A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

New Cyclic Pentapeptides from the Mangrove-Derived Aspergillus fumigatus GXIMD 03099.

Four new cyclic pentapeptides, avellanins D-G (1-4), together with four known compounds (5-8), were isolated from a mangrove-derived Aspergillus fumigatus GXIMD 03099 fungus from Acanthus ilicifolius L. Their structures were elucidated by analysis of HRESIMS, NMR, and ESI-MS/MS data. Their absolute configurations were determined by X-ray diffraction analysis and Marfey's method. Compounds 1-8 were screened for insecticidal and antibacterial activities. Compound 2 showed insecticidal activity against newly hatched larvae of Culex quinquefasciatus with an LC50 value of 86.6 µM; compound 4 had weak activity against Vibrio harveyi with an MIC value of 5.85 µM.

Etched stainless steel wire modified with conjugated microporous polymers-F6 for jacket-free stir bar sorptive extraction of benzoylureas in juice sample.

Benzoylurea (BU) insecticides have been widely used for pest control as third-generation insecticides. Considering that their residues in food may cause adverse effects on human health, the upper limits of BUs remaining in food have been set by the administration. Therefore, it is essential to develop a sensitive and efficient analytical method to determine the residues of BUs in food. Stir bar sorptive extraction (SBSE) is a novel sample preparation technique, and stainless steel wire (SSW) is an ideal substrate for an SBSE device. In this work, a novel SBSE device of SSW jacket-free stir bar with a dumbbell shape was designed and prepared. The conjugated microporous polymer CMP-F6, which possesses a porous structure, high hydrophobicity and rich fluorine-containing functional groups, was immobilized on the surface of SSW by the method of polyacrylonitrile glue adhesion. Compared with previous studies, which used SSW as a substrate, the method of etching partial SSW with hydrochloric acid, on the one hand, made the surface of SSW rough and easy to modify the extraction coating, and on the other hand, converted itself into a dumbbell-shaped structure, which is conducive to improving the extraction efficiency and stability of the SBSE device. The method of SBSE-HPLC-UV was established for determining five BUs. Owing to the hydrophobic interaction and F-F interaction between CMP-F6 and analytes, this method showed good extraction efficiency and had good linearity (R2 ≥ 0.9945) and high sensitivity (LODs in the range of 0.1-0.2 ng mL-1). It was used for the analysis of benzoylurea in an apple juice sample, and the recoveries were 74.3-117.9%.

Dihydroxyl modified UiO-66 as dispersive solid-phase extraction sorbent coupled with ultra-high performance liquid chromatography tandem mass spectrometry for detection of neonicotinoid insecticides.

The extensive usage of neonicotinoid insecticides (NIs) has raised many concerns about their potential harm to environment and human health. Thus, it is of great importance to develop an efficient and reliable method to determine NIs in food samples. In this work, three Zr4+-based metal-organic frameworks functionalized with various numbers of hydroxyl groups were fabricated with a facile one-pot solvothermal method. Among them, dihydroxy modified UiO-66 (UiO-66-(OH)2) exhibited best adsorption performance towards five target NIs. Then, a sensitive and efficient method for detection of NIs from vegetable and fruit samples was established based on dispersive solid phase extraction (dSPE) with UiO-66-(OH)2 as adsorbent coupled with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Key parameters affecting the dSPE procedure including amounts of adsorbent, adsorption time, eluent solvents and desorption time were investigated. Under the optimal conditions, rapid adsorption of NIs within five minutes was achieved due to the high affinity of UiO-66-(OH)2 towards NIs. The developed method exhibited high sensitivity with limits of detection (LODs) varied from 0.003 to 0.03 ng/mL and wide linearity range over 3-4 orders of magnitude from 0.01 to 500 ng/mL. Furthermore, the established method was applied for determining trace NIs from complex matrices with recoveries ranging from 74.6 to 99.6 % and 77.0-106.8 % for pear and tomato samples, respectively. The results indicate the potential of UiO-66-(OH)2 for efficient enrichment of trace NIs from complex matrices.

