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Article

Effects of Different Environmental Variables on the Ingestion of Microcystis aeruginosa by Moina mongolica

by
Hao Xing
1,2,†,
Ruitong Jiang
3,†,
Taoying Chen
1,4,*,
Hongtao Liu
1,2,
Yusu Yin
1,2,3,
Jianheng Zhang
1,2,
Wenhui He
1,2,3,
Chunyu Tang
1,2,5,* and
Peimin He
1,2,3,*
1
Water Environment and Ecology Engineering Research Center of the Shanghai Institution of Higher Education, Shanghai Ocean University, Shanghai 201306, China
2
College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China
3
Shanghai Engineering Research Center of River and Lake Biochain Construction and Resource Utilization, Shanghai 201702, China
4
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
5
Marine Science and Technology College, Zhejiang Ocean University, Zhoushan 316100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Mar. Sci. Eng. 2023, 11(3), 570; https://doi.org/10.3390/jmse11030570
Submission received: 9 February 2023 / Revised: 24 February 2023 / Accepted: 2 March 2023 / Published: 7 March 2023
(This article belongs to the Section Marine Biology)
Browse Figures
Versions Notes

Abstract

:
How to control the frequent occurrence of cyanobacteria, especially the outbreak of toxin-producing Microcystis aeruginosa, has been a subject of constant research. This investigation focused on the effect of Moina mongolica on restricting M. aeruginosa blooms under different variables (temperature, light intensity, and salinity) and its growth at the molecular level. The results of batch experiments showed that the range of M. mongolica feeding rates was from 4.02 ± 0.81 × 103~182.23 ± 5.37 × 103 cells/ind·h in the whole experiment, where the highest feeding rates of larva M. mongolica and adult M. mongolica were 133.21 ± 5.24 × 103 vs. 182.23 ± 5.37 × 103 cells/ind·h at 30 °C, 85.88 ± 0.44 × 103 vs. 143.15 ± 14.07 × 103 cells/ind·h at 3000 lx and 88.18 ± 0.32 × 103 vs. 84.49 ± 4.95 × 103 cells/ind·h at 0‰ salinity, respectively. The results of transcriptomics further demonstrated that the response of M. mongolica to M. aeruginosa toxicity was caused by the downregulation of relevant functional genes (cell components, cell processes, metabolic processes, and protein complexes) and related signaling pathways (apoptosis, phagosome, lysosome, ribosome, oxidative phosphorylation, amino and nucleoside sugar metabolism, and PPAR signaling pathways). The findings show that M. mongolica can be released to low-salinity lakes and coastal areas (the subtropic and temperate zones) to prevent and inhibit M. aeruginosa blooms in the early summer phase. Additionally, the results achieved by the investigation will provide the relevant technology for inhibiting cyanobacteria blooms because M. mongolica even resists the produced toxin by M. aeruginosa.

1. Introduction

It has been more than 140 years since harmful cyanobacterial blooms were first reported [1]. In recent years, with rapid developments of industry and urbanization, the acceleration of freshwater and marine pollution, eutrophication, and cyanobacteria outbreaks have destroyed and damaged many freshwater ecosystems [2]. There is also the specific risk of Microcystis aeruginosa blooming in enclosed waters with low salinity in the Jinshan coast, Shanghai, China [3,4]. Notably, Microcystis sp., as one of the general genera in cyanobacteria blooms, poses a great threat to ecosystems [5]. For example, Microcystis aeruginosa is able to kill fish, shrimp, and plankton by secreting toxins [5]. Accordingly, preventing or treating cyanobacteria blooms, especially toxic Microcystis aeruginosa, has constituted an urgent research task.
In response to this challenge, an increasing number of investigations have been preformed using biological manipulation to prevent and treat cyanobacteria blooms [4,6]. For example, Cladocera, as a vital link to sustaining the stability of water ecosystems, ingests planktonic algae and breaks down large filamentous cyanobacteria to reduce the growth of deleterious algal blooms, which plays a crucial role in controlling cyanobacteria blooms [7,8]. However, the toxin effect triggered by cyanobacteria on the growth of Cladocera must also be considered [9]. Pawlik–Skowrońska and Bownik [9] demonstrated that a mixture of a toxic substance (anabaenopeptin-B and microcystins) will inhibit Daphnia magna swimming behavior and hopping frequency. Consequently, once Cladocera is applied to control cyanobacteria blooms, it is also critical to pay attention to the growth of Cladocera itself at the gene level [6].
Moina mongolica, as one of the saltwater Cladocera [10], is a model organism for toxicity tests involving a wide range of salinity and temperature. The available literature reports that M. mongolica has stronger environmental tolerance to salinity ranging from 0.4~1.4‰ to 65.2~75.4‰ and a temperature between 3.2 to 36.0 °C [10]. On the other hand, M. mongolica, as a kind of small cladoceran, can filter algae with small particle diameters with an excellent treating efficiency [10]. Previous studies have focused on the influence of various environmental variables on its growth, reproduction, and feeding [11,12]. However, such investigations only addressed the condition of natural food (marine Chlorella), and literature on its influence on toxic M. aeruginosa in different environments is rare. The present investigation was performed to study the impact of environmental factors, e.g., temperature, light, salinity, and food, on the intake intensity of M. aeruginosa by M. mongolica under laboratory conditions. Additionally, the transcriptomics of M. mongolica are applied to elucidate the effects of M. aeruginosa on the growth of M. mongolica at the molecular level. The primary aim of the current study is to compare the feeding effect and growth status of M. mongolica in different environments and to provide essential data in support of future work concerning cyanobacteria bloom management.

