Mass Spectrometry Imaging Reveals the Morphology-Dependent Toxicological Effects of Nanosilvers on Multiple Organs of Adult Zebrafish (Danio rerio).

Nanosilvers with multifarious morphologies have been extensively used in many fields, but their morphology-dependent toxicity toward nontarget aquatic organisms remains largely unclear. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate the toxicological effects of silver nanomaterials with various morphologies on spatially resolved lipid profiles within multiple organs in adult zebrafish, especially for the gill, liver, and intestine. Integrated with histopathology, enzyme activity, accumulated Ag contents and amounts, as well as MSI results, we found that nanosilvers exhibit morphology-dependent nanotoxicity by disrupting lipid levels and producing oxidative stress. Silver nanospheres (AgNSs) had the highest toxicity toward adult zebrafish, whereas silver nanoflakes (AgNFs) exhibited greater toxicity than silver nanowires (AgNWs). Levels of differential phospholipids, such as PC, PE, PI, and PS, were associated with nanosilver morphology. Notably, we found that AgNSs induced greater toxicity in multiple organs, such as the brain, gill, and liver, while AgNWs and AgNFs caused greater toxicity in the intestine than AgNSs. Lipid functional disturbance and oxidative stress further caused inflammation and membrane damage after exposure to nanosilvers, especially with respect to sphere morphology. Taken together, these findings will contribute to clarifying the toxicological effects and mechanisms of different morphologies of nanosilvers in adult zebrafish.

Novel Phosphate Transporter-B PvPTB1;1/1;2 Contribute to Efficient Phosphate Uptake and Arsenic Accumulation in As-Hyperaccumulator Pteris vittata.

Arsenic (As) contamination in soil poses a potential threat to human health via crop uptake. As-hyperaccumulator Pteris vittata serves as a model plant to study As uptake and associated mechanisms. This study focuses on a novel P/AsV transport system mediated by low-affinity phosphate transporter-B 1 family (PTB1) in P. vittata. Here, we identified two plasma-membrane-localized PTB1 genes, PvPTB1;1/1;2, in vascular plants for the first time, which were 4.4-40-fold greater in expression in P. vittata than in other Pteris ferns. Functional complementation of a yeast P-uptake mutant and enhanced P accumulation in transgenic Arabidopsis thaliana confirmed their role in P uptake. Moreover, the expression of PvPTB1;1/1;2 facilitated the transport and accumulation of As in both yeast and A. thaliana shoots, demonstrating a comparable AsV uptake capacity. Microdissection-qPCR analysis and single-cell transcriptome analysis collectively suggest that PvPTB1;1/1;2 are specifically expressed in the epidermal cells of P. vittata roots. PTB1 may play a pivotal role in efficient P recycling during phytate secretion and hydrolysis in P. vittata roots. In summary, the dual P transport mechanisms consisting of high-affinity Pht1 and low-affinity PTB1 may have contributed to the efficient P/As uptake in P. vittata, thereby contributing to efficient phytoremediation for As-contaminated soils.

Sample preparation optimization of insects and zebrafish for whole-body mass spectrometry imaging.

Appropriate sample preparation is one of the most critical steps in mass spectrometry imaging (MSI), which is closely associated with reproducible and reliable images. Despite that model insects and organisms have been widely used in various research fields, including toxicology, drug discovery, disease models, and neurobiology, a systematic investigation on sample preparation optimization for MSI analysis has been relatively rare. Unlike mammalian tissues with satisfactory homogeneity, freezing sectioning of the whole body of insects is still challenging because some insect tissues are hard on the outside and soft on the inside, especially for some small and fragile insects. Herein, we systematically investigated the sample preparation conditions of various insects and model organisms, including honeybees (Apis cerana), oriental fruit flies (Bactrocera dorsalis), zebrafish (Danio rerio), fall armyworms (Spodoptera frugiperda), and diamondback moths (Plutella xylostella), for MSI. Three cutting temperatures, four embedding agents, and seven thicknesses were comprehensively investigated to achieve optimal sample preparation protocols for MSI analysis. The results presented herein indicated that the optimal cutting temperature and embedding agent were -20 °C and gelatin, respectively, providing better tissue integrity and less mass spectral interference. However, the optimal thickness for different organisms can vary with each individual. Using this optimized protocol, we exploited the potential of MSI for visualizing the tissue-specific distribution of endogenous lipids in four insects and zebrafish. Taken together, this work provides guidelines for the optimized sample preparation of insects and model organisms, facilitating the expansion of the potential of MSI in the life sciences and environmental sciences.

