All peels in the experiment were purchased from the market and were as follows: mangosteen (Garcinia mangostana), durian (Durio zibethinus), apple (Malus pumila), mango (Mangifera indica), passion fruit (Passiflora edulis), dragon fruit (Hylocereus undulatus), papaya (Chaenomeles sinensis), jujube (Ziziphus jujuba), grapefruit (Citrus maxima), orange (Citrus reticulata), orange (Citrus sinensis), lemon (Citrus limon), pineapple (Ananas comosus), banana (Musa nana), and pomegranate (Punica granatum). The method of preparation of the peel extracts was amended according to the extraction methods described by the previous researchers (Daud et al., 2010; Kato-Noguchi and Tanaka, 2003). All of the fruit peels were washed with deionized water, cut into a single layer of ca. 1 cm, and placed in a clean salver of a freeze dryer overnight. The condenser temperature was −82°C, and vacuum of 130 μB was applied, after which all of the frozen samples were sliced separately and ground into powder and stored in dark and dry conditions for further use. A part of the peel powder was taken out and mixed according to the ratio of powder (dry weight, the same as the whole text) to deionized water to 0.1 kg/L and leached for 3 days in the dark at 4°C. The rude extracts were filtered using a qualitative filter paper, stored in brown glass reagent bottles, and kept at 4°C. Each peel was disposed using the abovementioned method.

A. catenella (strain MEL90, GeneBan accession number MW386192) was obtained from the Marine Ecology Research Center, First Institute of Oceanography, Ministry of Natural Resources, and cultivated in modified f/2 medium (Guillard, 1975). The cultured conditions of microalgae were as follows: all samples were cultured at 20 ± 1°C under 12/12 light–dark cycle with radiance of 80 μm photon m^-2 s^-1 provided by a fluorescent lamp.

To screen out the peel extracts with potent inhibition effect on A. catenella, the algae in the exponential growth phase (5,000 cells/mL) was treated separately with the abovementioned 15 peel extracts at a final concentration of 5 g/L (the amount of raw material of peel contained in 1 L of culture medium was the same as in the whole text). A control group was set up (without the addition of peel extracts). The experiments were conducted in triplicate in 500-mL sterilized flasks containing 250 mL of medium. The effective algicidal extracts were confirmed based on the following three indicators:

The algicidal rate of different peel extracts at 48 h of the experiment was calculated. All of the samples were counted three times. The algicidal rate was calculated according to the algal cell density using the following formula:

where N_c represents the algal cell density of the control group (cells/mL) and N_t represents the algae cell density of the experimental group (cells/mL).

The spontaneous chlorophyll fluorescence of algal cell that can be used to characterize the growth of algal cells was detected in situ by the FL-3 (>670 nm) of flow cytometry (FCM, Accuri TM C6 Plus).

FCM can also be used to detect the forward-scattered light (FSC) related to cell size and the side-scattered light (SSC) indicating the fine structure and granular nature of the cell (complexity of cell contents). Each group was sampled (2 mL) and filtered by using a 300-mesh sieve silk after 48 h of experiment and then moved into the sample tube for on-machine detection (flow rate: 35 μL/s, number of cells: 5,000 cells). Gated analysis was performed based on the histograms of detected cell signals.

Based on the results of the experiments above, mango peel extracts (MPE) were used for further study as the effective algicidal material. The algal cell density and the culture conditions required in the experiment groups were the same as those described in Section 2.2. MPE was added into the culture medium of A. catenella to make the final concentrations of the peel extracts at 1.2, 2.5, 3.8, and 5 g/L. The group without MPE was the control group, and all experimental groups and control groups were in triplicate. The algal cells of all groups were observed and counted at 48, 96, and 144 h of experiment, respectively. The algicidal rate was calculated according to the algal cell density, and the calculation method is the same as the formula that appeared above.