2. Materials and Methods

2.1. Cultivation of Moina mongolica and Microcystis aeruginosa

Moina mongolica was provided from Dalian Ocean Univeristy, which was first collected from the saltwater area of Xiaochi in the south of Shanxi Province in 1982 [13,14] and now preserved in the Laboratory of Marine Biology of China-ASEAN, “The Belt and Road” Joint Laboratory of Marine Culture Technology (Shanghai), College of Fisheries and Life Sciences, Shanghai Ocean University. Four months before the experiment, M. mongolica was cultured in filtered and bodied brackish water with a salinity of 8‰ at the temperature of 21~28 °C and on a diet of Chlorella salina. In the feeding test, the larva M. mongolica was a healthy animal with a larva age of 1 d and more than three generations of reproduction from the same mother. The adult M. mongolica was M. mongolica with an age of more than 12 d and non-oviposition.
Toxic M. aeruginosa FACHB 905 originated from the Institute of Hydrobiology, the Chinese Academy of Sciences. M. aeruginosa was cultured in the medium of BG-11 under sterile condition. The cultivation conditions were set as 25 ± 1 °C, 3000 lx (L:D = 12 h:12 h), 8‰, and pH = 7.2. Finally, M. aeruginosa of the logarithmic stage was used as the experimental material. The algae cell density was calculated with a blood count board. Chlorella sp. was cultured by the medium of f/2 under a sterile condition.

2.2. Batch Experiments

2.2.1. Batch Temperature Experiments

Twenty (20) healthy larva and adult M. mongolica of the same size were placed into a beaker containing 100 mL of M. aeruginosa liquid, respectively. The group with M. mongolica-omitted was set as the control group. All groups were conducted in triplicate. The initial algal density of M. aeruginosa was 1 × 106 cells/mL. Two different kinds of M. mongolica (larva and adult) were cultured at five temperatures (T = 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C) with a salinity of 8 and a pH of 7.2 in a light incubator with 2500 lx intensity (L: D = 12 h: 12 h). No feeding was done 8 h before the experiment, and the algal density of M. aeruginosa was measured at 0, 4, 8, 16, 20, and 24 h, respectively.

2.2.2. Batch Light Experiments

Five (5) light regimes (light intensity of 1000, 3000, 5000, 7000, and 9000 lx) were set in the trial and carried out in a light incubator (temperature of 25 °C, L: D = 12 h: 0 h, salinity of 8, pH 7.2). The batch experiments were conducted under light conditions once every 4 h, lasting 12 h in total. Other test designs were consistent with the temperature settings.

2.2.3. Batch Salinity Experiments

Six (6) salinity gradients (0‰, 2‰, 4‰, 6‰, 8‰, 10‰) were set up in the light incubator (temperature 25 °C, light intensity 2500 lx, L:D = 12 h:12 h, pH 7.2). Other test procedures were in accordance with those of the batch temperature experiments.

2.3. Transcriptomics of M. mongolica

On the basis of the above results, three optimal conditions were chosen to conduct transcriptomics to assess the resistance of M. mongolica to M. aeruginosa. The materials and methods are provided in the Supporting Information Section.

2.4. Data Analysis

The filtering and feeding rates of M. mongolic were calculated according to the method proposed by Muyssen et al. [15]:
F = V N × l n C t − l n c t ′ t
G = F × c t ′ − c 0   l n c t ′ − l n c 0
where F is the filtration rate of M. mongolica; N is the total number of M. mongolica in the test; V is the volume of experimental algal liquid; Ct is the algal biomass of the control group (without M. mongolica) at time t; Ct’ is the algal biomass of the test group at time t; tis the test time; and G is the feeding rate of M. mongolica.
All of the graphs were drawn by GraphPad Prism 7.04. One-way ANOVA was carried out by SPSS 25.0, where p < 0.05 was taken as a significant difference in the values between the two groups.

