Biopesticide Spinosad: unraveling ecotoxicological effects on zebrafish, Danio rerio

Diogo B.S., Antunes S.C., Pinto I., Amorim J., Teixeira C., Teles L.O., Golovko O., Žlábek V., Carvalho A.P., Rodrigues S.

Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 μg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 μg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 μg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

Li X., Yang Y., Zhang B., Lin X., Fu X., An Y., Zou Y., Wang J., Wang Z., Yu T.

The current understanding of lactate extends from its origins as a byproduct of glycolysis to its role in tumor metabolism, as identified by studies on the Warburg effect. The lactate shuttle hypothesis suggests that lactate plays an important role as a bridging signaling molecule that coordinates signaling among different cells, organs and tissues. Lactylation is a posttranslational modification initially reported by Professor Yingming Zhao’s research group in 2019. Subsequent studies confirmed that lactylation is a vital component of lactate function and is involved in tumor proliferation, neural excitation, inflammation and other biological processes. An indispensable substance for various physiological cellular functions, lactate plays a regulatory role in different aspects of energy metabolism and signal transduction. Therefore, a comprehensive review and summary of lactate is presented to clarify the role of lactate in disease and to provide a reference and direction for future research. This review offers a systematic overview of lactate homeostasis and its roles in physiological and pathological processes, as well as a comprehensive overview of the effects of lactylation in various diseases, particularly inflammation and cancer.

Li C., Zhang C., Zhu C., Zhang J., Xia Q., Liu K., Zhang Y.

Triptolide is a major compound isolated from the Tripterygium wilfordii Hook that is mainly used for the treatment of autoimmune disorders and inflammatory diseases. Though triptolide-induced hepatotoxicity has been widely reported, the hepatic effects when the patients are in an inflammatory state are not clear. In this study, we used low-dose Lipopolysaccharides (LPS) to disrupt the inflammation homeostasis in the liver of zebrafish and explored the hepatotoxicity of triptolide under an inflammatory state. Compared with the Triptolide group, LPS-Triptolide cotreatment exacerbate the liver injury with a remarkable decrease of liver size and liver-specific fluorescence intensity, accompanied by significant elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Liver cell damages were further demonstrated by histological staining and scanning electron microscopy observation. Lipid metabolism was severely impaired as indicated by delayed yolk sac absorption, accumulated triglycerides in the liver, and dysregulation of the related genes, such as ppar-α, cpt-1, mgst, srebf1/2, and fasn. Oxidative stress could be involved in the molecular mechanism as the Nrf2/keap1 antioxidant pathways were down-regulated when the zebrafish in an inflammatory state. Moreover, the expression of autophagy-related genes such as beclin, atg5, map1lc3b, and atg3 was also dysregulated. Finally, apoptosis was significantly induced in responses to LPS-Triptolide co-treatment. We speculate that triptolide could exacerbate the immune response and impair lipid metabolism, resulting in enhanced sensitivity of the zebrafish liver to triptolide-induced toxic effects through disruption of the antioxidant system and induction of apoptosis.

Niedobová J., Ouředníčková J., Hamřík T., Mészáros M., Skalský M.

Spinosad is one of the world's most widely applied natural bioinsecticides and its use has been growing rapidly in the past decade. Biologically based plant protection products can also have unexpected effects on beneficial arthropods. Previous studies examining the lethal and sublethal effects of spinosad on different groups of spiders have shown widely diverging results. This work aims at an extension of knowledge related to the effects of spinosad on the generalist predator of genus Pardosa. We focused on the effects of fresh wet, fresh dry, and dry 3-day-old residues of the commercial plant protection product Spintor®. We examined changes in predatory activity at different prey densities after contacts with those residues and also on the mortality of spiders. For the density of six prey (flies), we found differences in spiders' predatory activity in the short term and the long term after their 1 hr contact with fresh wet residue. The fresh wet residues significantly decrease the number of killed flies. There was no significant effect of treatment on the mortality of spiders over short periods. Over long periods, the fresh wet residues caused a significant increase in spider mortality while dry residue had no effects.

Silva V., Yang X., Fleskens L., Ritsema C.J., Geissen V.

