Natural pyrethrins induced developmental toxicity of zebrafish swim bladder in vivo and genotoxicity of lung cells in vitro.

Natural pyrethrins (NPs) are insecticidal compounds isolated and extracted from pyrethrum flowers and are primarily use to control sanitary pests. The lungs become the main target after exposure, and its use may pose potential hazards to respiratory health. Therefore, in this paper, the toxic effects of NPs on human lung cells A549 were investigated and the risk of respiratory toxicity of NPs was studied using zebrafish swim bladder as a model. The results showed that NPs induced cytotoxicity, caused oxidative DNA damage and triggered mitochondria-mediated apoptosis. In addition, exposure to NPs decreased zebrafish embryo survival, hatchability, and heartbeat, and may inhibit normal swim bladder development by disrupting Wnt and Hedgehog signaling pathways. In conclusion, our results suggest that NPs can induce cytotoxicity in A549 in vitro and developmental toxicity in zebrafish in vivo. This study provides a conceptual basis for understanding the mechanisms of toxicity of NPs and assessing respiratory health risks in humans.

The Effects of Natural Insecticides on the Green Peach Aphid Myzus persicae (Sulzer) and Its Natural Enemies Propylea quatuordecimpunctata (L.) and Aphidius colemani Viereck.

Botanical insecticides and soaps are frequently proposed as environmentally safer alternatives to synthetic insecticides. However, the efficacy and selectivity of these products are often only partially supported by empirical evidence. Here, we tested the effectiveness of five botanical insecticides, belonging to different categories, on the green peach aphid Myzus persicae (Sulzer) and their selectivity towards two natural enemies, the ladybird beetle Propylea quatuordecimpunctata (L.) and the parasitoid Aphidius colemani (Dalman). White thyme essential oil (EO), sweet orange EO, crude garlic extract and Marseille soap were tested and compared with a pyrethrin-based commercial product. Both direct spray assays and residual contact assays on treated cabbage leaf disks were carried out. The tested products had low efficacy against aphids when compared to pyrethrins but were in general less detrimental to ladybird beetle larvae, meaning that if applied against other pests, they have a lower chance of harming this agent of aphid biocontrol. Some of the products (soap, orange EO) did, however, show direct exposure toxicity toward ladybird larvae, and thyme EO had extensive phytotoxic effects on cabbage leaves, possibly indirectly leading to higher mortality in ladybird adults. These results underline the necessity for case-by-case evaluations of botanical insecticides.

The low-lethal concentrations of rotenone and pyrethrins suppress the population growth of Rhopalosiphum padi.

As an important pest on winter wheat, Rhopalosiphum padi (L.) causes damage to the wheat yield by sucking plant nutrients, transmitting plant viruses and producing mildew. R. padi has been reported to develop resistance to pyrethroids and neonicotinoids. To explore potential alternative approaches for R. padi control, the activity of 10 botanical insecticides was evaluated. Results suggested that the toxicity of rotenone and pyrethrins to R. padi were the highest and near to the commonly used chemical insecticides. When exposed to the low-lethal concentrations (LC10, LC30) of rotenone or pyrethrins for 24 h, the lifespan and fecundity of adults in F0 generation decreased significantly compared to control. The negative effect could also be observed in the F1 generation, including the decreased average offspring, longevity of adult, and prolonged nymph period. The population parameters in F1 generation of R. padi were also inhibited by exposing to the low-lethal concentrations of rotenone or pyrethrins, including the decreased net reproductive rate, intrinsic rate of natural increase, finite rate of population increase, and gross reproduction rate. Co-toxocity factor results showed that mixtures of rotenone and thiamethoxam, pyrethrins and thiamethoxam showed synergistic effect. Our work suggested that rotenone and pyrethrins showed negative effect on the population growth under low-lethal concentrations. They are suitable for R. padi control as foliar spraying without causing population resurgence.

Natural pyrethrins induce cytotoxicity in SH-SY5Y cells and neurotoxicity in zebrafish embryos (Danio rerio).