3. Results

3.1. Changes of M. aeruginosa Ingested by M. mongolica at Different Temperatures

The results of M. mongolica feeding and water filtering rates to M. aeruginosa at two age stages (larva and adult) at different temperatures (15, 20, 25, 30, and 35 °C) are displayed in Table 1 and Table 2. The results obtained under the different cultivation periods at the same temperature showed that the feeding and water filtering rates of M. mongolica, either as a larva or an adult, to M. aeruginosa generally exhibited a decreasing trend with the cultivation periods at the temperatures of 20 °C, 25 °C, 30 °C, and 35 °C. The feeding water filtering rates of M. mongolica slightly increased at 15 °C in the later stage. Meanwhile, the feeding and water filtering rates of M. mongolica cultured with the same cultivation period at different temperatures were also compared, and the results show that the feeding and water filtering rates of M. mongolica at the early period (4 h) and the late period (24 h) were strongly different. In detail, the feeding and water filtering rates of M. mongolica at the early period were the highest at the temperature of 30 °C, and the feeding rate reached up to 100.91 ± 5.24 × 103 cells/ind·h by larva and 137.67 ± 5.37 × 103 cells/ind·h by adult, whereas, the water filtering rate was up to 0.10 ± 0.00 mL/ind·h by larva and 0.15 ± 0.00 mL/ind·h by adult. In addition, the highest feeding and water filtering rates of M. mongolica at the late period occurred at the temperature of 30 °C, which was 18.94 ± 1.50 × 103 cells/ind·h and 0.01 ± 0.00 mL/ind·h by larva, and 31.03 ± 1.25 × 103 cells/ind·h and 0.03 ± 0.00 mL/ind·h by adult, respectively. Moreover, the results of M. mongolica feeding and water filtering rates to M. aeruginosa at two age stages (larva and adult) at different temperatures and cultivation periods were compared. The results clearly demonstrated that the feeding and water filtering rates of adults were nearly significantly higher than those of larvae in the same condition (temperature and cultivation period) (p < 0.05), except at the temperature of 15 °C.

3.2. Changes of M. aeruginosa Ingested by M. mongolica under Different Light Intensities

The results of M. mongolica feeding and water filtering rates to M. aeruginosa at two age stages (larva and adult) under different light conditions (1000, 3000, 5000, 7000, and 9000 lx) are displayed in Table 3 and Table 4. Under the different cultivation periods treated by the same light condition, the results showed that the feeding and water filtering rates of M. mongolica, either as a larva or an adult, to M. aeruginosa decreased with increased cultivation periods, except under the condition of 3000 lx. Meanwhile, the comparison of the feeding and water filtering rates of M. mongolica between different light intensities under the same cultivation period revealed that the highest feeding and water filtering rates generally appeared under the condition of 3000 lx, irrespective of the length of the cultivation period, whereas, the highest values of feeding and water filtering rates were up to 85. 88 ± 0.44 × 103 cells/ind·h and 0.09 ± 0.00 mL/ind·h, and 143.15 ± 14.07 × 103 cells/ind·h and 0.14 ± 0.02 mL/ind·h after 4 h, respectively. The results of M. mongolica feeding and water filtering rates between larva and adult were also compared. The findings showed that the resistance of adults within lower light intensities (1000, 3000, 5000, and 7000 lx) was markedly greater than that of larva under the early periods (p < 0.05), while at a higher light intensity (9000 lx), the resistance of adults was significantly weaker than of larvae (p < 0.05).

3.3. Changes in the Intake of M. aeruginosa by M. mongolica under Different Salinity Conditions

The final batch experiments assessed different salinity conditions(Table 5 and Table 6). First, the response of larva and adult to different cultivation periods with the same salinity condition implied that the trend of feeding and water filtering rates generally decreased, except when exposed to 8‰ and 10‰(Table 5). Second, the effect of different salinities on M. mongolica feeding and water filtering rates at the early and late periods was considered. The results showed that the highest rates were all detected under the condition of 0‰ after 4 h. Moreover, the highest rates were detected under the condition of 8‰ after 24 h. In a comparison of the feeding and water filtering rates between larvae and adults, it was found that the feeding and water filtering rates of adults were significantly higher than those of larvae during all of the periods under the condition of 6‰ (p < 0.05). However, the comparison of the feeding and water filtering rates between larvae and adults showed no obvious trend by other treatments(p > 0.05).

3.4. Transcriptomic Analysis of Ingestion of Toxic Microcystis aeruginosa by Moina mongolica

3.4.1. Population Growth Changes of M. mongolica under Different Food Conditions

The population growth of M. mongolica fed with two different foods for 1 week is shown in Table S1. The results demonstrated that the survival and newborn numbers of M. mongolica fed with Chlorella sp. were significantly higher than those fed with toxic M. aeruginosa (p < 0.05). One week (1 w) after ingesting toxic M. aeruginosa, the survival number of M. mongolica greatly decreased with cultivation periods (p < 0.05), and there was no reproductive trend. Furthermore, the survival rate of M. mongolica-fed Chlorella sp. decreased slowly with an increased cultivation period (p > 0.05).

