Optimizing liquid fermentation for Wolfiporia cocos: gene expression and biosynthesis of pachymic acid and mycelial biomass.

Wolfiporia cocos, a versatile fungus acclaimed for its nutritional and therapeutic benefits in Traditional Chinese Medicine, holds immense potential for pharmaceutical and industrial applications. In this study, we aimed to optimize liquid fermentation techniques and culture medium composition to maximize mycelial biomass (MB) yield, pachymic acid (PA) concentration, and overall PA production. Additionally, we investigated the molecular basis of our findings by quantifying the expression levels of genes associated with PA and MB biosynthesis using quantitative real-time polymerase chain reaction. Under the optimized fermentation conditions, significant results were achieved, with maximum MB reaching 6.68 g l-1, PA content peaking at 1.25 mg g-1, and a total PA yield of 4.76 g l-1. Notably, among the four examined genes, squalene monooxygenase, exhibited enhanced expression at 0.06 ratio under the optimized conditions. Furthermore, within the realm of carbohydrate-active enzymes, the glycoside hydrolases 16 family displayed elevated expression levels at 21 ratios, particularly during MB production. This study enhances understanding of genetic mechanism governing MB and PA production in W. cocos, highlighting the roles of squalene monooxygenase and glycoside hydrolases 16 carbohydrate-active enzymes.

Genome sequencing of Colletotrichum gloeosporioides ES026 reveals plausible pathway of HupA.

BACKGROUND: Colletotrichum gloeosporioides ES026, isolated as an endophytic fungal strain, was found to produce the important medicinal compound HuperzineA (HupA). In a genetic context, ES026 showed potential in elucidating the biosynthetic pathway of HupA. METHODS AND RESULTS: The ES026 strain was sequenced using de-novo Illumina sequencing methods in this study. Assembling the cleaned data resulted in 58,594,804bp, consisting of 404 scaffolds. The G + C mol % content of this genome was 52.53%. The genome progressive-alignment with other 4 Colletotrichum strains revealed that ES026 showed closer relation with 030206, SMCG1#C and Nara gc5. More than 60 putative biosynthetic clusters were predicted with the fungal version antiSMASH4.0 program. More than 33 types I polyketide-related biosynthetic gene clusters were distributed, containing PKS and PKS-NRPS (polyketide-nonribosomal peptides) hybrid gene clusters. Another 8 NRPS biosynthetic gene clusters were distributed among the genome of ES026. The prenyltransferases, probably involved in aromatic prenyl-compounds and terpenoid biosynthesis, were analyzed using bioinformatics tools like MEGA. CONCLUSION: We predicted a new possible biosynthetic pathway for the HupA from the pipecolic acid, based on the published HupA biosynthesis proposed pathway, the biosynthesis and pipecolic acid-derived compounds. We hypothesize that a hybrid PKS-NRPS mega-enzyme was probably involved in the biosynthesis of HupA with the pipecolic acid, the building block of rapamycin, as a HupA precursor. The rapamycin is produced from a polyketide biosynthesis pathway, and the domain incorporating the pipecolic acid is studied.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

The Current Developments in Medicinal Plant Genomics Enabled the Diversification of Secondary Metabolites' Biosynthesis.

Medicinal plants produce important substrates for their adaptation and defenses against environmental factors and, at the same time, are used for traditional medicine and industrial additives. Plants have relatively little in the way of secondary metabolites via biosynthesis. Recently, the whole-genome sequencing of medicinal plants and the identification of secondary metabolite production were revolutionized by the rapid development and cheap cost of sequencing technology. Advances in functional genomics, such as transcriptomics, proteomics, and metabolomics, pave the way for discoveries in secondary metabolites and related key genes. The multi-omics approaches can offer tremendous insight into the variety, distribution, and development of biosynthetic gene clusters (BGCs). Although many reviews have reported on the plant and medicinal plant genome, chemistry, and pharmacology, there is no review giving a comprehensive report about the medicinal plant genome and multi-omics approaches to study the biosynthesis pathway of secondary metabolites. Here, we introduce the medicinal plant genome and the application of multi-omics tools for identifying genes related to the biosynthesis pathway of secondary metabolites. Moreover, we explore comparative genomics and polyploidy for gene family analysis in medicinal plants. This study promotes medicinal plant genomics, which contributes to the biosynthesis and screening of plant substrates and plant-based drugs and prompts the research efficiency of traditional medicine.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

Network Pharmacology and Molecular Docking Combined to Analyze the Molecular and Pharmacological Mechanism of Pinellia ternata in the Treatment of Hypertension.