Natural pyrethrins are widely used in agriculture because of their good insecticidal activity. Meanwhile, natural pyrethrins play an important role in the safety evaluation of pyrethroids as precursors for structural development of pyrethroid insecticides. However, there are fewer studies evaluating the neurological safety of natural pyrethrins on non-target organisms. In this study, we used SH-SY5Y cells and zebrafish embryos to explore the neurotoxicity of natural pyrethrins. Natural pyrethrins were able to induce SH-SY5Y cells damage, as evidenced by decreased viability, cycle block, apoptosis and DNA damage. The apoptotic pathway may be related to the involvement of mitochondria and the results showed that natural pyrethrins induced a rise in Capase-3 viability, Ca2+ overload, a decrease in adenosine triphosphate (ATP) and a collapse of mitochondrial membrane potential in SH-SY5Y cells. Natural pyrethrins may mediate DNA damage in SH-SY5Y cells through oxidative stress. The results showed that natural pyrethrins induced an increase in reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and catalase (CAT) activity, and induced a decrease in glutathione peroxidase (GPx) activity in SH-SY5Y cells. In vivo, natural pyrethrins induced developmental malformations in zebrafish embryos, which were mainly characterized by pericardial edema and yolk sac edema. Meanwhile, the results showed that natural pyrethrins induced damage to the Huc-GFP axis and disturbed lipid metabolism in the head of zebrafish embryos. Further results showed elevated ROS levels and apoptosis in the head of zebrafish embryos, which corroborated with the results of the cell model. Finally, the results of mRNA expression assay of neurodevelopment-related genes indicated that natural pyrethrins exposure interfered with their expression and led to neurodevelopmental damage in zebrafish embryos. Our study may raise concerns about the neurological safety of natural pyrethrins on non-target organisms.

Natural Pyrethrin-Induced Oxidative Damage in Human Liver Cells through Nrf-2 Signaling Pathway.

Natural pyrethrins (NPs), one kind of bio-pesticide, have been widely used in organic agriculture and ecological environment studies. Studies have shown that NPs may affect the metabolism of rat liver and human hepatocytes; nevertheless, the toxic effects of NPs on the liver and the related mechanisms are still incompletely understood. In this research, we utilized three types of human liver cells to investigate the mechanism of NPs' induction of oxidative stress. The results showed that NPs exhibit noteworthy cytotoxic effects on human liver cells. These effects are characterized by the induction of LDH release, mitochondrial collapse, and an increased production of ROS and MDA content, subsequently activating the Kelch-like ECH-associated protein 1/Nuclear factor erythroid 2- related factor 2 (Keap1/Nrf-2) pathway. The ROS inhibitor N-acetyl-L-cysteine (NAC) can alleviate ROS/Nrf2-mediated oxidative stress. In addition, the siRNA knockdown of Nrf-2 exacerbated the injury, including ROS production, and inhibited cell viability. In summary, the ROS-mediated Keap1/Nrf-2 pathway could be an important regulator of NP-induced damage in human liver cells, which further illustrates the hepatotoxicity of NPs and thereby contributes to the scientific basis for further exploration.

Pyrethrins and Pyrethroids: A Comprehensive Review of Natural Occurring Compounds and Their Synthetic Derivatives.

This comprehensive scientific review provides an in-depth analysis of both the natural compounds, pyrethrins, and their synthetic derivatives, pyrethroids, focusing on their classification, biosynthesis, mechanism of action, general and pharmaceutical uses, as well as their toxicity and environmental impact. Pyrethrins, derived from certain plant species, have long been recognized for their potent insecticidal properties. The review begins by examining the classification of pyrethrins and pyrethroids, elucidating their structural characteristics and unique features within the field of natural and synthetic compounds. The biosynthetic pathways responsible for producing pyrethrins in plants are discussed, highlighting the enzymatic reactions and genetic regulation involved. In addition, the synthesis of pyrethroid derivatives is explored, including both natural and synthetic sources and potential optimization strategies. Understanding the mechanisms of action by which pyrethrins and pyrethroids exert their insecticidal effects is a crucial aspect of this review. Complex interactions with the nervous systems of target organisms are examined, providing insights into their selective toxicity and modes of action. In addition, the various applications of these compounds are explored, from their use in agriculture for pest control to their incorporation into household insecticides and potential pharmaceutical applications. The review also critically evaluates the potential toxicity of pyrethrins and pyrethroids to human health. By consolidating current knowledge and research findings, this review provides a comprehensive understanding of the properties and applications of pyrethrins and pyrethroids, highlighting their benefits and risks, and the importance of responsible and sustainable use in various areas.