Insecticidal Activities and Phenological Variations of Characteristic Component from Michelia yunnanensis.

Volatile secondary metabolites of plants interact with environments heavily. In this work, characteristic components of Michelia yunnanensis essential oils (EOs) were isolated, purified and identified by column chromatography, GC-MS and NMR. Leaves of M. yunnanensis were collected monthly and extracted for EOs to investigate chemical and insecticidal activity variations as well as potential influencing environments. Different organs were employed to reveal distribution strategies of characteristic components. Results of insecticidal activities showed that all EOs samples exerted stronger contact activity to Lasioderma serricorne, but repellent effect was more efficient on Tribolium castaneum. One oxygenated sesquiterpene was isolated from EOs, basically it could be confirmed as (+)-cyclocolorenone (1). It exerted contact toxicity to L. serricorne (LD 50 = 28.8 µg/adult). Chemical analysis showed that M. yunnanensis leaves in reproductive period would produce and accumulate more 1 than in vegetative period. Moreover, reproductive organs (flowers and fruits) contained more 1 than vegetative organs (leaves and twigs). Partial correlation analysis indicated that temperature-related elements positively correlated with the relative content of 1.

Alien species water hyacinth realizes waste into treasure: The preparation of biomass sorbent to determine benzoylurea insecticides in tea products.

A fast and effective analytical method with biomass solid-phase microextraction sorbent combined with a high-performance liquid chromatography-ultraviolet detector was proposed for the determination of benzoylurea (BU) insecticides in tea products. The novel sorbent was prepared by activating and then carbonizing water hyacinth with a fast growth rate and low application value as raw material and showed a high specific surface area and multiple interactions with analytes, such as electrostatic action, hydrogen bonding, and π-π conjugation. After optimizing the three most important extraction parameters (pH [X1], sample loading rate [X2], and solution volume [X3]) by Box-Behnken design, the as-established analytical method showed good extraction performance: excellent recovery (80.13%-106.66%) and wide linear range (1-400 µg/L) with a determination coefficient of 0.9992-0.9999, a low limit of detection of 0.02-0.1 µg/L and the satisfactory practical application results in tea products. All these indicate that the water hyacinth-derived material has the potential as a solid-phase extraction sorbent for the detection and removal of BU insecticides from tea products, and at the same time, it can also achieve the effect of rational use of biological resources, maintaining ecological balance, turning waste into treasure, and achieving industrial production.

Determination of insecticide residues in beverages based on MIL-100(Fe) dispersive solid-phase microextraction in combination with dispersive liquid-liquid microextraction followed by HPLC-MS/MS.

A novel dispersive solid-phase microextraction method based on a metal-organic framework (MIL-100(Fe)) combined with a dispersive liquid-liquid microextraction technique was proposed for the extraction and enrichment of four insecticides in beverages. The qualitative and quantitative analysis of these insecticides was conducted using HPLC-MS/MS. To optimize the extraction process, several parameters were investigated, and the main variables were optimized using CCD-based RSM. The developed method displayed a wide linear range of 1.000-1000 ng/L and R2 values >0.993 for all four calibration curves. The method demonstrated high sensitivity, with LODs and LOQs of 0.3-0.6 ng/L and 0.8-1.0 ng/L, respectively. In addition, the greenness of the proposed method was assessed using the Complex GAPI tool, and the results showed that the proposed method exhibits benefits, such as minimal usage of organic solvents and negligible matrix influence, making it a suitable method for the detection of insecticide residues in beverages.

Gas Chromatography-Mass Spectrometry Chemical Profiling of Commiphora myrrha Resin Extracts and Evaluation of Larvicidal, Antioxidant, and Cytotoxic Activities.