3.4.2. Sequencing Data Statistics and Quality Assessment

By evaluating the base error rate, base distribution range, and the quality of the base of the sample itself, a statistical table for the quality evaluation of the sequencing results was prepared (Table S2). The percentage of the number of bases with a Phred score greater than 30 in the total number of bases in six samples of M. mongolica was up to 94.41%, which indicates that the sequencing result was excellent.

3.4.3. Assembly and Splicing of Transcript Sequencing and Gene Function Annotation

The numbers of successfully assembled unigenes in sequencing, transfers, average length, and the size of N50 were 21,074, 35,713, 1600.64 bp, and 3439 bp, respectively (Table S3). Table S3 also shows that the gene sequence length of 200–500 and 500–1000 accounted for a large proportion. Mapping comparison results were obtained by comparison with all clean reads using Trinity software, and the analysis comparison rates ranged from 87.60% to 89.57%. Table S4 presents the results of transcript function annotation. A total of 21,074 unigenes were assembled; the most annotations were in the GO library with a total of 8737, accounting for 41.46%, while the fewest annotations were in the KEGG library with a total of 6773, accounting for 32.14%. In the COG functional modules of research annotation, there were 8270 gene annotations distributed in 23 available categories of COG (Table S5). These functional classifications were rich in intracellular trafficking, secretion, vesicular transport, and signal transmission mechanisms.
According to the spliced unigene, the obtained genes were annotated and classified in terms of three categories, including cell components, biological processes, and molecular functions in the GO database, the results of which are shown in Figure 1a. The results showed that the cellular process, metabolic process, and biological regulation were mainly enriched in the biological process. The cell part, membrane part, and organelle were primarily enriched in cell components. In terms of molecular function, it was found that binding and catalytic activity were the most enriched. The annotated KEGG metabolic pathways can be divided into six categories (Figure 1b). The annotation results revealed that 919 genes were enriched in the signal transduction pathway, 609 genes were enriched in the cancer overview pathway, and 570 genes were enriched in the translation pathway.

3.4.4. Differentially Expressed Genes Analysis

According to the screening principle of |log2FC| ≥ 1 and p < 0.05, the C_ Group and E_ Group differences were analyzed, and 572 different genes were obtained. There were 233 upregulated and 339 downregulated genes, respectively. As shown in Figure 1a,b, 20 genes each were significantly upregulated and downregulated in GO classification.
According to GO analysis, it was found that the biological processes were mainly metabolic processes, cellular processes, and biological regulation processes. The figure shows 20 functional pathways of upregulated (Figure 2a) and downregulated (Figure 2b) genes in GO classification. Among the 120 significantly upregulated genes annotated by GO (Figure 2a) in the biological process, 39 genes were enriched in the metabolic process, 23 genes were enriched in the cellular process, and 20 genes were enriched in the biological regulation. In terms of cell composition, 30 genes were located in the membrane part and 27 genes were located in the cell part. Regarding molecular function, 57 genes were enriched in the catalytic reaction, and 57 genes were enriched in catalytic activity. Among the downregulated genes of the TOP20, 84 genes were enriched in the metabolic process and 80 genes were enriched in the cellular and biological processes. Concerning cell components, 82 genes were enriched in the cell part, 51 genes were enriched in the organelle part, and 48 genes were enriched in the membrane part. In terms of molecular function, 114 downregulated genes were enriched in catalytic reaction (binding) and 86 were enriched in catalytic activity.
According to KEGG analysis, among the top-20 pathways that were significantly upregulated, it was found that the pathways related to metabolism, growth, and metabolism of M. mongolica were starch and sucrose metabolism (Figure 3a), lysosome, the neuroactive ligand-receptor interaction, and the sphingolipid-signaling pathway. Pancreatic secretion, carbohydrate digestion and absorption, protein digestion and absorption, and the B cell receptor-signaling pathway were primarily related to the digestion of M. mongolica, in which the first 20 that were significantly enriched and downregulated according to KEGG belonged to five major categories (Figure 3b). Among them, ribosome, oxidative phosphorylation, starch and sucrose metabolism, amino acid, and nucleotide sugar metabolism were related to the metabolism of M. mongolica. Among these, ribosome was the most significant downregulation gene. Apoptosis, phagosomes, and lysosomes directly affected the cell growth and development of M. mongolica, and the most important was apoptosis. The most significant pathways in the biological system with animals were antigen processing and PPAR signaling pathway.
The analysis of the transcriptional response of M. mongolica to M. aeruginosa toxicity revealed that the toxic effects are achieved through molecular mechanisms involving the modulation of genes and protein pathways. The toxic effects include the destruction of ribosome protein synthesis, the inhibition of protein phosphate, the alteration of energy metabolic pathways, and the damage to neural networks, resulting in severe damage to the organism. Specifically, the results of transcriptomics showed that the response of M. mongolica to M. aeruginosa toxicity is primarily due to changes in the expression of relevant functional genes, such as uricase (related to purine metabolism pathway) and 60S acidic ribosomal protein (related to protein synthesis function) genes. These genes were significantly downregulated (Figure 3b), leading to a decline in the survival rate, growth, and reproductive ability of the organism. On the other hand, lipase 3-like (related to lipid metabolism pathway) and trypsin-like peptidase genes (related to digestion and degradation function) were significantly upregulated (Figure 3a), enabling a certain number of individuals to survive in the short term.