Exploring thermal isomerisation in gas chromatography analyses using natural pyrethrins: Comparison of comprehensive two-dimensional and one-dimensional gas chromatography.

This study aims to assess and qualitatively compare the visual presentation of chromatographic data from the isomerisation of natural pyrethrins - a group of pesticides derived from Chrysanthemum flowers - using one-dimensional gas chromatography (1DGC) and comprehensive two-dimensional gas chromatography (GC×GC). Molecular structural changes, such as thermal isomerisation in this case, occur during gas chromatography injection and separation, to provide characteristic patterns which may not be routinely recognised on the 1D chromatogram. To demonstrate the influence of analytical method parameters on isomerisation processes, variations in oven temperature (isothermal vs. temperature programmed analysis), inlet mode (split vs. splitless), inlet temperature, and carrier gas flow rate were investigated. Increasing oven temperature was the most significant factor affecting isomerisation. Splitless injection mode and increasing inlet temperature promoted isopyrethrin formation, while the effect of inlet temperature appeared minimal with a split injection technique, most likely due to the short residence time in the inlet. Increased carrier gas flow rates in a temperature programmed analysis reduced retention time and minimised isomerisation. The unique presentation of isopyrethrin peaks on a GC×GC contour plot allows for facile recognition of isomerisation especially at low concentrations, simplifies chromatogram interpretation, and aids in analyte identification. It also confirms that the isomerisation process is irreversible since the pyrethrin I and II compounds are absent throughout the bridge formation. These benefits support the use of GC×GC over 1DGC to study isomerisation. Additionally, due to limited data in the literature, Kováts retention indices and linear retention indices of the natural pyrethrins, including isopyrethrins, were experimentally determined on four columns: DB-5 ms UI, Rxi-17Sil MS, SLB-IL60i, and SLB-IL111i.

Four undescribed pyrethrins from seeds of Pyrethrum cinerariifolium and their aphidicidal activity.

Four undescribed pyrethrins C-F (1-4) as well as four known pyrethrins (5-8) were isolated from seeds of Pyrethrum cinerariifolium Trev. The structures of compounds 1-4 were elucidated by UV, HRESIMS, and NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC and ROESY), among which the stereostructure of compound 4 was determined by calculated ECD. Furthermore, compounds 1-4 were evaluated for their aphidicidal activities. The insecticidal assay results showed that 1-4 exhibited moderate aphidicidal activities at the concentration of 0.1 mg/mL with the 24 h mortality rates ranging from 10.58 to 52.98%. Among them, pyrethrin D (2) showed the highest aphidicidal activity, with the 24 h mortality rate of 52.98%, which was slightly lower than the positive control (pyrethrin II, 83.52%).

TcbHLH14 a Jasmonate Associated MYC2-like Transcription Factor Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium.

Natural pyrethrins have high application value, and are widely used as a green pesticide in crop pest prevention and control. Pyrethrins are mainly extracted from the flower heads of Tanacetum cinerariifolium; however, the natural content is low. Therefore, it is essential to understand the regulatory mechanisms underlying the synthesis of pyrethrins through identification of key transcription factors. We identified a gene encoding a MYC2-like transcription factor named TcbHLH14 from T. cinerariifolium transcriptome, which is induced by methyl jasmonate. In the present study, we evaluated the regulatory effects and mechanisms of TcbHLH14 using expression analysis, a yeast one-hybrid assay, electrophoretic mobility shift assay, and overexpression/virus-induced gene silencing experiments. We found that TcbHLH14 can directly bind to the cis-elements of the pyrethrins synthesis genes TcAOC and TcGLIP to activate their expression. The transient overexpression of TcbHLH14 enhanced expression of the TcAOC and TcGLIP genes. Conversely, transient silencing of TcbHLH14 downregulated the expression of TcAOC and TcGLIP and reduced the content of pyrethrins. In summary, these results indicate that the potential application of TcbHLH14 in improving the germplasm resources and provide a new insight into the regulatory network of pyrethrins biosynthesis of T. cinerariifolium to further inform the development of engineering strategies for increasing pyrethrins contents.