Nanoscale Three-Dimensional Imaging of Drug Distributions in Single Cells via Laser Desorption Post-Ionization Mass Spectrometry.

Exploring the three-dimensional (3D) drug distribution within a single cell at nanoscale resolution with mass spectrometry imaging (MSI) techniques is crucial in cellular biology, yet it remains a great challenge due to limited lateral resolution, detection sensitivities, and reconstruction problems. Herein, a microlensed fiber laser desorption post-ionization time-of-flight mass spectrometer (MLF-LDPI-TOFMS) was developed for the 3D imaging of two anticancer drugs within single cells at a 500 × 500 × 500 nm3 voxel resolution. Nanoscale desorption was obtained with a microlensed fiber (MLF), and a 157 nm post-ionization laser was introduced to enhance the ionization yield. Furthermore, a new type of alignment method for 3D reconstruction was developed on the basis of our embedded uniform circular polystyrene microspheres (PMs). Our findings demonstrate that this 3D imaging technique has the potential to provide information about the 3D distributions of specific molecules at the nanoscale level.

High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving Noncovalent Interactions.

Increasing charge state of protein complexes from native solutions while preserving noncovalent interactions in native mass spectrometry (MS) offers great opportunity to gain deeper insights into gas-phase protein structures. Several previous studies have disclosed the possibility of high pressure in supercharging small proteins, whereas its capability to supercharge large protein assemblies under native conditions and how it might affect protein structures remain open questions. Herein, we demonstrated that the high-pressure-induced supercharging strategy affords unique advantages of supercharging protein complexes with the highest charge state surpassing the Rayleigh limit (ZR) and concurrently preserving native-like topology. By examining 32 proteins and protein complexes with molecular weights (MWs) ranging from 8.58 to 801 kDa, we demonstrated that the increased average charge states of macromolecular ions have a strong dependence on the surface areas of native protein conformations and MWs. Factors that might contribute to the high-pressure-induced supercharging capability toward macromolecular ions were discussed. Furthermore, using collision cross section (CCS) variation as a function of charge state, we investigate the effects of gas pressure and charge states on gas-phase structures of proteins and protein complexes. Smaller proteins have the largest CCS variations once supercharged, while macromolecular protein complexes are less affected. The results revealed that both surface density of charge and charged surface basic residues contribute to the observed CCS-charge disciplines for all the macromolecules investigated. Taken together, the results presented here indicate that increasing gas pressure in the ion source affords a rapid, simple, and controlled supercharging method, offering the potency of facilitating further applications of native top-down MS analysis with improved transmission, fragmentation, and detection efficiency.

Perspective on Advances in Laser-Based High-Resolution Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) has become a powerful tool in diverse fields, such as chemistry, physics, materials, and life science. Although such research is getting more prevalent, it is limited by high-resolution (HR) instrumental development. Recently, unceasing efforts have been made toward taking spatial resolution from the microscale to nanoscale, especially for near-field based techniques. These emerging and unique features behind high-resolution mass spectrometry imaging (HR-MSI) provide new possibilities in various aspects of biological researches. Since many technological advances provided a forefront for HR-MSI, compelling challenges including sensitivity, specificity, multimodal imaging, data acquisition, and processing follow undoubtedly. Gratifyingly, with attempts and experiences in the infancy, the emerging techniques move toward much more mature to deal with underlying conundrums in the future. Not only confined to commercial instruments, this perspective highlights recent innovations covering all emerging HR-MSI (with spatial resolution below 5 µm) techniques. Besides, vital limitations and handicaps at this stage are presented while corresponding schemes are proposed. This perspective also gives the authors' personal outlooks of developments and applications for HR-MSI in coming years.

Micro-Lensed Fiber Laser Desorption Mass Spectrometry Imaging Reveals Subcellular Distribution of Drugs within Single Cells.