Hypertension is a cardiovascular disease that causes great harm to health and life, affecting the function of important organs and accompanied by a variety of secondary diseases, which need to be treated with drugs for a long time. P. ternata alone or combination with western medicine has played an important role in traditional Chinese medicine. Although P. ternata is used clinically to treat hypertension, its functional molecular mechanism and pharmacological mechanism have not been elucidated. Therefore, in this study, the potentially effective components, and targets of P. ternata in the treatment of hypertension were screened by the method of network pharmacology, and the mechanism of P. ternata in the treatment of hypertension was analyzed by constructing a component-target relationship network, PPI interaction network, targets' function analysis, and molecular docking. In the study, 12 potentially effective components and 88 targets were screened, and 3 potential protein modules were found and analyzed after constructing a PPI network using targets. In addition, 10 targets were selected as core targets of the PPI network. After that, the targets were analyzed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the molecular docking method is used to study the interaction between the targets and the active components. The above evidence shows that the mechanism of P. ternata in the treatment of hypertension is complicated, as it acts in many ways, mainly by affecting nerve signal transmission, cell proliferation, and apoptosis, calcium channels, and so on. The binding between targets and active components mainly depends on Pi bonds and hydrogen bonds. Using the method of network pharmacology and molecular docking to analyze the mechanism of P. ternata in the treatment of hypertension will help to provide a better scientific basis for the combined use of traditional Chinese medicine and western medicine, and will better help to improve the quality of P. ternata and point out its direction.

Full-length transcriptome sequencing and modular organization analysis of oleanolic acid- and dammarane-type saponins related gene expression patterns in Panax japonicus.

The saponins found in Panax japonicus, a traditional medicinal herb in Asia, exhibit high degrees of structural and functional similarity. In this study, metabolite analysis revealed that oleanolic acid-type and dammarane-type saponins were distributed unevenly in three tissues (rhizome_Y, rhizome_O, and secRoot) of P. japonicus. Single-molecule real-time (SMRT) sequencing and next generation sequencing (NGS) data revealed distinct and tissue-specific transcriptomic patterns relating to the production of these two types of saponins. In the co-expression network and hierarchical clustering analyses, one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and two 1-deoxy-D-xylulose-5-phosphate synthase (DXS) etc. transcripts were found to be key genes associated with the biosynthesis of oleanolic acid and dammarane-type saponins in P. japonicus, respectively. In addition, cytochrome p450 (CYP) and UDP-glucuronosyltransferase (UGT) family proteins that serve as regulators of saponin biosynthesis-related genes were also found to exhibit tissue-specific expression patterns. Together these results offer a comprehensive metabolomic and transcriptomic overview of P. japonicus.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

Endophytic Fungal Community of Huperzia serrata: Diversity and Relevance to the Production of Huperzine A by the Plant Host.

As the population ages globally, there seem to be more people with Alzheimer's disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer's disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.

Identification of a novel endophytic fungus from Huperzia serrata which produces huperzine A.

Huperzine A is isolated from Huperzia serrata and is used for treatment of Alzheimer's disease, due to its low toxicity and long effective period. The decrease in H. serrata sources means that natural huperzine A cannot meet the needs of clinical therapy. In this study, >200 endophytic fungal strains were isolated from H. serrata, and screened using high-performance liquid chromatography. Strain ES026 produced huperzine A. Production was identified and quantified by liquid chromatography-tandem mass spectrometry, and the yield of huperzine A was 1 µg/g dried mycelium. ES026 strain was identified as Colletotrichum gloeosporioides by morphology, polymerase chain reaction with species-specific primers and rDNA internal transcribed spacer sequence. ES026 contributes to the breeding of cultivated strains with high yield of huperzine A. Meanwhile, the strain was suitable for the study of biosynthesis of huperzine A.

Chromosome-scale genome assembly of medicinal plant Tinospora sagittata (Oliv.) Gagnep. from the Menispermaceae family.

