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.

Fine Mapping and Functional Analysis of Major Regulatory Genes of Soluble Solids Content in Wax Gourd (Benincasa hispida).

Soluble solids content (SSC) is an important quality trait of wax gourd, but reports about its regulatory genes are scarce. In this study, the SSC regulatory gene BhSSC2.1 in wax gourd was mined via quantitative trait locus (QTL) mapping based on high-density genetic mapping containing 12 linkage groups (LG) and bulked segregant analysis (BSA)-seq. QTL mapping and BSA-seq revealed for the first time that the SSC QTL (107.658-108.176 cM) of wax gourd was on Chr2 (LG2). The interpretable phenotypic variation rate and maximum LOD were 16.033% and 6.454, respectively. The QTL interval contained 13 genes. Real-time fluorescence quantitative expression analysis, functional annotation, and sequence analysis suggested that Bch02G016960, named BhSSC2.1, was a candidate regulatory gene of the SSC in wax gourd. Functional annotation of this gene showed that it codes for a NADP-dependent malic enzyme. According to BhSSC2.1 sequence variation, an InDel marker was developed for molecular marker-assisted breeding of wax gourd. This study will lay the foundation for future studies regarding breeding and understanding genetic mechanisms of wax gourd.

Fine mapping and identification of the candidate gene BFS for fruit shape in wax gourd (Benincasa hispida).

KEY MESSAGE: Non-synonymous mutations in the BFS gene, which encodes the IQD protein, are responsible for the shape of wax gourd fruits. Fruit shape is an important agronomic trait in wax gourds. Therefore, in this study, we employed bulked segregant analysis (BSA) to identify a candidate gene for fruit shape in wax gourds within F2 populations derived by crossing GX-71 (long cylindrical fruit, fruit shape index = 4.56) and MY-1 (round fruit, fruit shape index = 1.06) genotypes. According to BSA, the candidate gene is located in the 17.18 Mb region on chromosome 2. Meanwhile, kompetitive allele-specific PCR (KASP) markers were used to reduce it to a 19.6 Kb region. Only one gene was present within the corresponding region of the reference genome, namely Bch02G016830 (designated BFS). Subsequently, BFS was sequenced in six wax gourd varieties with different fruit shapes. Sequence analysis revealed two non-synonymous mutations in the round wax gourd and one non-synonymous mutation in the cylindrical wax gourd. Quantitative real­time PCR (qRT-PCR) analysis further showed that the expression of BFS in round fruits was significantly higher than in long cylindrical fruits at the ovary formation stage. Therefore, BFS is a candidate gene for determination wax gourd shape. The predicted protein encoded by the BFS gene belongs to the IQ67-domain protein family, which have the structural characteristics of scaffold proteins and coordinate Ca2+ CaM signaling from the membrane to the nucleus. Ultimately, two derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to facilitate marker-assisted selection for wax gourds breeding.

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.

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.

Stereoselective toxicity mechanism of neonicotinoid dinotefuran in honeybees: New perspective from a spatial metabolomics study.

Development of stereoisomeric neonicotinoid pesticides with lower toxicity is key to preventing global population declines of honeybees, whereas little is known about the in situ metabolic regulation of honeybees in response to stereoisomeric pesticides. Herein, we demonstrate an integrated mass spectrometry imaging (MSI) and untargeted metabolomics method to disclose disturbed metabolic expression levels and spatial differentiation in honeybees (Apis cerana) associated with stereoisomeric dinotefuran. This method affords a metabolic network mapping capability regarding a wide range of metabolites involved in multiple metabolic pathways in honeybees. Metabolomics results indicate more metabolic pathways of honeybees can be significantly affected by S-(+)-dinotefuran than R-(-)-dinotefuran, such as tricarboxylic acid (TCA) cycle, glyoxylate and dicarboxylate metabolism, and various amino acid metabolisms. MSI results demonstrate the cross-regulation and spatial differentiation of crucial metabolites involved in the TCA cycle, purine, glycolysis, and amino acid metabolisms within honeybees. Taken together, the integrated MSI and metabolomics results indicated the higher toxicity of S-(+)-dinotefuran arises from metabolic pathway disturbance and its inhibitory role in the energy metabolism, resulting in significantly reduced degradation rates of detoxification mechanisms. From the view of spatial metabolomics, our findings provide novel perspectives for the development and applications of pure chiral agrochemicals.