The visualization of temporal and spatial changes in the intracellular environment has great significance for chemistry and bioscience research. Mass spectrometry imaging (MSI) plays an important role because of its unique advantages, such as being label-free and high throughput, yet it is a challenge for laser-based techniques due to limited lateral resolution. Here, we develop a simple, reliable, and economic nanoscale MSI approach by introducing desorption laser with a micro-lensed fiber. Using this integrated platform, we achieved 300 nm resolution MSI and successfully visualized the distribution of various small-molecule drugs in subcellular locations. Exhaustive dynamic processes of anticancer drugs, including releasing from nanoparticle carriers entering nucleus of cells, can be readily acquired on an organelle scale. Considering the simplicity and universality of this nanoscale desorption device, it could be easily adapted to most of laser-based mass spectrometry applications.

Chemical and Topographical Single-Cell Imaging by Near-Field Desorption Mass Spectrometry.

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near-field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser-based mass spectrometry (MS) methods and multimodal single-cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single-cell imaging were achieved. This technique offers more in-depth cellular information than micrometer-range laser-based MS imaging methods. Considering the simplicity and compact size of the near-field device, this technique can be introduced to MALDI-MS, expanding the multimodal abilities of MS at nanoscale resolutions.

Approaching Standardless Quantitative Elemental Analysis of Solids: Microsecond Pulsed Glow Discharge and Buffer-Gas-Assisted Laser Ionization Time-of-Flight Mass Spectrometry.

Among various ionization sources for mass spectrometry, microsecond pulsed glow discharge (MP-GD) and buffer-gas-assisted laser ionization (BGA-LI) sources have the potential for direct quantitative elemental analysis of solids without the requirement of standard reference materials. The analytical potential of these two ionization sources has been evaluated by coupling them to orthogonal time-of-flight mass spectrometry (MS). A straightforward method was proposed to achieve the quantitative result: if a spectrum contains little interference and elemental peak currents are proportional to their concentrations, then the molar concentration of each element is equal to its ion current proportion in the total ion current. Two series of metal standards were applied for the evaluation. Explicit spectra with little interference can be acquired by both techniques. The interferences contribute only a very small portion to the total ion current for MP-GD-MS and BGA-LI-MS; therefore, their influence on the quantitative result can be ignored. All metal elements can be determined quite accurately by the proposed quantitation method, while gaps exist for nonmetal elements due to the high ionization potentials or gas species interference. Between the two techniques, BGA-LI-MS offers a more accurate quantitative result, primarily due to its higher plasma temperature.

Confirmatory surface analysis of equivocal documents with pigment-based gel inks via laser desorption laser postionization mass spectrometry imaging.

Laser desorption laser postionization time-of-flight mass spectrometry (L2MS) was applied for unambiguous discrimination of pigment-based inks in blue, black, and red gel pens and molecular imaging of equivocal documents in a quasi-non-destructive way. In comparison to laser desorption mass spectrometry (LD-MS), additional discriminatory information on ink components is acquired uniquely, facilitating the distinct differentiation of various pigmented gel inks. More importantly, diversified images of additional characteristic ions achieved using L2MS offer reliable support to discriminate forged documents and decipher important hidden contents. Apart from minimized matrix effect and maximized ionization yield, direct and confirmatory identification of forged documents is achieved successfully without solvent or matrix involved, not only eliminating unwanted damage and contamination to the samples but significantly shortening the overall analysis time. In addition, L2MS is a minimally destructive approach with tiny analyte consumption. With these appealing qualities, L2MS imaging is poised to be a powerful tool for confirmatory surface analysis of complex pigment-based samples. Graphical Abstract Weight and see: Highly distinct and comprehensive images of counterfeit documents with blue-pigmented gel inks are achieved successfully, due to the high sensitivity and increased ion yield of laser desorption laser postionization time-of-flight mass spectrometry. The hidden important contents of the obliterated documents are visually deciphered with the help of the additional chemical information.

Microtrace Analysis of Rare Earth Element Residues in Femtogram Quantities by Laser Desorption and Laser Postionization Mass Spectrometry.