The cyto-ultrastructure of A. catenella was observed with a transmission electron microscope (TEM, JEOL-2100 Ltd., Japan). Next, 80 mL of treated cell culture was collected and centrifuged (4,700 r/min, 7 min, 4°C). The obtained algal cell was fixed with 3.5% glutaraldehyde at 4°C for 4 h. The cells were then rinsed three more times with 0.1 mol/L potassium phosphate buffer (pH 7.2), fixed with 1.0% OsO₄ at 4°C for 2 h, and subsequently dehydrated using a gradient of ethanol and embedded in epoxy resin. Ultra-thin sections (70 nm) were obtained using a Reichert-Jung E ultramicrotome and double-stained with uranyl acetate and lead citrate. For the detailed operation, refer to the method described by Xue et al. (2023).

A total of 120 mL of treated cell culture was collected and centrifuged to obtain algal cells. The cell was stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 mM) (Solarbio, reactive oxygen species test kit) to assess the alteration of the reactive oxygen species (ROS) level of A. catenella. As DCFH-DA enters the algal cell and reacts with ROS, it was converted into DCF, the fluorescence intensity of which could reflect the ROS level of A. catenella. The treated algal cells were detected with a molecular fluorescence spectrophotometer (The excitation wavelength is 488 nm and the emission wavelength is 525 nm) (F-7100, Japan).

Malondialdehyde (MDA) representing the degree of lipid peroxidization was measured. Then, 80 mL of algal culture was harvested via gentle centrifugation (4,500 r/min, 10 min) to obtain cell pellets, and then the pellets were washed twice with sterile 0.1 M PBS (pH 7.2–7.4) (Solarbio Inc., Beijing, China). The washed algae cells were re-suspended in sterile 0.1 M PBS and sonicated at below 4°C (180 W, ultrasonic time: 1 s, rest time: 3 s) until no intact individual cell was observed with a microscope. Subsequently, the crude enzyme solution was obtained by centrifugation (12,000 rpm for 10 min at 4°C). The MDA content was measured using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. All assays were performed in triplicate.

According to the results of the previous experiments, 36 h was selected as the experimental node to collect samples (120 mL cell culture) for transcriptome analysis. Equal amounts of total RNA for each sample were extracted by using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quality and integrality of the extracted total RNA were assessed by using NanoDrop 2000 and agarose gel electrophoresis. Then, the cleaved RNA fragments were reverse-transcribed to create the final cDNA library. The cDNA library was sequenced on an Illumina platform (HiSeq X Ten, USA). FastQC was used to estimate the quality of the paired-end reads for each sample. The gene expression level was analyzed using the TPM of each gene based on gene length. Differentially expressed genes (DEGs) analysis was performed using DESeq2. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was also completed using the cluster Profiler R software package. For pathway enrichment analysis, cluster Profiler was used to test the statistical enrichment of DEGs in KEGG pathways. All DEGs were mapped to categories in KEGG and searched for significantly enriched pathways in DEGs by comparing with the whole transcriptome background. The p-value was calculated using the hypergeometric test and went through multiple testing corrections. The corrected p-value (q-value) ≤0.05 was considered as the pathway that was significantly enriched.

A total of 10 DEGs were randomly selected for quantitative real-time PCR (qRT-PCR) in all groups to verify the expression patterns observed in the RNA-seq results. The reference gene, assay genes, and specific primers are listed in Supplementary Table 1 . Reverse transcription and PCR were carried out in a system volume of 100 μL (0.5 μg RNA, 2 µL 5× TransScrip tall-in-SuperMix for qRT-PCR, 0.5 μL gDNA remover, and 90 µL nuclease-free H₂O) and a reaction system volume of 10 μl (1 µL cDNA, 5 µL 2× PerfectStart™ Green qRT-PCR SuperMix, 0.2 µL forward primer, 0.2 µL reverse primer, and 3.6 µL nuclease-free H₂O), respectively. Actin was chosen to be the housekeeping gene to normalize the expression levels of mRNA through the 2^−ΔΔCt method (Livak and Schmittgen, 2001; Wang et al., 2021).