Are biopesticides safe for the environment? Effects of pyrethrum extract on the non-target species Daphnia magna.

Biopesticides are natural compounds considered more safe and sustainable for the environment. However, it is also important to evaluate the potential risk in non-target organisms. Pyrethrum extract (PE) is a biopesticide, widely used for agriculture, veterinary, and aquaculture. This work aimed to evaluate acute (0.6 - 40.0 µg/L; 96 h; E(L)C50 toxicity) and sub-chronic (0.7 - 1.1 µg/L; 10 d; life-history parameters) effects of PE on Daphnia magna. Moreover, a biomarkers approach using antioxidant and biotransformation capacity, lipid peroxidation (LPO), neurotoxicity, and energy reserves content were evaluated. Acute effects (mortality, changes in swimming behavior, oxidative stress, lipid peroxidation, neurotoxicity) were recorded with the increase in PE concentration. Sub-chronic assay showed an increase in energy reserves content, antioxidant parameters, and LPO demonstrating that PE unbalances oxidative metabolism. This study can conclude that PE potentiates toxic effects in D. magna and demonstrates the vulnerability of a non-target organism to PE that is considered environmentally safe.

TcbZIP60 positively regulates pyrethrins biosynthesis in Tanacetum cinerariifolium.

Pyrethrins, synthesized in the perennial plant Tanacetum cinerariifolium, are a class of terpene mixtures with high insecticidal activity and low human toxicity, which are widely used in plant-derived pesticides. Numerous studies have identified multiple pyrethrins biosynthesis enzymes, which can be enhanced by exogenous hormones such as methyl jasmonate (MeJA). However, the mechanism by which hormone signaling regulates pyrethrins biosynthesis and the potential involvement of certain transcription factors (TFs) remain unclear. In this study, we found that the expression level of a TF in T. cinerariifolium was significantly increased after treatment with plant hormones (MeJA, abscisic acid). Subsequent analysis identified this TF as a member of the basic region/leucine zipper (bZIP) family and was thus named TcbZIP60. TcbZIP60 was localized in the nucleus, suggesting that it is involved in the transcription process. The expression profiles of TcbZIP60 were similar to those of pyrethrins synthesis genes in different flower organs and at different flowering stages. Furthermore, TcbZIP60 could directly bind to the E-box/G-box motifs in the promoters of the pyrethrins synthesis genes TcCHS and TcAOC to activate their expression. Transient overexpression of TcbZIP60 increased the expression levels of pyrethrins biosynthesis genes, leading to the significant accumulation of pyrethrins. Silencing of TcbZIP60 significantly downregulated pyrethrins accumulation and the expression of related genes. Overall, our results reveal a novel TF, TcbZIP60, that regulates both the terpenoid and jasmonic acid pathways of pyrethrins biosynthesis in T. cinerariifolium.

Piperonylbutoxide as a Dubious Cause of Cardiac Manifestations in Pyrethroid Insecticide Poisoning; a Letter to Editor.

There is little evidence regarding pyrethroid poisoning manifesting with cardiac problems in the literature. Many authors, however, adopted that pyrethroids have a direct effect on heart tissue and can cause cardiotoxicity. Interestingly, no experimental studies have yet determined its mechanism of toxicity on cardiac muscle cells. This letter aims to describe the probable cause of clinical manifestations attributed to piperonylbutoxide, an ignored ingredient in pesticide poisoning, which is added to many pesticide products to increase their insecticidal potency. We think that cardiac manifestations in some cases of pyrethroid poisoning are due to the concomitant piperonylbutoxide toxicity and its possible effect on norepinephrine release from adrenal gland, which might explain changes in cardiac findings. Thus, it is necessary for all clinical toxicologists to determine suspicious ingredients when they are facing a doubtful manifestation.

