolontha melolontha(Coleoptera: Scarabaeidae) larvae, stability in culture, and the superior isolate used to produce an oil-in-water mycoinsecticide. The phenotypic and genotypic characters of four isolates with high virulence were evaluated for their stability after repeating 12 cycles ofin vitrosubculture. Repeated subculture did not affect the germination of conidia; however, the morphology of some isolates changed significantly. Three isolates lost their virulence, whereas the KTU-2 isolate remained highly pathogenic. Therefore, KTU-2 was selected as the superior isolate for mycoinsecticide production. After the conidia of KTU-2 were produced by solid-state fermentation using cracked rice as the substrate, the conidia were formulated as an oil-in-water emulsion and its efficacy was assessed. The formulation caused 80% mortality onMe. melolonthalarvae even at the lowest application rate (1 × 105conidia/mL) in pot experiments, and complete mortality was obtained with the concentration of 1 × 107conidia/mL. Lethal concentrations that kill 50% and 95% ofMe. melolonthalarvae present were estimated as 9.29 × 103and 2.1 × 106conidia/mL, respectively. Oil-in-water mycoinsecticide could be a potential candidate for the commercial control ofMe. melolonthaand other white grubs.

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite.

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid β-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha β-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions.

SOIL-INSECT toolbox: A new chamber for analysing the behaviour of herbivorous insects and tri-trophic interactions in soil

A chamber, named SOIL-INSECT toolbox, was developed to analyse the effect of various factors on the behaviour of soil-dwelling insects. It is equipped with sensors that continuously monitor the concentration of CO2 in the different compartments of the chamber without disturbing the air balance in the soil. The chamber can be adapted to study different stimuli, including volatile compounds, both in the presence and absence of plants. The chamber was tested using the larvae of Melolontha spp., which confirmed its suitability for carrying out complex studies on insect-insect and insect-plant-microbiome interactions in a complex environment such as soil. The results of behavioural experiments using L3 larvae of Melolontha spp. in sterilized and natural soils revealed that the soil condition affected the behaviour of the larvae, likely due to its effect on the soil microbiome and physico-chemical characteristics.

Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

Effectiveness of the Entomopathogenic Fungal Species Metarhizium anisopliae Strain NCAIM 362 Treatments against Soil Inhabiting Melolontha melolontha Larvae in Sweet Potato (Ipomoea batatas L.).

The effect of fungal entomopathogen M. anisopliae strain NCAIM 362 against M. melolontha larvae in sweet potato was tested under open field conditions when crop management included compost supply and soil cover (agro-foil or agro-textile). Additionally, the effect of M. anisopliae same strain against M. melolontha was compared with the effect of α-cypermethrin under greenhouse conditions. Soil microbial community using Illumina sequencing and soil biological activity were tested as possible parameter influencing M. anisopliae effect. According to the results, compost supply and textile cover may enhance the effectiveness of M. anisopliae under open field conditions, while no effect of fungal treatment was detected under greenhouse conditions. Even if soil parameters (chemical composition, bacterial, and biological activity) were identical, the effect of α-cypermethrin against M. melolontha larvae was significant: lower ratio of larval survival and less damaged tubers were detected after the chemical treatment. Our results suggest that M. anisopliae strain NCAIM 362 is not effective to control M. melolontha larvae, further pieces of research are needed to test other species of the Metarhizium genus to find an effective agent for sustainable pest control in sweet potato.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Aboveground phytochemical responses to belowground herbivory in poplar trees and the consequence for leaf herbivore preference

Belowground (BG) herbivory can influence aboveground (AG) herbivore performance and food preference via changes in plant chemistry. Most evidence for this phenomenon derives from studies in herbaceous plants but studies in woody plants are scarce. Here we investigated whether and how BG herbivory on black poplar (Populus nigra) trees by Melolontha melolontha larvae influences the feeding preference of Lymantria dispar (gypsy moth) caterpillars. In a food choice assay, caterpillars preferred to feed on leaves from trees that had experienced attack by BG herbivores. Therefore, we investigated the effect of BG herbivory on the phytochemical composition of P. nigra trees alone and in combination with AG feeding by L. dispar caterpillars. BG herbivory did not increase systemic AG tree defences like volatile organic compounds, protease inhibitors and salicinoids. Jasmonates and salicylic acid were also not induced by BG herbivory in leaves but abscisic acid concentrations drastically increased together with proline and few other amino acids. Leaf coating experiments with amino acids suggest that proline might be responsible for the caterpillar feeding preference via presumptive phagostimulatory properties. This study shows that BG herbivory in poplar can modify the feeding preference of AG herbivores via phytochemical changes as a consequence of root-to-shoot signaling.