Tinospora sagittata (Oliv.) Gagnep. is an important medicinal tetraploid plant in the Menispermaceae family. Its tuber, Radix Tinosporae, used in traditional Chinese medicine, is rich in diterpenoids and benzylisoquinoline alkaloids (BIAs). To enhance our understanding of medicinal compounds' biosynthesis and Menispermaceae's evolution, we herein report assembling a high-quality chromosome-scale genome with both PacBio HiFi and Illumina sequencing technologies. PacBio Sequel II generated 2.5 million circular consensus sequencing (CCS) reads, and a hybrid assembly strategy with Illumina sequencing resulted in 4483 contigs. The assembled genome size was 2.33 Gb, consisting of 4070 scaffolds (N50 = 42.06 Mb), of which 92.05% were assigned to 26 pseudochromosomes. T. sagittata's chromosomal-scale genome assembly, the first species in Menispermaceae, aids Menispermaceae evolution and T. sagittata's secondary metabolites biosynthesis understanding.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

A telomere-to-telomere reference genome provides genetic insight into the pentacyclic triterpenoid biosynthesis in Chaenomeles speciosa.

Chaenomeles speciosa (2n = 34), a medicinal and edible plant in the Rosaceae, is commonly used in traditional Chinese medicine. To date, the lack of genomic sequence and genetic studies has impeded efforts to improve its medicinal value. Herein, we report the use of an integrative approach involving PacBio HiFi (third-generation) sequencing and Hi-C scaffolding to assemble a high-quality telomere-to-telomere genome of C. speciosa. The genome comprised 650.4 Mb with a contig N50 of 35.5 Mb. Of these, 632.3 Mb were anchored to 17 pseudo-chromosomes, in which 12, 4, and 1 pseudo-chromosomes were represented by a single contig, two contigs, and four contigs, respectively. Eleven pseudo-chromosomes had telomere repeats at both ends, and four had telomere repeats at a single end. Repetitive sequences accounted for 49.5% of the genome, while a total of 45 515 protein-coding genes have been annotated. The genome size of C. speciosa was relatively similar to that of Malus domestica. Expanded or contracted gene families were identified and investigated for their association with different plant metabolisms or biological processes. In particular, functional annotation characterized gene families that were associated with the biosynthetic pathway of oleanolic and ursolic acids, two abundant pentacyclic triterpenoids in the fruits of C. speciosa. Taken together, this telomere-to-telomere and chromosome-level genome of C. speciosa not only provides a valuable resource to enhance understanding of the biosynthesis of medicinal compounds in tissues, but also promotes understanding of the evolution of the Rosaceae.

Biosynthetic pathways of triterpenoids and strategies to improve their Biosynthetic Efficiency.

"Triterpenoids" can be considered natural products derived from the cyclization of squalene, yielding 3-deoxytriterpenes (hydrocarbons) or 3-hydroxytriterpenes. Triterpenoids are metabolites of these two classes of triterpenes, produced by the functionalization of their carbon skeleton. They can be categorized into different groups based on their structural formula/design. Triterpenoids are an important group of compounds that are widely used in the fields of pharmacology, food, and industrial biotechnology. However, inadequate synthetic methods and insufficient knowledge of the biosynthesis of triterpenoids, such as their structure, enzymatic activity, and the methods used to produce pure and active triterpenoids, are key problems that limit the production of these active metabolites. Here, we summarize the derivatives, pharmaceutical properties, and biosynthetic pathways of triterpenoids and review the enzymes involved in their biosynthetic pathway. Furthermore, we concluded the screening methods, identified the genes involved in the pathways, and highlighted the appropriate strategies used to enhance their biosynthetic production to facilitate the commercial process of triterpenoids through the synthetic biology method.

Rhizosphere microbial diversity in rhizosphere of Pinellia ternata intercropped with maize.