Identification and Application of BhAPRR2 Controlling Peel Colour in Wax Gourd (Benincasa hispida).

Peel color is an important factor affecting commodity quality in vegetables; however, the genes controlling this trait remain unclear in wax gourd. Here, we used two F2 genetic segregation populations to explore the inheritance patterns and to clone the genes associated with green and white skin in wax gourd. The F2 and BC1 trait segregation ratios were 3:1 and 1:1, respectively, and the trait was controlled by nuclear genes. Bulked segregant analysis of both F2 plants revealed peaks on Chr5 exceeding the confidence interval. Additionally, 6,244 F2 plants were used to compress the candidate interval into a region of 179 Kb; one candidate gene, Bch05G003950 (BhAPRR2), encoding two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2), which is involved in the regulation of peel color, was present in this interval. Two bases (GA) present in the coding sequence of BhAPRR2 in green-skinned wax gourd were absent from white-skinned wax gourd. The latter contained a frameshift mutation, a premature stop codon, and lacked 335 residues required for the protein functional region. The chlorophyll content and BhAPRR2 expression were significantly higher in green-skinned than in white-skinned wax gourd. Thus, BhAPRR2 may regulate the peel color of wax gourd. This study provides a theoretical foundation for further studies of the mechanism of gene regulation for the fruit peel color of wax gourd.

Spatiotemporal Visualization of Insecticides and Fungicides within Fruits and Vegetables Using Gold Nanoparticle-Immersed Paper Imprinting Mass Spectrometry Imaging.

Food safety issues caused by pesticide residue have exerted far-reaching impacts on human daily life, yet the available detection methods normally focus on surface residue rather than pesticide penetration to the internal area of foods. Herein, we demonstrated gold nanoparticle (AuNP)-immersed paper imprinting mass spectrometry imaging (MSI) for monitoring pesticide migration behaviors in various fruits and vegetables (i.e., apple, cucumber, pepper, plum, carrot, and strawberry). By manually stamping food tissues onto AuNP-immersed paper, this method affords the spatiotemporal visualization of insecticides and fungicides within fruits and vegetables, avoiding tedious and time-consuming sample preparation. Using the established MSI platform, we can track the migration of insecticides and fungicides into the inner region of foods. The results revealed that both the octanol-water partition coefficient of pesticides and water content of garden stuffs could influence the discrepancy in the migration speed of pesticides into food kernels. Taken together, this nanopaper imprinting MSI is poised to be a powerful tool because of its simplicity, rapidity, and easy operation, offering the potential to facilitate further applications in food analysis. Moreover, new perspectives are given to provide guidelines for the rational design of novel pesticide candidates, reducing the risk of food safety issues caused by pesticide residue.

Insights into the degradation and toxicity difference mechanism of neonicotinoid pesticides in honeybees by mass spectrometry imaging.

Honeybees are essential for the pollination of a wide variety of crops and flowering plants, whereas they are confronting decline around the world due to the overuse of pesticides, especially neonicotinoids. The mechanism behind the negative impacts of neonicotinoids on honeybees has attracted considerable interest, yet it remains unknown due to the limited insights into the spatiotemporal distribution of pesticides in honeybees. Herein, we demonstrated the use of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for the spatiotemporal visualization of neonicotinoids, such as N-nitroguanidine (dinotefuran) and N-cyanoamidine (acetamiprid) compounds, administered by oral application or direct contact, in the whole-body section of honeybees. The MSI results revealed that both dinotefuran and acetamiprid can quickly penetrate various biological barriers and distribute within the whole-body section of honeybees, but acetamiprid can be degraded much faster than dinotefuran. The degradation rate of acetamiprid is significantly decreased when piperonyl butoxide (PBO) is applied, whereas that of dinotefuran remains almost unchanged. These two factors might contribute to the fact that dinotefuran affords a higher toxicity to honeybees than acetamiprid. Moreover, the toxicity and degradation rate of acetamiprid can be affected by co-application with tebuconazole. Taken together, the results presented here indicate that the discrepant toxicity between dinotefuran and acetamiprid does not lie in the difference in their penetration of various biological barriers of honeybees, but in the degradation rate of neonicotinoid pesticides within honeybee tissues. Moreover, new perspectives are given to better understand the causes of the current decline in honeybee populations posed by insecticides, providing guidelines for the precise use of conventional agrochemicals and the rational design of novel pesticide candidates.