Plant extracts and essential oils can be alternative environmentally friendly agents to combat pathogenic microbes and malaria vectors. Myrrh is an aromatic oligum resin that is extracted from the stem of Commiphora spp. It is used in medicine as an insecticide, cytotoxic, and aromatic. The current study assessed the effect of Commiphora myrrha resin extracts on the biological potency of the third larval stage of Aedes aegypti, as well as its antioxidant and cytotoxic properties against two types of tumor cells (HepG-2 and Hela cell lines). It also used GC-MS to determine the chemical composition of the C. myrrha resin extracts. Fifty components from the extracted plant were tentatively identified using the GC-MS method, with curzerene (33.57%) typically listed as the primary ingredient, but other compounds also make up a significant portion of the mixture, including 1-Methoxy-3,4,5,7-tetramethylnaphthalene (15.50%), ß-Elemene (5.80%), 2-Methoxyfuranodiene (5.42%), 2-Isopropyl-4,7-Dimethyl-1-Naphthol (4.71%), and germacrene B (4.35%). The resin extracts obtained from C. myrrha exhibited significant efficacy in DPPH antioxidant activity, as evidenced by an IC50 value of 26.86 mg/L and a radical scavenging activity percentage of 75.06%. The 50% methanol extract derived from C. myrrha resins exhibited heightened potential for anticancer activity. It demonstrated substantial cytotoxicity against HepG-2 and Hela cells, with IC50 values of 39.73 and 29.41 µg mL-1, respectively. Notably, the extract showed non-cytotoxic activity against WI-38 normal cells, with an IC50 value exceeding 100 µg mL-1. Moreover, the selectivity index for HepG-2 cancer cells (2.52) was lower compared to Hela cancer cells (3.40). Additionally, MeOH resin extracts were more efficient against the different growth stages of the mosquito A. aegypti, with lower LC50, LC90, and LC95 values of 251.83, 923.76, and 1293.35 mg/L, respectively. In comparison to untreated groups (1454 eggs/10 females), the average daily number of eggs deposited (424 eggs/L) decreases at higher doses (1000 mg/L). Finally, we advise continued study into the possible use of C. myrrha resins against additional pests that have medical and veterinary value, and novel chemicals from this extract should be isolated and purified for use in medicines.

Insecticidal bisindole alkaloids from leaves of Tabernaemontana divaricata 'Dwaft'.

Six undescribed bisindole alkaloids, namely taberdisines A-F (1-6), were isolated from the leaves of Tabernaemontana divaricata 'Dwaft'. Among them, alkaloids 1 and 2 were the first examples of strychnos-iboga type alkaloid with both C-C linkage patterns. Alkaloid 3, a new type of aspidosperma-iboga with a furan-ring, as well as other three undescribed ones was disclosed. Their structures were elucidated by comprehensive spectroscopic analyses. Alkaloids 1 and 5 showed insecticide activity on Sf9 cell and eggs of Spodoptera frugiperda in vivo, which might explain the potential of the plants for insect resistance.

Limonoids from the fruits of Chisocheton erythrocarpus and their mosquito larvicidal activities.

Phytochemical study of the fruits of Chisocheton erythrocarpus (Hiern) allowed the identification of eight undescribed limonoids, namely erythrocarpines O - V (1-6, 7a and 7b), along with seven known compounds. The structures of these compounds were elucidated based on spectroscopic and HRMS data, along with electronic circular dichroism to configure the absolute configuration. Erythrocarpines O and P are γ-hydroxybutenolide analogs of mexicanolide-type limonoids while erythrocarpine Q - V are phragmalin-type limonoids possessing a 1,29-oxymethylene bridge with either benzoyl or cinnamoyl moiety in their structures. Mosquito larvicidal activity revealed that crude DCM extract of C. erythrocarpus possessed a good larvicidal effect against Aedes aegypti larvae in 48 h (LC50 = 153.0 ppm). Subsequent larvicidal activity of isolated compounds indicated that erythrocarpine G (10) and 14-deoxyxyloccensin K (11) were responsible for the enhanced larvicidal effect of the extract, reporting LC50 values of 18.55 ppm and 41.16 ppm, respectively. Moreover, residual activity testing of the crude DCM extract revealed that the duration of its larvicidal effects is up to 14 days, where it maintained a 98 % larval mortality throughout the test period, under laboratory conditions.