The recently released Farm to Fork Strategy of the European Union sets, for the first time, pesticide reduction goals at the EU level: 50% reduction in overall use and risk of chemical pesticides and a 50% use reduction of more hazardous pesticides. However, there is little guidance provided as to how to achieve these targets. In this study, we compiled the characteristics of all 230 EU-approved, synthetic, open-field use active substances (AS) used as herbicides, fungicides and insecticides, and explored the potential of seven Farm to Fork-inspired pesticide use reduction scenarios to achieve the 50% reduction goals. The pesticide reduction scenarios were based on recommended AS application rates, pesticide type, soil persistence, presence on the candidate for substitution list, and hazard to humans and ecosystems. All 230 AS have been found to cause negative effects on humans or ecosystems depending on exposure levels. This is found despite the incomplete hazard profiles of several AS. 'No data available' situations are often observed for the same endpoints and specific organisms. The results of the scenarios indicate that only severe pesticide use restrictions, such as allowing only low-hazard substances, will result in the targeted 50% use and risk reductions. Over half of the 230 AS considered are top use or top hazard substances, however, the reduction actions depend on the still to be defined EC priority areas and action plans, also for other recent and related strategies. Broader scenario implications (on productivity, biodiversity or economy) and the response of farmers to the pesticide use restrictions should be explored in those plans to define effective actions. Our results emphasize the need for a re-evaluation of the approved AS and of their representative uses, and the call for open access to AS, crop and region-specific use data to refine scenarios and assess effective reductions.

Huang T., Jiang H., Zhao Y., He J., Cheng H., Martyniuk C.J.

In this critical review, we synthesize data from peer-reviewed literature reporting on triazole fungicide exposures in the zebrafish model. Based on their mode of action in plants (potent inhibitors of ergosterol synthesis), we focused attention on mechanisms related to cellular, lipid, and steroid metabolism. Evidence from several studies reveals that zebrafish exposed to triazoles present with impaired mitochondrial oxidative phosphorylation and oxidative stress, as well as dysregulation of lipid metabolism. Such metabolic disruptions are expected to underscore developmental delays, deformity, and aberrant locomotor activity and behaviors often observed following exposure. We begin by summarizing physiological and behavioral effects observed with triazole fungicide exposure in zebrafish. We then discuss mechanisms that may underlie adverse apical effects, focusing on mitochondrial bioenergetics and metabolism. Using computational approaches, we also identify novel biomarkers of triazole fungicide exposure. Extracting and analyzing data contained in the Comparative Toxicogenomics Database (CTD) revealed that transcriptional signatures responsive to different triazoles are related to metabolism of lipids and lipoproteins, biological oxidations, and fatty acid, triacylglycerol, and ketone body metabolism among other processes. Pathway and sub-network analysis identified several transcripts that are responsive in organisms exposed to triazole fungicides, several of which include lipid-related genes. Knowledge gaps and recommendations for future investigations include; (1) targeted metabolomics for metabolites in glycolysis, Krebs cycle, and the electron transport chain; (2) additional studies conducted at environmentally relevant concentrations to characterize the potential for endocrine disruption, given that studies point to altered cholesterol (precursor for steroid hormones), as well as altered estrogen receptor alpha and thyroid hormone expression; (3) studies into the potential role for lipid peroxidation and oxidation of lipid biomolecules as a mechanism of triazole-induced toxicity, given the strong evidence for oxidative damage in zebrafish following exposure to triazole fungicides.

Meng S., Chen X., Song C., Fan L., Qiu L., Zheng Y., Chen J., Xu P.

The chronic effect of environmental methomyl on the antioxidant system in testis of Nile tilapia (Oreochromis niloticus) and its recovery pattern was investigated. Tilapia were exposed to sublethal concentrations of 0.2, 2, 20 and 200 μgL−1 methomyl for 30 days and thereafter moved to methomyl-free water for 18 days. Antioxidant levels in testis, including glutathione peroxidase, catalase, glutathione-S-transferase, glutathione reductase, superoxide dismutase, reduced glutathione, oxidized glutathione were measured every 6 days during the period of exposure, and at 18 days after being transferred to methomyl-free water. The results showed that lower methomyl concentration (0.2 μgL−1) had no effect on the above antioxidants, thus 0.2 μgL−1 could be seen as NOAEL for methomyl to tilapia. However, higher methomyl concentration of 2, 20 and 200 μgL−1 could significantly influence the above antioxidants. Glutathione peroxidase and oxidized glutathione increased significantly. On the contrary, reduced glutathione decreased significantly. Catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase increased at lower methomyl (2 and 20 μgL−1), but decreased at higher methomyl (200 μgL−1). The recovery test showed that oxidative damage caused by lower methomyl of 2 and 20 μgL−1 was reversible, and oxidative damage caused by higher methomyl of 200 μgL−1 was irreversible within 18 days of recovery period.