TcMYB8, a R3-MYB Transcription Factor, Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium.

Pyrethrins are a mixture of terpenes, with insecticidal properties, that accumulate in the aboveground parts of the pyrethrum (Tanacetum cinerariifolium). Numerous studies have been published on the positive role of MYB transcription factors (TFs) in terpenoid biosynthesis; however, the role of MYB TFs in pyrethrin biosynthesis remains unknown. Here, we report the isolation and characterization of a T. cinerariifolium MYB gene encoding a R3-MYB protein, TcMYB8, containing a large number of hormone-responsive elements in its promoter. The expression of the TcMYB8 gene showed a downward trend during the development stage of flowers and leaves, and was induced by methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA). Transient overexpression of TcMYB8 enhanced the expression of key enzyme-encoding genes, TcCHS and TcGLIP, and increased the content of pyrethrins. By contrast, transient silencing of TcMYB8 decreased pyrethrin contents and downregulated TcCHS and TcGLIP expression. Further analysis indicated that TcMYB8 directly binds to cis-elements in proTcCHS and proTcGLIP to activate their expression, thus regulating pyrethrin biosynthesis. Together, these results highlight the potential application of TcMYB8 for improving the T. cinerariifolium germplasm, and provide insight into the pyrethrin biosynthesis regulation network.

Natural and Synthetic Pyrethrins Act as Feeding Deterrents against the Black Blowfly, Phormia regina (Meigen).

Pyrethrum is a botanical insecticide derived from pyrethrum flowers. Feeding deterrence caused by pyrethrum has been reported in several sucking insects; however, there is no account of the cause of deterrence-whether from a single component or the combination of six active ingredients, called pyrethrins. We determined the feeding deterrence of natural pyrethrins, their two main components (pyrethrins I and II), and pyrethroid insecticides on the blowfly, Phormia regina. In a dual-choice feeding assay that minimized tarsal contact with food sources but allowed feeding through proboscises, natural pyrethrins, synthetic pyrethrins I/II, and allethrin were observed to induce deterrence at a concentration 16 times lower than the lowest concentration at which the knockdown rate increased. Feeding bouts were interrupted by intensive grooming of the proboscis at the deterring concentration, but no such grooming was observed to occur while feeding on the unpalatable tastants-NaCl, quinine, and tartaric acid. The underlying mode of action for the feeding deterrence of pyrethrins at sub-lethal concentrations probably occurs on the fly oral gustatory system, while differing from that of unpalatable tastants. The potent feeding deterrence of pyrethrins may provide effective protection for pyrethrum plants by rapidly deterring insects from feeding, before insecticidal activities occur.

Overexpression of chrysanthemyl diphosphate synthase (CDS) gene in Tagetes erecta leads to the overproduction of pyrethrin.

Pyrethrins are widely accepted as natural insecticides and offers several advantages of synthetic compounds, i.e., rapidity of action, bioactivity against a wide range of insects, comparatively lesser costs and the like. A significant source of pyrethrin is Chrysanthemum cinerariaefolium; cultivated in restricted areas, as a result; natural pyrethrins are not produced in a large amount that would meet the ongoing global market demand. However, increasing its content and harnessing the desired molecule did not attract much attention. To enhance the production of pyrethrins in Tagetes erecta, the Chrysanthemyl diphosphate synthase (CDS) gene was overexpressed under the promoter CaMV35S. Hypocotyls were used as explant for transformation, and direct regeneration was achieved on MS medium with 1.5 mg L-1 BAP and 5.0 mg L-1 GA3. Putative transgenics were screened on 10 mgL-1 hygromycin. After successful regeneration, screening and rooting process, the transgenic plants were raised inside the glass house and PCR amplification of CDS and HYG-II was used to confirm the transformation. Biochemical analysis using HPLC demonstrated the expression levels of the pyrethrin, which was approx. twenty-six fold higher than the non-transformed Tagetes plant.