A newly developed laser desorption and laser postionization time-of-flight mass spectrometer (LD-LPI-TOFMS) for the direct microtrace determination of rare earth elements (REEs) in residues has been presented. Benefiting from spatially and temporally separated processes between desorption and ionization, LD-LPI-TOFMS plays a dual role in alleviating the barriers of deteriorating spectral resolution at high irradiance, serious matrix effects and elemental fractionation effects at low irradiance. Compared with the conventional laser desorption/ionization (LDI) method, this technique offers unambiguous full-elemental determination of 15 REEs with more uniform relative sensitivity coefficients (RSCs) ranging from 0.5 to 2.5 for all REEs investigated, satisfying the semiquantitative analysis criteria. More importantly, a highly sensitive analysis of REEs with very little consumption was achieved by getting the utmost out of desorbed neutral atoms instead of increasing the amount of the sample, resulting in outstanding relative and absolute limits of detection (LODs and ALODs) of ∼ng/mL and ∼femtogram. The results presented here indicate that LD-LPI-TOFMS offers great potential in microtrace determination for elements in solution samples with minor sample preparation.

Pulsed microdischarge with inductively coupled plasma mass spectrometry for elemental analysis on solid metal samples.

Pulsed microdischarge employed as source for direct solid analysis was investigated in N2 environment at atmospheric pressure. Compared with direct current (DC) microdischarge, it exhibits advantages with respect to the ablation and emission of the sample. Comprehensive evidence, including voltage-current relationship, current density (j), and electron density (ne), suggests that pulsed microdischarge is in the arc regime while DC microdischarge belongs to glow. Capability in ablating metal samples demonstrates that pulsed microdischarge is a viable option for direct solid sampling because of the enhanced instantaneous energy. Using optical spectrometer, only common emission lines of N2 can be acquired in DC mode, whereas primary atomic and ionic lines of the sample are obtained in the case of pulsed mode. Calculations show a significant difference in N2 vibrational temperatures between DC and pulsed microdischarge. Combined with inductively coupled plasma mass spectrometry (ICPMS), pulsed microdischarge exhibits much better performances in calibration linearity and limits of detection (LOD) than those of DC discharge in direct analysis of samples of different matrices. To improve transmission efficiency, a mixture of Ar and N2 was employed as discharge gas as well as carrier gas in follow-up experiments, facilitating that LODs of most elements reached ng/g.

Thermal diffusion desorption for the comprehensive analysis of organic compounds.

Comprehensive analysis of organic compounds is crucial yet challenging considering that information on elements, fragments, and molecules is unavailable simultaneously by current analytical techniques. Additionally, many compounds are insoluble or only dissolve in toxic solvents. A solvent- and matrix-free strategy has been developed which allows the organic compound analyzed in its original form. It utilizes thermal diffusion desorption with the solid analyte irradiated with high energy laser. It is capable of providing explicit elemental, fragmental, and molecular information simultaneously for a variety of organic compounds. Thermal diffusion desorption has many advantages compared to the electrospray and MALDI techniques. The protons that form the protonated molecular ions originate from the analyte itself. All the elements and fragments are also derived from the analyte itself, which provides abundant information and expedites the identification of organic compounds.

An improved technology for monitoring groundwater flow velocity and direction in fractured rock system based on colloidal particles motion.

The colloidal borescope, using colloidal particle motion, is used to monitor the flow velocities and directions of groundwater. It integrates advanced techniques such as microscopy, high-speed photography, and big data computing and enjoys high sensitivity at the micron level. However, In the same well, the groundwater flow velocity monitored by colloidal hole mirror is varies greatly from that obtained by conventional hydrogeological monitoring, such as pumping test. In order to solve this problem, the stability catcher and stratified packer are designed to control the interference of the vertical flow in drilling, and to monitor the flow velocity and direction of groundwater velocity at the target aquifer and target fracture. Five wells with different aquifers and different groundwater types were selected for monitoring in south-central China. The instantaneous velocity and direction are converted into east-west component and north-south component, the average velocity and direction is calculated according to the time of 10 min, and the particle trajectory diagram is established. Based on these results, it proposed a concept of cumulative flow velocity. Using curve-fitting equations, the limits of cumulative flow velocities as the monitoring time tends to infinity were then calculated as the actual flow velocities of the groundwater. The permeability coefficient of aquifer is calculated by using the fissure ratio of aquifer, hydraulic slope and flow velocity, and compared with the permeability coefficient obtained by pumping test. The results are as follows: (1) The variation coefficient of the instantaneous flow velocity measured at the same depth in the same well at different times is greater than that of the time average flow velocity and greater than that of the cumulative flow velocity. The variation coefficient of the actual velocity is the smallest, indicating that the risk of using the actual flow velocity is lower. (2) The variation coefficient of the flow rate monitored at different depths in the same well is mainly controlled by the properties of the aquifer. The more uniform water storage space in the aquifer, the smaller the variation coefficient. (3) The comparison between the permeability coefficient obtained by monitoring and the permeability coefficient obtained by pumping test shows that the flow of structural fissure water controlled by planar fissure is more surface flow, and the results are consistent. When the groundwater flow is controlled by pores and solution gaps, the flow channel is complicated, which is easy to produce turbulent flow, and the result consistency is poor. (4) According to different research accuracy requirements, different monitoring and calculation methods can be selected for different aquifers and groundwater types. Researches show that, the permeability coefficient calculated for the actual flow velocity in well DR01 is the same as that calculated for the pumping test. The aquifer characteristics reflected by the coefficient of variation of the actual flow velocity in the same aquifer are more realistic. The pumping test method obtains the comprehensive parameters of a certain aquifer, and this method can be used to monitor a certain fissure. In this paper, the new technology developed for monitoring, and the new algorithm established for data processing, can accurately obtain the flow velocity and direction of groundwater, using capsule hole mirror monitoring method. The key parameters of hydrogeology can be obtained by using one well, which can reduce the time and cost input and improve the work efficiency.