Lammertyn S., Masín C.E., Zalazar C.S., Fernandez M.E.

• Response of E. fetida earthworms exposed to soil contaminated with atrazine. • The number of cocoons and the rate of production were affected by the herbicide. • At atrazine amounts resembling field conditions, the enzymes’ response was low. • Acetylcholinesterase activity was stimulated at low herbicide concentration. • E. fetida could perform safely as vermiremediator at low atrazine concentrations. Atrazine is one of the most used herbicides and its over-application in fields can affect the soils and their associated biota. In this work, earthworms ( Eisenia fetida ) were exposed to different concentrations of atrazine to assess possible sublethal harmful effects. In the search for reliable biomarkers of these effects, acetylcholinesterase (AChE), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were measured as well as behaviour and population biological parameters such as biomass, growth and production of cocoons. It was found that biomass changes were not significantly affected by atrazine presence and after 28 days the increment was around 18.3–27.3%. Protein content in earthworms was reduced in the presence of atrazine after 28 days. The lower atrazine concentration (2 mg Kg −1 ) affected in the shortest term (7 days) the rate of cocoon production and increased LDH activity and, especially, the AChE activity in around 40%. The presence of a higher concentration (10 mg Kg −1 ) of the herbicide reduced one third of the number of total cocoons, affecting the reproduction. ALP was the least sensitive biomarker to atrazine exposure. When exposed to up to 10 mg Kg −1 of atrazine, E. fetida showed a tendency to rebalance and maintained its general health.

Miao Z.Q., Tu Y.Q., Guo P.Y., He W., Jing T.X., Wang J.J., Wei D.D.

Psocids are a new risk for global food security and safety because they are significant worldwide pests of stored products. Among these psocids, Liposcelis bostrychophila has developed high levels of resistance or tolerance to heat treatment in grain storage systems, and thus has led to investigation of molecular mechanisms underlying heat tolerance in this pest. In this study, the time-related effects of thermal stress treatments at relatively high temperatures on the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST) and malondialdehyde (MDA), of L. bostrychophila were determined. Thermal stress resulted that L. bostrychophila had a significantly higher MDA concentration at 42.5 °C, which indicated that the heat stress increased lipid peroxidation (LPO) contents and oxidative stress in this psocid pest. Heat stress also resulted in significant elevation of SOD, CAT and GST activities but decreased POD activity. Our data indicates that different antioxidant enzymes contribute to defense mechanisms, counteracting oxidative damage in varying levels. POD play minor roles in scavenging deleterious LPO, while enhanced SOD, CAT and GST activities in response to thermal stress likely play a more important role against oxidative damage. Here, we firstly identified five LbHsps (four LbHsp70s and one LbHsp110) from psocids, and most of these LbHsps (except LbHsp70-1) are highly expressed at fourth instar nymph and adults, and LbHsp70-1 likely presents as a cognate form of HSP due to its non-significant changes of expression. Most LbHsp70s (except LbHsp70-4) are significantly induced at moderate high temperatures (<40 °C) and decreased at extreme high temperatures (40–45 °C), but LbHsp110-1 can be significantly induced at all high temperatures. Results of this study suggest that the LbHsp70s and LbHsp110 genes are involved in tolerance to thermal stress in L. bostrychophila, and antioxidant enzymes and heat shock proteins may be coordinately involved in the tolerance to thermal stress in psocids.

Bahl M.F., Brodeur J.C., Costa C.S., D’Andrea M.F., Sansiñena J.A., Marino D.J., Natale G.S.

This paper presents the first acute toxicity data of the natural insecticide spinosad in amphibians. The sensitivity of two neotropical sympatric anuran species, Boana pulchella and Rhinella arenarum, to spinosad-based formulation Tracer™ was evaluated. Lethal effects are reported in tadpoles of B. pulchella stage 25 between 2.81 and 35.44 mg spinosad/L, while for the same concentration range no lethal effects were detected in tadpoles of R. arenarum of the same stage. In addition, Tracer™ produced sublethal effects at the individual level on the swimming activity, morphology (growth and presence of abnormalities), and development of B. pulchella from 2.81 to 5.78 mg spinosad/L, while in R. arenarum effects were only detected in the swimming activity and growth from 2.78 and 6.22 mg/L, respectively. At the biochemical level, Tracer™ produced inhibition of different enzymatic activities, among them, catalase activity at 2.81 mg spinosad/L, glutathione S- transferase activity from 2.81 to 2.98 mg spinosad/L, and acetylcholinesterase activity at 2.81 mg spinosad/L. These findings allow us to conclude that B. pulchella is more sensitive than R. arenarum to spinosad-based formulation Tracer™. The effects demonstrated here are not consistent with those expected since spinosad is supposed to be an environmental healthy alternative. This paper provides useful and necessary information to implement regulations on the use of new compounds entering the market and its associated risks.