3.4.5. Validation of Transcriptomic Data by qRT-PCR

In order to verify the differentially expressed genes identified by transcriptome, 16 differentially expressed genes were randomly selected for qPCR analysis. The 2−ΔΔCt method was used to calculate the relative expression of genes. Furthermore, we compared the results with the expression of the transcriptome. The results demonstrated that the qPCR results of 16 genes were consistent with the transcriptome’s expression changes (Figure 4).

4. Discussion

4.1. Effect of Temperature on Feeding of M. aeruginosa by M. mongolica

In the investigation, temperature was found to have a significant effect on the feeding of M. aeruginosa by M. mongolica. According to relevant observations, 28–32 °C is generally reported as the most appropriate temperature range for the population growth of M. mongolica [11,12]. In the experiment, the most significant intake of M. aeruginosa by the two stages of M. mongolica occurred at 30 °C. This study also revealed that the most suitable temperature range for M. aeruginosa to be consumed by M. mongolica was 25–30 °C. With the increase in temperature, time, and life activity, the feeding behavior of M. mongolica was correspondingly promoted, which was reflected in the increase in feeding rate in the later period. A previous investigation reported that when the temperature of M. mongolica was 15 °C or lower, part of its reproductive mode could be changed from parthenogenesis to hermaphroditism, and its filtering ability was also decreased [16]. In the experiment, the feeding rate below 25 °C was low, which verified the above assertion.
The feeding rate of M. mongolica in different periods exhibited a decreasing trend, which may be associated with the starvation treatment before the experiment. The feeding rate of Pacific Spinoza was much higher than that of regular feeding after it was placed in a starving environment for a period of time [17]. Additionally, the feeding ability of Daphnia magna to Chlorellapyrenoidosa under starvation also increased significantly [18]. Moreover, Ferrão–Filho and Kozlowsky–Suzuki [19] demonstrated that some cladhorn species that are highly sensitive to starvation are also highly sensitive to M. aeruginosa. Their work indicated that M. mongolica might not be strong enough to restrict the growth of toxic cyanobacterial blooms like Daphnia magna. M. mongolica is a small cladoceran, and its feeding ability is slightly less than that of Daphnia magna. However, M. mongolica is a saltwater species and can survive in salt waters and low-salt coasts. Now, only M. mongolica can be used to restrict cyanobacteria blooming occurring in saltwaters and low-salt coasts, and we can select good strains from M. mongolica species and increase the efficiency for restricting toxic M. aeruginosa in the future.

4.2. Effect of Light Intensity on the Feeding of M. aeruginosa by M. mongolica

By setting different light intensities, the feeding intensity of M. mongolica on M. aeruginosa was significantly different. The results revealed a significant difference between high light, low light, and medium light in the period of M. mongolica feeding on M. aeruginosa. The reason that the high light appeared to increase first may be related to the sensitivity of M. mongolica to adapt to high-light intensity. Zhao et al. [20] reported that the highest intake of M. mongolica to Chlorellapyrenoidosa occurred at 5000–7000 lx. In contrast, in the current study, the best light intake of M. mongolica to M. aeruginosa was observed at 3000 lx. This may be ascribed to the growth of M. aeruginosa being affected by different light intensitiesand M. mongolica having the greatest degree of light avoidance among those in the range of high-light intensity. In addition, different strains might have different adaptabilitiesfor light intensity [20,21]. Abundant literature has proven that particular light levels can promote the development of M. aeruginosa and the release of microcystins [22,23]. This may be one of the primary reasons for the low intake of M. mongolica at a high light intensity.

4.3. Effect of Salinity on the Feeding of M. aeruginosa by M. mongolica

Although salinity significantly differed in M. mongolica feeding on salt-tolerant M. aeruginosa, no clear trend was found. This may be attributable to the broad salt characteristics of M. aeruginosa on the one hand and the response of M. aeruginosa to salt stress on the other hand. When the salinity was high, the feeding rate of M. mongolica was low, which may be associated with the effect of salt stress on the growth of M. aeruginosa [24]. It was reported that approximately 7‰ may be the salt tolerance range of most M. aeruginosa [25]. Overall, the higher the salt level, the stronger the growth stress. Furthermore, the higher the level of salt, the more M. aeruginosa cells would die, which is not conducive to feeding M. mongolica. Research also demonstrated that the feeding rate of M. mongolica was high at low salinity (5–10‰) [12]. M. mongolica is characterized as being of saltwater origin and possessing the ability of two-way hypertonic and hypotonic regulation. The hyperosmotic capability of M. mongolica in low salinity water can also promote its appetite. It was determined that the feeding rate of juvenile and adult M. mongolica changed significantly with salinity, indicating that M. mongolica was sensitive to salinity changes. Some studies also examined the effects of pH and salinity on the feeding of Daphnia magna in different growth stages and found that Daphnia magna at different ages exhibited a trend of low promotion and high inhibition in response to changes in the external environment. In addition, juvenile Daphnia magna were more sensitive to changes in salinity [26], which is in accordance with the results of this study.