Role of three-body recombination for charge reduction in MALDI process.

Ions in Matrix-Assisted Laser Desorption/Ionization (MALDI) are predominantly singly charged for small analyte molecules. With the estimated high number density and low temperature of electrons, the three-body recombination mechanism is attractive and should be considered as an important cause for the charge reduction in the plume. Theoretical calculations indicate that the rate coefficient of the three-body recombination is about 50 times higher than that of the two-body recombination if the analyte molecule has insufficient degrees of freedom. Experimental results show that, for small analyte molecules, the ratio of AH2(2+)/AH(+) is close to the theoretical 5% value from the three-body recombination modeling and this ratio declines with the increasing electron and matrix molecule number density caused by greater laser irradiance. The ratio of [A + 2](+)/[A + 1](+) is higher than the theoretical isotopic value, and the excess [A + 2](+) could exclusively result from the three-body recombination collisions. Further evidence demonstrates that [A + 2](+)/[A + 1](+) increases with electron number density, which is in correspondence with the model. All of these theoretical and experimental results indicate that three-body recombination is an essential charge reduction mechanism for small molecules in the MALDI plume.

Mechanism of Rotenone Toxicity against Plutella xylostella: New Perspective from a Spatial Metabolomics and Lipidomics Study.

The botanical pesticide rotenone can effectively control target pest Plutella xylostella, yet insights into in situ metabolic regulation of P. xylostella toward rotenone remain limited. Herein, we demonstrated metabolic expression levels and spatial distribution of rotenone-treated P. xylostella using spatial metabolomics and lipidomics. Specifically, rotenone significantly affected purine and amino acid metabolisms, indicating that adenosine monophosphate and inosine were distributed in the whole body of P. xylostella with elevated levels, while guanosine 5'-monophosphate and tryptophan were significantly downregulated. Spatial lipidomics results indicated that rotenone may significantly destroy glycerophospholipids in cell membranes of P. xylostella, inhibit fatty acid biosynthesis, and consume diacylglycerol to enhance fat oxidation. These findings revealed that high toxicity of rotenone toward P. xylostella may be ascribed to negative effects on energy production and amino acid synthesis and damage to cell membranes, providing guidelines for the toxicity mechanism of rotenone on target pests and rational development of botanical pesticide candidates.

Metabolic profiling reveals genotype-associated alterations in carotenoid content during banana postharvest ripening.

Banana fruits have attracted considerable attention for health-promoting effects attributed to ubiquitous functional metabolites. However, genotype-dependent accumulation patterns of carotenoids in banana remain largely unclear. Here, we performed a systematic metabolomic investigation of 18 banana cultivars of the AAA, AAB, or ABB genome groups. Our results indicate that the levels of soluble sugars increase during postharvest ripening regardless of genotype, whereas amino acids (AAs) and tricarboxylic acid (TCA) cycle-derived organic acids display genotype-dependent patterns. The levels of AAs derived from the glycolytic pathway increased, whereas those derived from the TCA cycle significantly decreased during ripening. The carotenoid composition in banana pulp was genotype-specific, and the contents of α-carotene were the highest in AAA-genome bananas. Moreover, high α-carotene and ß-carotene contents in banana were correlated with elevated levels of TCA cycle-derived AAs and decreased levels of glycolysis-derived AAs. Taken together, these findings provide a comprehensive understanding of genotype-associated carotenoid accumulation, thereby facilitating the breeding of future high carotenoid banana cultivars.