Hoseinifar S.H., Yousefi S., Van Doan H., Ashouri G., Gioacchini G., Maradonna F., Carnevali O.

In fish, like other organisms, the lack of balance between the production of reactive oxygen species (ROS) and antioxidant defense system (so-called oxidative stress) can cause DNA hydroxylation, p...

Santos V.S., Limongi J.E., Pereira B.B.

The association of low concentrations of pyriproxyfen and Spinosad, a naturally-occurring insecticide, was evaluated as an environment-friendly strategy to rationalize Aedes aegypti control programs by reducing larvicide consumption, saving financial costs and increasing residual effect against mosquitoes development. Firstly, the ecotoxicological parameters of the mixture was performed on non-target species Daphnia magna and the results confirmed that the low concentrations used in this larvicide mixture were not able to alter the reproductive parameters of chronically exposed microcrustaceans. In contrast, the mixture altered the behavior and development of Aedes aegypti , effectively inhibiting the emergence of adult insects for a long period. The results confirm the hypothesis that even at very low concentrations, the combination of the Spinosad and Pyriproxyfen larvicides offers an opportunity for Aedes aegypti public control programs to be more efficient. • An environment-friendly strategy to control Aedes aegypti was purposed. • Association of low concentrations of larvicides was tested against Aedes aegypti . • Ecotoxicological parameters of the mixture was performed on Daphnia magna . • The tested mixture only altered the behavior and development of Aedes aegypti . • Results present an alternative way for public control programs to be more efficient.

Cherkas A., Holota S., Mdzinarashvili T., Gabbianelli R., Zarkovic N.

A human organism depends on stable glucose blood levels in order to maintain its metabolic needs. Glucose is considered to be the most important energy source, and glycolysis is postulated as a backbone pathway. However, when the glucose supply is limited, ketone bodies and amino acids can be used to produce enough ATP. In contrast, for the functioning of the pentose phosphate pathway (PPP) glucose is essential and cannot be substituted by other metabolites. The PPP generates and maintains the levels of nicotinamide adenine dinucleotide phosphate (NADPH) needed for the reduction in oxidized glutathione and protein thiols, the synthesis of lipids and DNA as well as for xenobiotic detoxification, regulatory redox signaling and counteracting infections. The flux of glucose into a PPP—particularly under extreme oxidative and toxic challenges—is critical for survival, whereas the glycolytic pathway is primarily activated when glucose is abundant, and there is lack of NADP+ that is required for the activation of glucose-6 phosphate dehydrogenase. An important role of glycogen stores in resistance to oxidative challenges is discussed. Current evidences explain the disruptive metabolic effects and detrimental health consequences of chronic nutritional carbohydrate overload, and provide new insights into the positive metabolic effects of intermittent fasting, caloric restriction, exercise, and ketogenic diet through modulation of redox homeostasis.

Cheng B., Zhang H., Jia K., Li E., Zhang S., Yu H., Cao Z., Xiong G., Hu C., Lu H.

Our study investigated the effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish. 10 h post-fertilization (hpf) zebrafish embryos were exposed to several concentrations of spinetoram (0, 5.0 mg/L, 7.5 mg/L, 10 mg/L) for up to 96 hpf, and their mortality, heart rate, number of innate and adaptive immune cells, oxidative stress, apoptosis and gene expression were detected. Studies indicated that the spinetoram exposed zebrafish embryos showed yolk sac edema, slow growth, decreased heart rate, decreased number of immune cells, delayed thymic development and cell apoptosis. In addition, there were also significant changes in oxidative stress related indicators in zebrafish, the content of ROS and MDA and the activity of CAT and SOD increased with the increase of spinetoram concentration. Moreover, we detected the expression of TLR4 related genes including TLR4, MYD88 and NF-κB p65 which were significantly up-regulated in the treated groups. Meanwhile, we also found that pro-inflammatory factors IL-6, IL-8, IFN-γ and CXCL-c1c were up-regulated, but anti-inflammatory factor IL-10 was down-regulated in the treated groups. Briefly, our results show that spinetoram induces the developmental toxicity and immunotoxicity of zebrafish to a certain extent, providing basis for the further research on the molecular mechanism of spinetoram exposure to aquatic ecosystems.