Biological activity of essential oils from Ferulago angulata and Ferula assa-foetida against food-related microorganisms (antimicrobial) and Ephestia kuehniella as a storage pest (insecticidal); an in vitro and in silico study.

Misuse of synthetic pesticides and antimicrobials in agriculture and the food industry has resulted in food contamination, promoting resistant pests and pathogen strains and hazards for humanity and the environment. Therefore, ever-increasing concern about synthetic chemicals has stimulated interest in eco-friendly compounds. Ferulago angulata (Schltdl.) Boiss. and Ferula assa-foetida L., as medicinal species with restricted natural distribution and unknown biological potential, aimed at investigation of their essential oil (EO) biological properties, were subjected. Z-ß-Ocimene and Z-1-Propenyl-sec-butyl disulfide molecules were identified as the major composition of the essential oil of the fruits of F. angulata and F. assa-foetida, respectively. In vitro antimicrobial activity and membrane destruction investigation by scanning electron microscopy imaging illustrated that F. angulata EO had potent antibacterial activity. Besides, the EOs of both plants exhibited significant anti-yeast activity against Candida albicans. In relation to insecticidal activity, both EOs indicated appropriate potential against Ephestia kuehniella; however, the F. assa-foetida EO had more toxicity on the studied pest. Among several insecticidal-related targets, acetylcholinesterase was identified as the main target of EO based on the molecular docking approach. Hence, in line with in vitro results, in silico evaluation determined that F. assa-foetida has a higher potential for inhibiting acetylcholinesterase and, consequently, better insecticide properties. Overall, in addition to the antioxidant properties of both EO, F. angulata EO could serve as an effective prevention against microbial spoilage and foodborne pathogens, and F. assa-foetida EO holds promise as a multi-purpose and natural biocide for yeast contamination and pest management particularly against E. kuehniella.

Chemical Composition, Repellent, and Phytotoxic Potentials of the Fractionated Resin Essential Oil from Araucaria heterophylla Growing in Tunisia.

The resin essential oil (REO) of the Tunisian Araucaria heterophylla trunk bark was investigated for its chemical composition. Then, it was evaluated for its insecticidal and allelopathic activities. The REO was obtained by hydrodistillation for 9 h (yield of 4.2 % w/w). Moreover, fractional hydrodistillation was carried out at 3-hour intervals, resulting in 3 fractions (R1-R3), to facilitate chemical identification and localization of the aforementioned biological activities. GC/MS analysis of the obtained samples allowed the identification of 25 compounds, representing between 91.2 and 96.3 % of their total constituents, which consisted predominantly of sesquiterpene hydrocarbons, oxygenated sesquiterpenes and diterpene hydrocarbons. α-Copaene (10.8 %), γ-muurolene (5.8 %), α-copaen-11-ol (7.8 %), spathulenol (10.5 %), 15-copaenol (8.2 %), ylangenal (10.3 %), dehydrosaussurea lactone (7.7 %), and sandaracopimaradiene (11.4 %) were identified as major compounds. The second part aimed to assess the impact of the A. heterophylla EO and its three fractions for their insecticidal and repellent activity against Tribolium castaneum (Herbst), a stored grain pest, of which a strong repellent activity was noted. In addition, the studied samples showed high phytotoxic effects against Lactuca sativa. The third fraction (R3) performed a total inhibitory potential on seed germination and seedling growth of the target plant. Furthermore, alongside this discovery, an estimation was conducted through molecular docking analysis. Wherein the main compounds of the studied samples were docked into the active pocket of protoporphyrinogen IX oxidase (PDB: 1SEZ), a key enzyme in chlorophyll biosynthesis. Thus, it is recommended to use the REO of A. heterophylla as a natural herbicide.