Impacts of root herbivory on seedlings of three species of boreal forest trees

Current meta-analysis has identified the shortage of information on responses of woody plants to root damage as the most critical gap in studies of belowground insect herbivory. We explored the impacts of natural and simulated root herbivory on seedlings of three boreal forest-forming trees, Pinus sylvestris, Picea abies and Betula pendula. We compared the effects of natural herbivory and mechanical damage by placing one larva of the common cockchafer, Melolontha melolontha, of different instars (to manipulate root loss) into a pot with a one-year-old seedling or we excised a portion of the roots by inserting a 13-mm diameter cork borer into the soil. During the experiment, which lasted from 2 to 8 weeks, the larvae consumed 53–73% of root biomass, whereas the mechanical damage treatment removed 30–40% of roots. Seedling mortality was low (5%) across all treatments, but the aboveground biomass decreased by 3–43% due to natural herbivory and by 6–30% due to mechanical damage. The M. melolontha larvae consumed a larger amount of roots from B. pendula than from either P. sylvestris or P. abies, but the larvae showed similar relative growth rates and mass gains, indicating a different efficiency of conversion of ingested food (ECI). The values of ECI calculated for larvae of M. melolontha (2.3–3.5%) appeared much lower than the 17% reported earlier for larvae of foliage-feeding beetles, presumably due to the high energetic cost of locomotion in the soil. Consequently, root-feeding insects consume much more plant biomass than leaf-feeding insects do to gain similar body mass, and therefore they inflict relatively more damage to plants per unit of mass gained. Based on the biomass of insect herbivores in European forests, we estimate that they consume 0.5% of the available fine root biomass. The effects of low levels of root damage on growth and reproduction of woody plants remain virtually unknown, emphasising the need for studies of plant- and ecosystem-level effects of minor but chronic root herbivory.

Root damage to apple plants by cockchafer larvae induces a change in volatile signals below‐ and above‐ground

AbstractVolatile organic compounds (VOCs) mediate communication between plants and insects. Plants under insect herbivore attack release VOCs either at the site of attack or systemically, indicating within‐plant communication. Some of these VOCs, which may be induced only upon herbivore attack, recruit parasitoids and predatory insects to feed on the attacking insects. Moreover, some plants are able to ‘eavesdrop’ on herbivore‐induced plant volatiles (HIPVs) to prime themselves against impending attack; such eavesdropping exemplifies plant–plant communication. In apple orchards, the beetle Melolontha melolontha L. (Coleoptera: Scarabaeidae) is an important insect pest whose larvae live and feed on roots for about 4 years. In this study, we investigated whether the feeding activity of M. melolontha larvae (1) alters the volatile profile of apple roots, (2) induces the release of HIPVs systemically in the leaves, and (3) whether infested plants communicate to neighbouring non‐infested conspecifics through HIPVs. To answer these questions, we collected constitutive VOCs from intact M9 roots as well as M. melolontha larvae‐damaged roots using a newly designed ‘rhizobox’, to collect root‐released volatiles in situ, without damaging the plant root system. We also collected VOCs from the leaf‐bearing shoots of M9 whose roots were under attack by M. melolontha larvae and from shoots of neighbouring non‐infested conspecifics. Gas chromatography‐mass spectrometry analysis showed that feeding activity of M. melolontha larvae induces the release of specific HIPVs; for instance, camphor was found in the roots only after larvae caused root damage. Melolontha melolontha also induced the systemic release of methyl salicylate and (E,E)‐α‐farnesene from the leaf‐bearing shoots. Methyl salicylate and (E,E)‐α‐farnesene were also released by the shoots of non‐infested neighbouring conspecifics. These phenomena indicate the induction of specific VOCs below‐ and above‐ground upon M. melolontha larvae feeding on apple roots as well as plant–plant communication in apple plants.