GIS model for geothermal advantageous target selection.

As the particularly popular green energy, geothermal resources are gradually favored by countries around the world, and the development model centered on geothermal dew point cannot meet the increasing geothermal demand. In this paper, a GIS model combining PCA and AHP is proposed, aiming to select the advantages of geothermal resources at the regional scale and analyze the main influencing indicators. Through the combination of the two methods, both data and empirical can be considered, then the geothermal advantage distribution on the area can be displayed through GIS software images. A multi-index evaluation system is established to qualitatively and quantitatively evaluate the mid-high temperature geothermal resources in Jiangxi Province, and carry out the evaluation of the dominant target areas and the analysis of geothermal impact indicators. The results show that it is divided into 7 geothermal resource potential areas and 38 geothermal advantage targets, and the determination of deep fault is the most critical index of geothermal distribution. This method is suitable for large-scale geothermal research, multi-index and multi-data model analysis and precise positioning of high-quality geothermal resource targets, which can meet the needs of geothermal research at the regional scale.

Integrated mass spectrometry imaging and metabolomics reveals sublethal effects of indoxacarb on the red fire ant Solenopsis invicta.

BACKGROUND: Indoxacarb, representing an efficient insecticide, is normally made into a bait to spread the poison among red fire ants so that it can be widely applied in the prevention and control of Solenopsis invicta. However, the potential toxicity mechanism of S. invicta in response to indoxacarb remains to be explored. In this study, we integrated mass spectrometry imaging (MSI) and untargeted metabolomics methods to reveal disturbed metabolic expression levels and spatial distribution within the whole-body tissue of S. invicta treated with indoxacarb. RESULTS: Metabolomics results showed a significantly altered level of metabolites after indoxacarb treatment, such as carbohydrates, amino acids and pyrimidine and derivatives. Additionally, the spatial distribution and regulation of several crucial metabolites resulting from the metabolic pathway and lipids can be visualized using label-free MSI methods. Specifically, xylitol, aspartate, and uracil were distributed throughout the whole body of S. invicta, while sucrose-6'-phosphate and glycerol were mainly distributed in the abdomen of S. invicta, and thymine was distributed in the head and chest of S. invicta. Taken together, the integrated MSI and metabolomics results indicated that the toxicity mechanism of indoxacarb in S. invicta is closely associated with the disturbance in several key metabolic pathways, such as pyrimidine metabolism, aspartate metabolism, pentose and glucuronate interconversions, and inhibited energy synthesis. CONCLUSION: Collectively, these findings provide a new perspective for the understanding of toxicity assessment between targeted organisms S. invicta and pesticides. © 2023 Society of Chemical Industry.

Metabolomics and mass spectrometry imaging reveal the chronic toxicity of indoxacarb to adult zebrafish (Danio rerio) livers.

Indoxacarb is a widely used insecticide in the prevention and control of agricultural pests, whereas its negative effects on non-target organisms remain largely unclear. Herein, we demonstrated the integrated metabolomics and mass spectrometry imaging (MSI) methods to investigate the chronic exposure toxicity of indoxacarb at environmentally relevant concentrations in adult zebrafish (Danio rerio) liver. Results showed that movement behaviors of zebrafish can be affected and catalase (CAT), glutamic oxalacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) activities were significantly increased after indoxacarb exposure for 28 days. Pathological analysis of zebrafish livers also showed that cavitation and pathological reactions occur. Metabolomics results indicated that metabolic pathways of zebrafish liver could be significantly affected by indoxacarb, such as tricarboxylic acid (TCA) cycle and various amino acid metabolisms. MSI results revealed the spatial differentiation of crucial metabolites involved in these metabolic pathways within zebrafish liver. Taken together, these integrated MSI and metabolomics results revealed that the toxicity of indoxacarb arises from metabolic pathways disturbance, which resulted in the decrease of liver detoxification ability. These findings will promote the current understanding of pesticide risks and metabolic disorders in zebrafish liver, which provide new insights into the environmental risk assessment of insecticides on aquatic organisms.