MPE was sequentially extracted with organic solvents (polarity: N-hexane < dichloromethane < ethyl acetate < water) at the ratio of 1:1 (v/v). The MPE and the organic solvent were mixed and repeatedly shaken for 20 min for extraction, and the aqueous phase and the organic phase were stratified after shaking. MPE was extracted three times per solvent, and the extracts were pooled together. The organic solvent was removed by rotary evaporation for each extraction solution, and the remaining extracts and solutions were baked in an oven at 60°C until constant weight. The powder of each phase of the same quality was weighed and rehydrated with an equal amount of water, The redissolved solution was added into the A. catenella medium (initial density of 5,000 cells/mL), and the final concentration was set at 5 g/L. Another group was set up to add an equal amount of MPE as the control, and the one without the addition of MPE was the blank control group. The samples were collected for observation, and the calculation of the algicidal rate was at 48 h of the experiment. The calculation method was the same as discussed above.

Chromatographic conditions: The N-hexane phase was passed through the organic needle filter (0.22 μm pore size) to obtain the samples to be tested on the machine. Analyses were performed on a Diane Ultimate 3000 liquid chromatograph (Thermo Fisher Scientific) equipped with a Symmetry C18 (2.1 × 150 mm, 5 µm, Waters). The column temperature was 35°C. The mobile phase consisted of 0.1% (v/v) formic acid solution (eluent A) and methanol (eluent B). The flow rate was 0.2 mL/min. The gradient program was optimized as follows: T/(min): 0-2-7-15-22-25-27-29, B/(%): 10-10-35-65-85-90-10-10. The run time was 29 min. The injection volume was 5 μL.

Mass spectrometry conditions: A Bruker maXis Q-TOF (Thermo Fisher Scientific) with an electrospray ionization (ESI) system was used. Sodium formate solution was used to correct. The scan mode was full scan; scan range (m/z): 50–1,500; drying gas temperature: 180°C; drying gas flow rate: 6 L/min; atomized gas: 1.0 bar.

All data were presented as means ± standard deviation (SD). Visualization was performed by using Origin 2024b (Origin Lab, USA). Statistical analysis was conducted using the SPSS software package (IBM SPSS Statistics 25.0). The original data was presented as mean ± standard deviation (SD) and statistically analyzed by one-way analysis of variance, followed by Duncan’s post-hoc multiple comparison test using SPSS 25.0 for Windows (SPSS, Chicago, IL, USA), and p <0.05 was considered significant.

The optimal algicidal extracts were determined based on the algicidal rate, morphological characteristics, and spontaneous chlorophyll fluorescence of A. catenella exposed to different peel extracts ( Figure 1 ). A total of 13 fruit peel extracts had an algicidal rate of more than 50% among the 15 extracts used in the experiment. Thereinto MPE exhibited the maximal algicidal effect (86.9 ± 1.61% at 48 h) ( Figure 1A ). The results of autofluorescence showed that orange peel, mango peel, durian peel, jujube peel, dragon peel, pineapple peel, papaya peel, and pomegranate peel extracts played a more significant inhibitory effect on the fluorescence value of A. catenella compared with the control group (p < 0.05) ( Figure 1B ). The mean spontaneous chlorophyll fluorescence of algal cells treated with MPE was 4.61 ± 0.15, which was the greatest influence on the spontaneous chlorophyll fluorescence of A. catenella. The morphological characteristics of algal cells were reflected by the values of the FSC and SSC. The results showed that the SSC values of the algal cells in the experimental groups decreased in varying degrees compared with the control group ( Figure 1C ). Given the combined results of the three indicators above, a conclusion could be obtained—that MPE had the best algicidal effect, Therefore, MPE was selected as the experimental material for subsequent research and application.