Dry tubers of Pinellia ternata (Thunb.) Breit are used in traditional Chinese medicine. Commonly known as "banxia" in China, the tubers contain valuable compounds, including alkaloids and polysaccharides that are widely used in pharmaceuticals. The quantity and quality of these important compounds are affected by whether P. ternata is grown as a sole crop or as an intercrop, and P. ternata cultivation has become challenging in recent years. By intercropping P. ternata, its maximum yield, as well as large numbers of chemical components, can be realized. Here, a large data set derived from next-generation sequencing was used to compare changes in the bacterial communities in rhizosphere soils of P. ternata and maize grown as sole crops and as intercrops. The overall microbial population in the rhizosphere of intercropped P. ternata was significantly larger than that of sole-cropped P. ternata, whereas the numbers of distinct microbial genera, ranging from 552 to 559 among treatments, were not significantly different between the two rhizospheres. The relative abundances of the genera differed. Specifically, the numbers of Acidobacteria and Anaerolineaceae species were significantly greater, and those of Bacillus were significantly lower, in the intercropped P. ternata rhizosphere than in the sole-cropped rhizosphere. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03011-3.

Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids.

Chinese goldthread (Coptis chinensis Franch.), a member of the Ranunculales, represents an important early-diverging eudicot lineage with diverse medicinal applications. Here, we present a high-quality chromosome-scale genome assembly and annotation of C. chinensis. Phylogenetic and comparative genomic analyses reveal the phylogenetic placement of this species and identify a single round of ancient whole-genome duplication (WGD) shared by the Ranunculaceae. We characterize genes involved in the biosynthesis of protoberberine-type alkaloids in C. chinensis. In particular, local genomic tandem duplications contribute to member amplification of a Ranunculales clade-specific gene family of the cytochrome P450 (CYP) 719. The functional versatility of a key CYP719 gene that encodes the (S)-canadine synthase enzyme involved in the berberine biosynthesis pathway may play critical roles in the diversification of other berberine-related alkaloids in C. chinensis. Our study provides insights into the genomic landscape of early-diverging eudicots and provides a valuable model genome for genetic and applied studies of Ranunculales.

Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim.

A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS-PAGE, with an apparent molecular mass of approximately 13kDa. The molecular mass of Trichosanthrip was 10,964.617Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC(50) of 1.6ng/ml. Liquid chromatography-tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.

[Damage and control of Agrotis ypsilon on Scrophularia ningpoensis].

OBJECTIVE: To study the damage of Agrotis ypsilon on Scrophularia ningpoensis and the control method, so as to provide scientific basis for its integrated pests management (IPM). METHOD: The field investigation and the field controlling trial were carried out for the research. RESULT: There is obvious relationship between the pre-season crops and the damage degree of S. ningpoensis. The damage rate of the fields which had planted maize and tobacco in the last planting season was much higher than that of the other fields. The average damage rate could reach 12.43% and 15.68%. The result of five pesticides against A. ypsilon in field trial showed that the controlling effect of 10% beta-cypermethrin EC 2000 times and 40% chlorpyrifos EC 1500 times were 92.53% and 91.69%, respectively. CONCLUSION: A. ypsilon could be well controlled while 10% beta-cypermethrin EC or 40% chlorpyrifos EC are sprayed during the period of seedling.

Larvicidal activity of Magnolia denudata seed hydrodistillate constituents and related compounds and liquid formulations towards two susceptible and two wild mosquito species.

BACKGROUND: Anopheles sinensis, Aedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit malaria, dengue fever and West Nile virus diseases respectively. This study was conducted to determine the toxicity of 17 constituents from Magnolia denudata seed hydrodistillate (MD-SHD) and four experimental MD-SHD liquid formulations (10-50 mg L(-1) liquid) to third-instar larvae from insecticide-susceptible Cx. p. pallens and Ae. aegypti as well as wild Ae. albopictus and An. sinensis. RESULTS: 2,4-Di-tert-butylphenol was the most toxic constituent (LC50 1.98-3.90 mg L(-1)), followed by linoleic acid (7.19-10.49 mg L(-1)), towards larvae of the four mosquito species. High toxicity was also produced by nerolidol, (±)-limonene, α-terpinene and γ-terpinene (LC50 9.84-36.42 mg L(-1)). The toxicity of these compounds was virtually identical towards larvae of the four mosquito species, even though An. sinensis larvae were resistant to deltamethrin and temephos. The MS-SHD 50 mg L(-1) liquid resulted in 92-100% control towards larvae of the four mosquito species, while commercial temephos 200 g L(-1) emulsifiable concentrate was almost ineffective towards An. sinensis larvae (30% mortality). CONCLUSION: Reasonable mosquito control in the aquatic environment can be achieved by MD-SHD 50 mg L(-1) liquid as a potential larvicide.