Wang X., Zhou J., Shen M., Shen J., Zhang X., Jin Y.

Chlorpyrifos (CPF) is a widely used insecticide in pest control, and it can affect aquatic animals by contaminating the water. In this study, larval zebrafish were exposed to CPF at concentrations of 30, 100 and 300 μg/l for 7 days. In the CPF-treated group, lipid droplet accumulation was reduced in larval zebrafish. The levels of triglyceride (TG), total cholesterol (TC), and pyruvate were also decreased after CPF exposure. Cellular apoptosis were significantly increased in the heart tissue after CPF exposure compared with the control. Transcription changes in cardiovascular genes were also observed. Through transcriptome analysis, we found that the transcription of 465 genes changed significantly, with 398 upregulated and 67 downregulated in the CPF-treated group, indicating that CPF exposure altered the transcription of genes. Among these altered genes, a number of genes were closely related to the glucose and lipid metabolism pathways. Furthermore, we also confirmed that the transcription of genes related to fatty acid synthesis, TC synthesis, and lipogenesis were significantly decreased in larval zebrafish after exposure to CPF. These results indicated that CPF exposure induced lipid metabolism disorders associated with cardiovascular toxicity in larval zebrafish.

Jiang C.S., Schrader M.

Peroxisomes are ubiquitous, dynamic, oxidative organelles with key functions in cellular lipid metabolism and redox homeostasis. They have been linked to healthy ageing, neurodegeneration, cancer, the combat of pathogens and viruses, and infection and immune responses. Their biogenesis relies on several peroxins (encoded by PEX genes), which mediate matrix protein import, membrane assembly, and peroxisome multiplication. Defects in peroxins or peroxisomal enzymes can result in severe disorders, including developmental and neurological abnormalities. The drive to understand the role of peroxisomes in human health and disease, as well as their functions in tissues and organs or during development, has led to the establishment of vertebrate models. The zebrafish (Danio rerio) has become an attractive vertebrate model organism to investigate peroxisomal functions. Here, we provide an overview of the visualisation of peroxisomes in zebrafish, as well as the peroxisomal metabolic functions and peroxisomal protein inventory in comparison to human peroxisomes. We then present zebrafish models which have been established to investigate peroxisomal disorders. These include model zebrafish for peroxisome biogenesis disorders/Zellweger Spectrum disorders, and single enzyme deficiencies, particularly adrenoleukodystrophy and fatty acid beta-oxidation abnormalities. Finally, we highlight zebrafish models for deficiencies of dually targeted peroxisomal/mitochondrial proteins. Advantages for the investigation of peroxisomes during development and approaches to the application of zebrafish models for drug screening are discussed.

El-Wahed S.I., Ibrahim H.A.

Moreira A., Nogueira V., Bouguerra S., Antunes S.C., Rodrigues S.

Vermelho A.B., Moreira J.V., Akamine I.T., Cardoso V.S., Mansoldo F.R.

Pesticide use in crops is a severe problem in some countries. Each country has its legislation for use, but they differ in the degree of tolerance for these broadly toxic products. Several synthetic pesticides can cause air, soil, and water pollution, contaminating the human food chain and other living beings. In addition, some of them can accumulate in the environment for an indeterminate amount of time. The agriculture sector must guarantee healthy food with sustainable production using environmentally friendly methods. In this context, biological biopesticides from microbes and plants are a growing green solution for this segment. Several pests attack crops worldwide, including weeds, insects, nematodes, and microorganisms such as fungi, bacteria, and viruses, causing diseases and economic losses. The use of bioproducts from microorganisms, such as microbial biopesticides (MBPs) or microorganisms alone, is a practice and is growing due to the intense research in the world. Mainly, bacteria, fungi, and baculoviruses have been used as sources of biomolecules and secondary metabolites for biopesticide use. Different methods, such as direct soil application, spraying techniques with microorganisms, endotherapy, and seed treatment, are used. Adjuvants like surfactants, protective agents, and carriers improve the system in different formulations. In addition, microorganisms are a tool for the bioremediation of pesticides in the environment. This review summarizes these topics, focusing on the biopesticides of microbial origin.