TcGLIP GDSL Lipase Substrate Specificity Co-determines the Pyrethrin Composition in Tanacetum cinerariifolium.

Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.

Evaluation of the Bioefficacy and Insecticide Compatibility of Entomopathogens for Management of Whitefly (Hemiptera: Aleyrodidae) on Upland Cotton Under Laboratory and Polyhouse Conditions.

Entomopathogens (EPFs) are potential alternatives to chemical insecticides for managing Bemisia tabaci (Genn.), an invasive pest of the cotton crop. EPFs alone may not always provide enough insect pest control, but combining EPFs with pesticides, provided both components are compatible, can make an integrated pest management program considerably more effective. Hence, the bioefficacy of EPFs against whitefly, their compatibility with pesticides, and the factors responsible for determining compatibility were studied. The highest nymphal mortality was recorded with the Beauveria bassiana strains Bb-4511 (95.1%) and Bb-4565 (89.9%), and Metarhizium anisopliae Ma-1299 (86.7%) at 1 × 106 conidia ml-1. Lower LC50 values were observed for Cordyceps javanica Cj-089 and Bb-4511, 0.2 × 104 and 0.5 × 104 conidia ml-1, respectively. The toxicity index values in insecticide sensitivity assays ranged from 19.4 to 119.6% among all the EPFs. Comparatively, all the EPFs except Bb-4543 and Bb-4565 showed compatible to moderately toxic reactions to neonicotinoids and spinosyns. Organophosphates (ethion) and pyrethrins (bifenthrin) were toxic to very toxic to all the EPFs except Bb-4511, Fv-083, and Ma-1299. Cj-102 and Cj-089 were compatible with 50% of the average recommended dose of bifenthrin and ethion, and the average recommended dose for the field application of neonicotinoids and spinosyns. Principal component analysis showed that spore production and toxicity index values correlate with each other and are responsible for determining the EPF compatibility with insecticides. The EPF spore production and toxicity index are important factors for determining chemical compatibility. Compatible EPFs can be used individually or in combination as promising and compatible biological alternatives to insecticides in the management of whitefly in cotton.

Overexpression of TcCHS Increases Pyrethrin Content When Using a Genotype-Independent Transformation System in Pyrethrum (Tanacetum cinerariifolium).

Pyrethrum (Tanacetum cinerariifolium) is one of the most important industrial crops for the extraction of pyrethrins, which are natural insecticidal compounds. Progress in pyrethrum molecular breeding with the objective of increasing pyrethrin content has been slow for lack of a suitable gene transfer system. Regeneration recalcitrance is a crucial barrier to establishing a genetic transformation system in pyrethrum. Therefore, in this study, an Agrobacterium-mediated transformation system in pyrethrum was developed using shoot apical meristems from germinated seedlings. Factors affecting transformation efficiency were optimized. Optimal conditions included explants at the "no true leaf" stage with a half apical meristem, an Agrobacterium tumefaciens cell density of OD600 = 0.5, two days of cocultivation, and the incorporation of 1.5 mg L-1 6-BA and 30 mg L-1 kanamycin into the selection medium. Under the optimized conditions, two expression cassettes (proTcCHS-GUS and proRbcS-TcCHS) were successfully transformed into pyrethrum. Polymerase chain reaction (PCR), Southern blotting, reverse-transcription quantitative PCR (RT-qPCR), and histochemical staining confirmed the identity of proTcCHS-GUS transgenic plants. PCR and RT-qPCR analyses confirmed the identity of proRbcS-TcCHS transgenic plants. The transformation efficiency was 0.83% (5 transgenic lines/600 infected explants). The relative concentration of pyrethrins in proRbcS-TcCHS transformants (OX T0-1: 1.50% or OX T0-2: 1.24%) was higher than that in nontransformed plants (WT: 0.76%). Thus, the genetic transformation system overcame the low regeneration efficiency and integrated a foreign gene into the pyrethrum genome. The new system is a suitable and effective tool for creating high-yielding cultivars of pyrethrum.