Different concentrations of MPE exhibited diverse effects on A. catenella ( Figure 2 ). At 48 h after the addition of MPE, the algicidal rates of the 1.2-, 2.5-, 3.8-, and 5-g/L groups were -1.93 ± 0.11%, 56.88 ± 0.03%, 82.95 ± 2.68%, and 89.01 ± 0.91%, respectively, and there were significant differences in the algicidal rates between the various treatment groups (p < 0.05). This result presented that MPE be of the property of “low promotion, high inhibition”. The algicidal rates of the 5-g/L treatment groups reached 93.32 ± 0.56% at 96 h, and a better algicidal rate remained (92.74 ± 0.89%) at 144 h (day 6), which exhibited that MPE had strong timeliness. The EC₅₀ (48 h) of MPE was about 2.5 g/L.

TEM was used to record the death process of algal cells at the subcellular level when A. catenella was exposed to MPE. Compared with the control group, the MPE-treated cells showed significant morphological differences and structural damage ( Figure 3 ). A. catenella is an armored dinoflagellate, the normal cell of which is enclosed by a layer of thecal plates. The membrane and lamella structure of chloroplasts and mitochondria are intact and clear. Compared with the control group, the mitochondria and chloroplasts lost the lamellar structure. The boundary of the chloroplast membrane especially was blurred when the algal cell was exposed to 5 g/L MPE for 12 h. Simultaneously, a large number of starch granules and peroxisomes appeared in the cells exposed to MPE. Furthermore, the structure inside the cell underwent significant changes, and the organelles began to lyse at 24 and 36 h of the experiment.

Compared to the control, the ROS levels in the 2.5- and 5-g/L groups increased significantly at 12 h after the MPE treatment (p < 0.05) ( Figure 4A ). As the experiment progressed, the ROS level in the 5-g/L treatment group increased significantly at 36 h (p < 0.05) and was 3.15 and 2.58 times higher than that of the control group and the 2.5-g/L treatment group, respectively. The results proved that A. catenella produced a large amount of ROS after exposure to MPE.

Compared with the control group in which the MDA content remained at a stable status (from 1.37 ± 0.06 to 1.44 ± 0.11 nmol/mg protein), the MDA content of A. catenella exposure to MPE exhibited an upward trend throughout the whole experiment period ( Figure 4B ). The MDA content in the 2.5-g/L MPE treatment group increased slowly and remained stable at 36 h, while the MDA content in the 5-g/L MPE group continued to increase and reached 2.56 ± 0.07. The results demonstrated that MPE caused the peroxidation of membrane lipids in cells, which implied that there was still a vast amount of ROS accumulated in the cells.

To investigate an overview of the molecular mechanisms of MPE in A. catenella, we constructed three comparison groups (H-36 h vs. C-36 h, L-36 h vs. C-36 h, and L-36 h vs. H-36 h). KEGG enrichment analysis was carried out to understand the biological functions and functional pathways of DEGs; the result is shown in Figure 5 . The pathways significantly enriched in the H-36 h vs. C-36 h groups contained the following (q-value ≤0.05): tryptophan metabolism, proteasomes, peroxisome, cysteine and methionine metabolism, etc., were significantly upregulated (q-value ≤0.05). On the contrary, photosynthesis, starch and sucrose metabolism, and carbohydrate digestion and absorption pathways showed a significantly downward regulation (q-value ≤0.05). In addition, the peroxisome pathway was significantly upregulated (q-value ≤0.05). Compared with the upregulated pathway that was significantly enriched in the H-36 h vs. C-36 h group, the citrate cycle (TCA cycle), valine, leucine, and isoleucine degradation, fatty acid degradation and amino acid biosynthesis, etc., were additionally enriched in the L-36 h vs. C-36 h group. However, in the enrichment results of the L-36 h vs. H-36 h group, glycine, serine, and threonine metabolism, phenylalanine metabolism, valine, leucine, and isoleucine degradation, tyrosine metabolism, fatty acid metabolism, fatty acid degradation, peroxisome, cysteine, and methionine metabolism, etc., were significantly downregulated (q-value ≤0.05).