Insights into the Metabolomic Capacity of Podaxis and Isolation of Podaxisterols A-D, Ergosterol Derivatives Carrying Nitrosyl Cyanide-Derived Modifications.

Cultures of a termite-associated and a free-living member of the fungal genus Podaxis, revived from spores maintained in century-old herbarium collections, were analyzed for their insecticidal and antimicrobial effects. Their secondary metabolomes were explored to uncover possible adaptive mechanisms of termite association, and dereplication of LC-HRMS/MS data sets led to the isolation of podaxisterols A-D (1-4), modified ergosterol derivatives that result from a Diels-Alder reaction with endogenous nitrosyl cyanide. Chemical structures were determined based on HRMS/MS and NMR analyses as well as X-ray crystallography. The putative origin of the endogenous fungal nitrosyl cyanide and ergosterol derivatives is discussed based on results obtained from stable isotope experiments and in silico analysis. Our "omics"-driven analysis of this underexplored yet worldwide distributed fungal genus builds a foundation for studies on a potential metabolic adaptations to diverse lifestyles.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Design, Semisynthesis, Insecticidal and Antibacterial Activities of a Series of Marine-Derived Geodin Derivatives and Their Preliminary Structure-Activity Relationships.

To enhance the biological activity of the natural product geodin (1), isolated from the marine-derived fungus Aspergillus sp., a series of new ether derivatives (2-37) was designed and semisynthesized using a high-yielding one-step reaction. In addition, the insecticidal and antibacterial activities of all geodin congeners were evaluated systematically. Most of these derivatives showed better insecticidal activities against Helicoverpa armigera Hübner than 1. In particular, 15 showed potent insecticidal activity with an IC50 value of 89 µM, comparable to the positive control azadirachtin (IC50 = 70 µM). Additionally, 5, 12, 13, 16, 30 and 33 showed strong antibacterial activity against Staphylococcus aureus and Aeromonas salmonicida with MIC values in the range of 1.15-4.93 µM. The preliminary structure-activity relationships indicated that the introduction of halogenated benzyl especially fluorobenzyl, into 1 and substitution of 4-OH could be key factors in increasing the insecticidal and antibacterial activities of geodin.

Secondary Metabolites from Marine Sponges of the Genus Oceanapia: Chemistry and Biological Activities.

In this review, we summarized the distribution of the chemically investigated Oceanapia sponges, including the isolation and biological activities of their secondary metabolites, covering the literature from the first report in 1989 to July 2019. There have been 110 compounds reported during this period, including 59 alkaloids, 33 lipids, 14 sterols and 4 miscellaneous compounds. Besides their unique structures, they exhibited promising bioactivities ranging from insecticidal to antibacterial. Their complex structural characteristics and diverse biological properties have attracted a great deal of attention from chemists and pharmaceuticals seeking to perform their applications in the treatment of disease.

Evaluation of the Potential of a Lectin Extracted from Polygonumpersicaria L. as a Biorational Agent against Sitophilusoryzae L.

Rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), is one of the most destructive stored-product pests that is resistant to a wide range of chemical insecticides. In the present study, we investigated whether a lectin extracted from Polygonum persicaria L. (PPA) can be used as a biorational agent to control such insect pests. Along with the lethal digestive assay, the sub-lethal insecticidal activities of PPA, including the effects on digestive, detoxifying, and antioxidant enzyme activities, were evaluated against S. oryzae adults. The effect of feeding a diet containing PPA and carob extract as a food attractant on the mortality of S. oryzae adults was also investigated. Feeding on the diet containing PPA resulted in a significant mortality of S. oryzae adults with a LC50 (Lethal Concentration to kill 50% of insects) of 3.68% (w/w). The activity of digestive enzymes, including α-amylase, α-glucosidase, TAG-lipase, trypsin, chymotrypsin, elastase, and carboxy- and aminopeptidase, were decreased by the sub-lethal concentration of PPA. Detoxifying and antioxidant enzymes, including esterase, superoxide dismutase, catalase, glutathione-S-transferase, ascorbate peroxidase, glucose 6-phosphate dehydrogenase, and malondialdehyde, were activated in adults affected by PPA. These findings indicated that PPA, in addition to causing digestive disorders, leads to oxidative stress in S. oryzae. The presence of carob extract had no effect on the PPA-induced mortality of the insect. According to the results of the present study, PPA has promising insecticidal efficiency against S. oryzae. In addition, the usage of PPA with a food attractant carob extract in bait traps can be recommended as a new biorational formulation in S. oryzae management.