4.4. Molecular Mechanism of the Decrease of Survival Rate of M. mongolica under Toxic Microcystis Stress

Most Microcystis spp. can produce the hepatotoxin microcystin (MC) [6,27,28], and MC would inhibit protein phosphatases [29,30] and damage DNA through the promotion of oxidative stress [31]. Lyu et al. [6] studied the transcriptional analysis of Daphnia clonal variation in tolerance to toxic Microcystis and reported that major MoAs (modes of action), such as glutathione metabolism, protein processing in endoplasmic reticulum, amino sugar/nucleotide sugar metabolism, and arachidonic acid metabolism were linked to tolerance fitness in Daphnia similoides. Most Daphnia would be negatively affected by toxic Microcystis, as toxic Microcystis could contribute to enhanced mortality, abnormal development, and lower reproduction [32,33].
The study systematically explained the molecular mechanism of the decline in the survival rate of M. mongolica under the stress of toxic microcystins at the transcriptome level. Through transcriptome data, it was determined that in the GO library classification, with the toxic microcystins FACHB-905 as the bait, the differential gene changes in M. mongolica mainly belonged to three categories. Combined with KEGG significant enrichment analysis, it primarily belonged to six categories (except drug development). The above results indicate that the inhibition of the population growth of M. mongolica was closely related to the internal gene and protein pathway, revealing the molecular mechanism of M. mongolica in response to the stress of M. aeruginosa from the molecular level. These findings provide helpful information support for the research of cladoceran ecotoxicology, further achieving the early warning function for the monitoring of microcystin blooms.
As the test organism of potential water environment pollution monitoring, the paper also demonstrates the feasibility of M. mongolica as a model organism. The available literature has shown that the toxic effects of M. aeruginosa on cladoceran mainly lie in the destruction of ribosomal protein synthesis, interference with the digestion pathway, and inhibition of the protein phosphorylation pathway [34]. Liu et al. [35] observed that polystyrene nanomaterials function to induce functional genes of Daphniapulex to change in oxidative stress, immune defense, glucose metabolism, etc., depending on RNA sequencing. In this experiment, with the assistance of high-throughput sequencing technology, we investigated the molecular mechanism of M. mongolica in response to the stress of the microcystins toxin. A total of five pathways were found to be significantly upregulated after enrichment. They mainly comprised protein digestion and absorption, pancreatic secretion, starch and sucrose metabolism, and lysosome and carbohydrate absorption. Among them, in the carbohydrate digestion and metabolic absorption pathway, Alpha-amylase (α-amylase) was significantly upregulated under the stress of toxic microcystins. In carbohydrate metabolism, the tricarboxylic acid (TCA) cycle is the final oxidative decomposition pathway of carbohydrates, lipids, and amino acids.In the protein digestion and absorption pathway, to cope with stress, M. mongolica sustained its growth by absorbing protein and other nutrients, as shown in the significant upregulation of trypsin, which speeds up the operation of the digestive system of M. mongolica. Klumpen et al. [36] even reported that the white matter biosynthesis gene was downregulated and the carbohydrate metabolism gene was upregulated in Daphniapulex under starvation conditions.
Relevant studies have confirmed that toxic microcystins have low nutritional value and are not a high-quality food, and, thus, that it is necessary to strengthen carbohydrate absorption [37,38]. A-amylase can synthesize high-energy substances in numerous ways, commonly known as ATP. Therefore, the polysaccharide enzymes and starch-related proteins were significantly upregulated, which may be related to the stimulation of microcystin. M. mongolica needs to consume more energy and absorb nutrients to resist interference of the external environment. Therefore, it needs to obtain more carbohydrates and protein, which can be converted into nutrients to promote its growth to resist external stress. In the lipid metabolism pathway, lysosomal aspartic protease was significantly downregulated, while sphingomyelin phosphodiesterase was significantly upregulated [39]. Aspartase is an acid protein enzyme that can degrade proteins, promote antigens, and activate enzymes. It is mainly reflected in the pathological immune process [40]. Sphingolipid phosphodiesterase is a very important hydrolase in animal metabolism. It can induce apoptosis and cell differentiation under external stimuli and plays an essential role in preventing animal poisoning. On the basis of the enriched KEGG pathway, we can find that ribosomes are significantly downregulated genes. Ribosomes serve an essential function in cells, and their primary function is translating into proteins. Their downregulation will inhibit the protein synthesis of M. mongolica. In addition, some studies have found that the decline of ribosomal function may be an important way for the body to cope with the lack of energy supply due to mitochondrial damage during aging, which may realize healthy aging through the redistribution of energy [41]. Therefore, the significant downregulation of ribosome pathway-related genes may constitute one of the stress modes of M. mongolica in response to the toxicity of M. aeruginosa. Since the endoplasmic reticulum plays a role in transporting proteins, it can effectively prevent proteins from being misfolded [42]. Protein disulfide isomerase is mainly used to secrete and catalyze proteins, promoting disulfide bond enzyme folding to the correct position. Heat shock proteins and their partners can also repair some affected misfolding proteins on this basis. However, it was found that protein disulfide isomerase and heat shock protein (Hsp70) were significantly downregulated in the toxic M. aeruginosa group, resulting in the aggregation of misfolded proteins in the cell body. According to the latest literature, ROS is an early (pre-injury) stress signal, and protein defect is a late (post-injury) stress signal, which can trigger the heat stress response [43]. ROS is induced in large quantities that are far beyond the scavenging capacity of the body’s antioxidant system [44]. The oxidative phosphorylation pathway was also downregulated, which may be caused by microcystin. MC toxin is water-soluble and stable in molecular structure. Once it enters the organism, it will combine with protein phosphatase and inhibit protein phosphorylation. ROS is induced by MC in large quantities, causing lipids to be in a state of oxidation, resulting in redox damage and then interfering with the cell signal pathway.
Moreover, the KEGG pathway shows that the genes related to porphyrin and chlorophyll metabolism were significantly upregulated. Although chlorophyll does not exist in M. mongolica, the porphyrin metabolism pathway may be activated under the action of M. aeruginosa. The porphyrin molecule can be used to synthesize heme, whereas heme generally exists in cladoceran, which plays a role in regulating oxygen content in the body. The upregulation of related genes may be an adaptive mechanism of M. mongolica to cope with oxidative stress. Nucleotides and other energy synthesis come directly from ATP. The downregulation of ATP lipid-binding protein is unfavorable to nucleotide synthesis and DNA and RNA synthesis [45].
The amino sugar and nucleoside sugar metabolism pathway is also a significantly enriched down-regulation pathway, mainly related to the catabolism of sugars. The downregulation of this pathway indicates that M. mongolica cannot perform standard sugar decomposition when feeding on toxic M. aeruginosa, and thus there are few ways to obtain energy, damaging the growth and metabolism of animals. In the process of amino sugar and nucleoside sugar metabolism, chitinase directly affects animal molting behavior, and its downregulation will also influence animal development and growth [46]. Furthermore, the down-regulation pathway includes apoptosis, phagosome, antigen processing and display, PPAR signal pathway, etc. These pathways are primarily related to the cellular processes of M. mongolica, in which phagocytosis is directly related to the animal immune system. First, phagosomes are formed in the body, and their phagocytic response is to resist the invasion of toxic substances from the outside world. However, the downregulation of the phagosome pathway directly affects the body’s immune system [47]. The PPAR-signaling pathway is mainly used to protect and immunize adipocytes, avoid lipid peroxidation, and protect the body from damage [48]. Therefore, the blockage of the PPAR-signaling pathway will inhibit the natural immunity and growth ability of M. mongolica. These down-regulation pathways will have various genetic information, and the long-term growth of the population will also be affected.