We furtherly analyzed the regulation and expression trends of 36 DEGs involved in the photosynthetic and antioxidant system of A. catenella from the transcription level. The specific results are presented in Figure 6 . The result about the photosynthetic system showed that there were no significant DEGs found in the L-36 h vs. H-36 h group, which indicated that a low dose of MPE could significantly affect and inhibit the expression of photosynthesis-related genes of A. catenella (q-value ≤0.05). This study found that, of the 28 DEGs involved in photosynthesis, 27 DEGs were downregulated in the H-36 h vs. C-36 h group, of which 14 were significantly different (q-value ≤0.05). The expression levels of psbA gene encoding the D1 protein and psbD gene encoding the D2 protein were significantly downregulated (q-value ≤0.05), and the same tendency occurred in the result of genes (psbB and psbC) encoding core antenna complexes. Furthermore, the reaction center pigment proteins (psaA and psaB) of PSI are also significantly downregulated (q-value ≤0.05). The gene petF encoding ferredoxin and cytochrome b6 (petB) in the photosynthetic chain of the photosynthetic system were significantly downregulated (q-value ≤0.05).

In the result of analyzing DEGs involved in the antioxidant system, the expression of GSR gene associated with the deoxidation of GSSG to GSH was upregulated. SOD1 (Cu–Zn) and SOD2 (Fe-Mn) gene-encoded SOD, CAT gene-encoded CAT, GPX gene-encoded GPX, and GSR gene-encoded glutathione reductase were all significantly upregulated in the treatment group (H-36 h and L-36 h) (q-value ≤0.05). We also found PXMP4 (peroxisome membrane protein 4) and MPV17 (mitochondrial inner membrane protein MPV17) genes to be significantly upregulated in the treatment group (q-value ≤0.05).

The results of the qRT-PCR validation are shown in Supplementary Figure 1 . Overall, nine DEGs had the same trend in RNA-seq and qRT-PCR, and one DEG had a different pattern in RNA-seq profiling of control and treatment samples. The 90% compliance rate between RNA-seq and qRT-PCR indicates that RNA-seq has high accuracy, indicating that the identified pathway and candidate DEGs are reliable pathways for mango peel leaching effect.

We also found that amino acids and fatty acids in A. catenella were gradually degraded with the increase of MPE according to the KEGG enrichment result. We further analyzed the pathways concerning the fatty acid and amino acid degradation. The specific information is displayed in Figure 7 . The results showed that 108, 133, and 149 genes were separately involved in fatty acid degradation. Valine, leucine, and isoleucine degradation and peroxisome were significantly upregulated in L-36 h vs. C-36 h group (q-value ≤ 0.05). In the comparison of L-36 h and H-36 h, the pathways of fatty acid metabolism, fatty acid degradation, valine, leucine, and isoleucine degradation, and peroxisome were downregulated. There were 36, 25, 39, and 30 genes that were significant in the pathways mentioned above, respectively (q-value ≤0.05), which demonstrated that organic substances such as amino acids necessary for the growth of algal cells were more utilized in the H-36 h group.

The algicidal rates of the different extraction phases from MPE are shown in Figure 8 . The results showed that the N-hexane phase had the highest algicidal rate (82.38 ± 0.41%), which implied that the main algae-inhibiting substances in the MPE may be in the N-hexane phase, and the N-hexane extraction component was used for the next experiment.

The results of the LC-QTOF analysis showed that there were 11 phytochemical constituents in the N-hexane extracts ( Supplementary Table 2 ). The main phytocomponents of MPE were analyzed by their peak time (Pt), name of compounds (NCs), molecular formula (MF), mass-to-charge ratio (m/z), and peak area (PA, %). The main substances detected comprised seven esters, one aromatic acid, one phenolic compound, one amino acid, and one sugar. The main composition observed was 3-O-talopyranosylmannopyranoside, which presented the highest intensity and peak area (4.69%), followed by amino acid (4.57%). However, esters were the most abundant, and their total peak area was the highest (9%).