Bioguided Phytochemical Study of Ipomoea cairica Extracts with Larvicidal Activity against Aedes aegypti.

Vector-borne diseases, such as those transmitted by Aedes aegypti, are a constant threat to inhabitants of tropical regions of the planet. Synthetic chemicals are commonly used as a strategy to control them; however, these products are known to persist in ecosystems and drive the appearance of resistance genes in arthropod vectors. Thus, the use of natural products has emerged as an environmentally friendly alternative in integrated vector control strategies. The present bioguided study investigated the larvicidal potential of Ipomoea cairica extracts, fractionated using thin-layer and open-column chromatography, because this species has been shown to exert larvicidal effects on the genus Aedes. The objective of this study was to evaluate the nonvolatile components in ethanolic extract of I. cairica stems as a potential natural larvicidal, and coumarins, such as 7-hydroxy-6-methoxychromen-2-one (scopoletin) and 7-hydroxychromen-2-one (umbelliferone), were identified as major compounds; however, they were not shown to be responsible for the larvicidal activity. Based on the results of the larvicidal action tests, these coumarins are not directly responsible for the larvicidal activity, but this activity might be attributed to a synergistic effect of all the compounds present in the most active secondary fraction, called F.DCM, which had an LC50 value of 30.608 mg/L. This type of study has yet not been conducted in the region; therefore, it is an important contribution to recognizing a natural and easy-to-cultivate source of vector control, such I. cairica.

Isolation and biological activity of azocine and azocane alkaloids.

Thousands of known alkaloids contain a nitrogen (N) heterocycle. While five-, six- and seven-membered N-heterocycles (ie: pyrroles, imidazoles, indoles, pyridines and azepines and their saturated variants) are common, those with an eight-membered N-heterocycle are comparatively rare. This review discusses the structure and bioactivity of alkaloids that contain an azocine (or saturated azocane) ring, and the array of sources whence they originate.

Discovery of mosquitocides from fungal extracts through a high-throughput cytotoxicity-screening approach.

BACKGROUND: Mosquitoes transmit a variety of diseases. Due to widespread insecticide resistance, new effective pesticides are urgently needed. Entomopathogenic fungi are widely utilized to control pest insects in agriculture. We hypothesized that certain fungal metabolites may be effective insecticides against mosquitoes. METHODS: A high-throughput cytotoxicity-based screening approach was developed to search for insecticidal compounds in our newly established global fungal extract library. We first determined cell survival rates after adding various fungal extracts. Candidate insecticides were further analyzed using traditional larval and adult survival bioassays. RESULTS: Twelve ethyl acetate extracts from a total of 192 fungal extracts displayed > 85% inhibition of cabbage looper ovary cell proliferation. Ten of these 12 candidates were confirmed to be toxic to Anopheles gambiae Sua5B cell line, and six showed > 85% inhibition of Anopheles mosquito cell growth. Further bioassays determined a LC50, the lethal concentration that kills 50% of larval or adult mosquitoes, of 122 µg/mL and 1.7 µg/mosquito, respectively, after 24 h for extract 76F6 from Penicillium toxicarium. CONCLUSIONS: We established a high-throughput MTT-based cytotoxicity screening approach for the discovery of new mosquitocides from fungal extracts. We discovered a candidate extract from P. toxicarium that exhibited high toxicity to mosquito larvae and adults, and thus were able to demonstrate the value of our recently developed approach. The active fungal extracts discovered here are ideal candidates for further development as mosquitocides.