Isolation and identification of entomopathogenic nematodes (Nematoda: Rhabditida) from the Eastern Black Sea region and their biocontrol potential against Melolontha melolontha (Coleoptera: Scarabaeidae) larvae

A survey was conducted for entomopathogenic nematodes in various agricultural fields in the Eastern Black Sea region of Turkey. A total of 77 soil samples were collected from 15 distinct geographic areas during 2010-2011. Seven entomopathogenic nematode isolates (ZET02, ZET04, ZET09, ZET28, ZET31, ZET35, and ZET76) were detected from the soil samples (9.1% positive) using the Galleria baiting technique. Morphological and molecular characterizations of the isolates were performed for species identification. Five isolates were identified as Heterorhabditis bacteriophora (ZET02, ZET04, ZET09, ZET28, and ZET35) and 2 isolates were identified as Steinernema feltiae (ZET31 and ZET76). The efficacy of all isolates was tested on Melolontha melolontha larvae in plastic boxes and pot experiments. Different concentrations of nematodes at 0, 500, 1000, or 2000 infective juveniles (IJs)/mL and 2 different temperature regimes (15 and 25 °C) were used. One hundred percent mortality was obtained from the ZET09 and ZET35 isolates at a concentration of 2000 IJs/mL at 25 °C. The same isolates also provided 100% protection with 100 IJs/cm^2 in strawberry planted pot experiments.

Sensing the underground--ultrastructure and function of sensory organs in root-feeding Melolontha melolontha (Coleoptera: Scarabaeinae) larvae.

IntroductionBelow ground orientation in insects relies mainly on olfaction and taste. The economic impact of plant root feeding scarab beetle larvae gave rise to numerous phylogenetic and ecological studies. Detailed knowledge of the sensory capacities of these larvae is nevertheless lacking. Here, we present an atlas of the sensory organs on larval head appendages of Melolontha melolontha. Our ultrastructural and electrophysiological investigations allow annotation of functions to various sensory structures.ResultsThree out of 17 ascertained sensillum types have olfactory, and 7 gustatory function. These sensillum types are unevenly distributed between antennae and palps. The most prominent chemosensory organs are antennal pore plates that in total are innervated by approximately one thousand olfactory sensory neurons grouped into functional units of three-to-four. In contrast, only two olfactory sensory neurons innervate one sensillum basiconicum on each of the palps. Gustatory sensilla chaetica dominate the apices of all head appendages, while only the palps bear thermo-/hygroreceptors. Electrophysiological responses to CO2, an attractant for many root feeders, are exclusively observed in the antennae. Out of 54 relevant volatile compounds, various alcohols, acids, amines, esters, aldehydes, ketones and monoterpenes elicit responses in antennae and palps. All head appendages are characterized by distinct olfactory response profiles that are even enantiomer specific for some compounds.ConclusionsChemosensory capacities in M. melolontha larvae are as highly developed as in many adult insects. We interpret the functional sensory units underneath the antennal pore plates as cryptic sensilla placodea and suggest that these perceive a broad range of secondary plant metabolites together with CO2. Responses to olfactory stimulation of the labial and maxillary palps indicate that typical contact chemo-sensilla have a dual gustatory and olfactory function.

Carbon utilization pattern as a potential quality control criterion for virulence of Beauveria brongniartii

The registered entomopathogenic fungus Beauveria brongniartii (BIPESCO 2) was tested for its virulence after one, five and 10 times sub-culturing on four types of selective synthetic nutrient media. Bioassays with third instar Melolontha melolontha larvae showed that sub-culturing negatively affects the virulence of the fungus after 10 transfers. With the Biolog™ SF-P2 and Biolog™ SF-N2 microtiter plate systems the sub-cultivated B. brongniartii conidia were monitored for any change in the carbon utilization pattern of 128 carbon sources. With the help of Spearman’s rank correlation, principal components analysis and canonical correspondence analysis, respectively, six carbon sources were identified as potential virulence indicators for BIPESCO 2 (pyruvic acid, maltose, glycyl- l-glutamic acid, malonic acid, glucuronamide and phenylethylamine). The Biolog™ microtiter plate system is suggested as a simple and inexpensive test-system for virulence determination of B. brongniartii strain BIPESCO 2 in routine quality control.