5. Conclusions

Restricting water M. aeruginosa blooms and reducing toxic microcystins have been urgent research aims. M. mongolica, as a saltwater Cladocera-feeding phytoplankton, was applied in the investigation to explore the optimal conditions for M. mongolica to control the outbreak of M. aeruginosa, and to explore the effect of M. aeruginosa on the growth of M. mongolica at the molecular level through transcriptomics. The detailed results are as follows: (1) At different temperatures, the feeding behavior of M. mongolica varied. The optimum feeding temperature range of M. mongolicaorella was 25–30 °C. Therefore, it can be inferred that in the high-temperature stage of cyanobacteria growth in summer, this temperature range is conducive to removing M. aeruginosa by M. mongolica; (2) Under different light conditions, the optimum light for M. mongolica to feed on M. aeruginosa was 3000 lx. This is inconsistent with the optimal light for feeding M. mongolica in the natural environment. The optimal light for providing marine Chlorella was 5000–7000 lx, which may influence the growth of M. aeruginosa and is closely related to the release of endotoxins; (3) Under different salinity conditions, two kinds of M. mongolica in different growth stages were more sensitive to salinity change. With the increase in salinity, the intensity of M. mongolica feeding on M. aeruginosa decreased. The growth of M. mongolica and M. aeruginosa decreased under salinity stress, with weak short-term adaptability and cell death, which is not conducive to the feeding of M. mongolica.
Meanwhile, the transcriptome data was analyzed to reveal the difference in gene expression of M. mongolica under the influence of M. aeruginosa. The molecular mechanism of M. mongolica in response to microcystin-induced stress was identified at the molecular level. The results of transcriptomics showed that that the toxic effects of M. aeruginosa on M. mongolica were attributed to the downregulation of key functional genes, such as uricase, ribosomal protein, estradiol dehydrogenase, and keratin-associated protein, (and related signaling pathways, such as apoptosis oxidative phosphorylation, amino and nucleoside sugar metabolism, and PPAR-signaling pathways). The findings presented by our research group provide a valuable contribution to the theoretical and practical knowledge base for the treatment of M. aeruginosa blooms using M. mongolica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse11030570/s1, Table S1: Changes in population growth parameters of M. mongolica under different food conditions after one week of feeding (statistical data are displayed as mean ± std); Table S2: Sequencing data quality evaluation statistics; Table S3: Spliced transcript length distribution; Table S4: Transcript function annotation statistics; Table S5: COG function classification.