Identification and pathogenicity of bacteria from European shot-hole borer,Xyleborus dispar Fabricius (Coleoptera: Scolytidae)

In this study, we have isolated and identified 5 bacterial isolates from European shot-hole borer,Xyleborus dispar (Coleoptera: Scolytidae), an important pest of hazelnut. After various morphological, physiological, biochemical and molecular characteristics were determined in detail, bacterial isolates were identified asPseudomonas fluorescens (Xd1),Bacillus megaterium (Xd2),Bacillus thuringiensis (Xd3),Pseudomonas rhizosphaerae (Xd4) andPantoea cedenensis (Xd5). Especially, theB. thuringiensis strain was identified in more detail asBacillus thuringiensis subsp.tenebrionis (H8ab). The insecticidal activities were determined against the larvae ofAgelastica alni L. (Col.: Chrysomelidae),Amphimallon solstitiale L. (Col.: Scarabaeidae),Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae), andMelolontha melolontha L. (Col.: Scarabaeidae) larvae at 1.8×109 CFU/ml dose, within five days. The highest insecticidal activities are 100, 100, 80 and 100% forBacillus thuringiensis subsp.tenebrionis (Xd3) onA. alni, A. solstitiale, L. decemlineata, andM. melolontha larvae, respectively. Our results indicate that especiallyB. thuringiensis subsp.tenebrionis (Xd3) may be valuable as a microbial control agent for coleopteran pests, respectively.

Identification and pathogenicity of entomopathogenic bacteria from common cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae)

Melolontha melolontha larvae are susceptible to several pathogens indigenous to the area in which these insects occur in Turkey. We isolated and identified seven bacterial strains from M. melolontha and evaluated their pathogenic activity during three hazelnut seasons from 2002 to 2004 on larvae of M. melolontha. Using various morphological, physiological, and biochemical characteristics in detail, bacterial isolates were identified as Pseudomonas sp., Bacillus thuringiensis, Pseudomonas sp., Enterobacter sp., B. sphaericus, Acinetobacter sp., and B. weihenstephanensis. The insecticidal activity of isolates at 1.8 × 109 bacteria/ml dose, within 10 days on the larvae of M. melolontha are 40% for Pseudomonas sp., 80% for Bacillus thuringiensis, 50% for Pseudomonas sp., 20% for Enterobacter sp., 60% for B. sphaericus, and 80% for B. weihenstephanensis. We also purified crystals from B. thuringiensis and B. sphaericus and tested the insecticidal activity on the larvae of M. melolontha. In crystal protein bioassays, the highest insecticidal effect detected was 70% with crystals of B. thuringiensis. Our results indicate that indigenousB. thuringiensis and B. weihenstephanensis isolates and crystal of B. thuringiensis may be valuable as biological control agents.

Persistence and efficacy of Beauveria brongniartii strains applied as biocontrol agents against Melolontha melolontha in the Valley of Aosta (northwest Italy)

To monitor and select genetically characterized strains of Beauveria brongniartii to be used as microbiological control agents against Melolontha melolontha in different climatic conditions of the Valley of Aosta (northwest Italy). Molecular random amplified polymorphic DNA markers allowed monitoring of five B. brongniartii strains (C2, F, K2, N3 and W2) in field trials. Ten sites were chosen at Jovençan, Saint-Pierre and Quart areas, where a mixture of the five strains colonizing rye kernels was applied to the soil of each M. melolontha infested site. Growth, persistence and virulence on M. melolontha larvae of five fungal strains were evaluated in two subsequent 24-month studies. Beauveria brongniartii grew best at the Jovençan sites. Not only did strain F persist better than the other strains in most soil samples but it was also the most virulent strain. Strain F was isolated the most frequently from infected M. melolontha larvae recovered from the test sites. A general decrease in the larvae rate was detected in the test field soil. Strain F of B. brongniartii was better than other strains in growth, persistence and virulence against M. melolontha larvae in the test site soil. Results obtained from preliminary field trials support the use of strain F as a biological control agent against M. melolontha in the Valley of Aosta even if further targeted studies are still necessary.

A new isolate of Melolontha melolontha entomopoxvirus in Turkey: Morphology, infectivity and prevalence in the field

A virus infection caused by Melolontha melolontha L. (Coleoptera: Scarabaeidae) entomopoxvirus (MmEPV) was recorded in 0.87% of 1,260 larvae collected in 2001-2002 in Trabzon, Turkey. The diseased larvae had dark brown or black and white spots, compared to the normal white-cream colour of healthy larvae. MmEPV infections reduced the larval life span from 6 mo to 15±5 d (Mean±S.D.). Based on light and electron microscopic observations, the average length of the spheroids was 5.45±2.34 μm (Mean±S.D.) and the average width was 2.89±1.13 μm (Mean±S.D.) (n=50). The presence of a virus was also demonstrated in a DNA-DNA hybridization assay. The bioassays carried out to determine the infectivity of MmEPV proved its effectiveness against the third-instar larvae of the M. melolontha. The highest insecticidal activity determined in the M. melolontha larvae was 93.3% at the concentration of 7.5×10 6 spheroids/ml. Our results suggest that a Turkish isolate of MmEPV is promising for biological control of the M. melolontha larvae.

Detecting predation and scavenging by DNA gut-content analysis: a case study using a soil insect predator-prey system

White grubs (larvae of Coleoptera: Scarabaeidae) are abundant in below-ground systems and can cause considerable damage to a wide variety of crops by feeding on roots. White grub populations may be controlled by natural enemies, but the predator guild of the European species is barely known. Trophic interactions within soil food webs are difficult to study with conventional methods. Therefore, a polymerase chain reaction (PCR)-based approach was developed to investigate, for the first time, a soil insect predator-prey system. Can, however, highly sensitive detection methods identify carrion prey in predators, as has been shown for fresh prey? Fresh Melolontha melolontha (L.) larvae and 1- to 9-day-old carcasses were presented to Poecilus versicolor Sturm larvae. Mitochondrial cytochrome oxidase subunit I fragments of the prey, 175, 327 and 387 bp long, were detectable in 50% of the predators 32 h after feeding. Detectability decreased to 18% when a 585 bp sequence was amplified. Meal size and digestion capacity of individual predators had no influence on prey detection. Although prey consumption was negatively correlated with cadaver age, carrion prey could be detected by PCR as efficiently as fresh prey irrespective of carrion age. This is the first proof that PCR-based techniques are highly efficient and sensitive, both in fresh and carrion prey detection. Thus, if active predation has to be distinguished from scavenging, then additional approaches are needed to interpret the picture of prey choice derived by highly sensitive detection methods.

Accurate determination of oosporein in fungal culture broth by differential pulse polarography.

A simple and accurate differential pulse polarographic method has been developed for the determination of oosporein in the culture broth of the fungus Beauveria brongniartii. This hydroxybenzoquinone derivative is the only major secondary metabolite secreted by this entomopathogenic fungus, which is used as biological pest control agent (BCA) against Melolontha melolontha larvae. It can be found in the host organism as well as in the formulated product. The polarographic behavior of oosporein was examined in various buffer systems over the pH range 3-10. In Britton-Robinson buffer/methanol solution (3:7 v/v, pH 5.5) the differential pulse polarograms exhibited reproducible peaks at E(p) = -0.18 V vs silver/silver chloride/potassium chloride (3 M). Under these conditions, a plot of peak height vs concentration of oosporein was found to be linear over the range 5.9 x 10(-)(7) to 2.5 x 10(-)(5) M (0.18-7.74 microg mL(-)(1); r = 0.9998). The detection limit was calculated to be 54 ng mL(-)(1). To evaluate the concentration of oosporein, the standard addition method was applied. The analysis of oosporein in the culture broth led to a mean value of 524.9 microg mL(-)(1) broth with a relative standard deviation (S(rel)) of +/-2.6%. The proposed polarographic method is accurate, not time-consuming, and it is of low cost because no separation steps are necessary.