Author Contributions

H.X. and R.J.: methodology, investigation, software, writing—original draft. H.X., R.J., T.C., H.L., Y.Y., J.Z., W.H. and P.H.: writing—review and editing. H.X., R.J., T.C., H.L., Y.Y., J.Z. and W.H.: formal analysis. T.C., C.T. and P.H.: project administration, funding acquisition, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Major Projects of Water Pollution Control and Management of China (2017ZX07205003), the National Sci-Tech Support Plan of China (2012BAC07B03), the Natural Science Foundation of Shanghai (21ZR1427400), the Evaluation of the Carbon Sink in the Coastal Sea of Shanghai (11N5500808182022401), the Shanghai Science and Technology Development Fund (20DZ2250700), Shanghai, China.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The GO annotations and KEGG pathway analysis of differentially expressed genes (DEGs). ((a) GO function classification map; (b) KEGG pathway classification map).
Figure 1. The GO annotations and KEGG pathway analysis of differentially expressed genes (DEGs). ((a) GO function classification map; (b) KEGG pathway classification map).
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Figure 2. GO classification of functional pathways ((a) up-regulated genes; (b) down-regulationgenes).
Figure 2. GO classification of functional pathways ((a) up-regulated genes; (b) down-regulationgenes).
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Figure 3. KEGG classification of significantly different proteins. (a) Upregulated pathway; (b) downregulated pathway.
Figure 3. KEGG classification of significantly different proteins. (a) Upregulated pathway; (b) downregulated pathway.
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Figure 4. qPCR analysis of 16 DEGs.
Figure 4. qPCR analysis of 16 DEGs.
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Table
Table 1. Feeding rates of M. mongolica at two age stages at different temperatures (data are listed as mean ± standard deviation; L and A represent larva and adult M. mongolica, respectively).
Table 1. Feeding rates of M. mongolica at two age stages at different temperatures (data are listed as mean ± standard deviation; L and A represent larva and adult M. mongolica, respectively).
TimeM. mongolica (103Cells/ind·h)
15 °C20 °C25 °C30 °C35 °C
LALALALALA
465.89 ± 9.1669.45 ± 9.9747.43 ± 5.2680.38 ± 10.4885.88 ± 0.44143.15 ± 14.07133.21 ± 5.24182.23 ± 5.3792.22 ± 2.56108.25 ± 4.35
841.14 ± 4.5532.16 ± 7.1631.98 ± 0.1833.74 ± 2.3149.70 ± 2.7271.25 ± 6.6938.68 ± 2.2387.38 ± 2.2139.12 ± 1.5842.22 ± 2.52
1227.16 ± 1.6911.82 ± 1.6941.04 ± 0.1952.41 ± 2.3047.00 ± 3.2058.80 ± 5.5427.24 ± 2.9364.27 ± 4.5426.44 ± 1.7263.26 ± 3.44
1613.56 ± 2.176.33 ± 1.2511.36 ± 1.1727.10 ± 1.3240.60 ±1.1045.87 ± 2.1324.93 ± 2.0849.80 ± 2.1524.56 ± 1.4343.58 ± 2.28
2011.44 ± 2.067.82 ± 2.122.48 ± 0.7615.94 ± 1.4722.99 ± 1.3129.59 ± 0.6217.94 ± 2.9933.38 ± 1.4416.17 ± 1.7333.17 ± 1.77
2419.55 ± 1.2613.03 ± 1.552.28 ± 0.7911.42 ± 2.8213.23 ± 1.2715.14 ± 1.4317.26 ± 1.5033.38 ± 1.2516.22 ± 1.3934.32 ± 1.13
Table
Table 2. Water filtration rates of M. mongolica at two age stages at different temperatures (data are listed as mean ± standard deviation).
Table 2. Water filtration rates of M. mongolica at two age stages at different temperatures (data are listed as mean ± standard deviation).
TimeM. mongolica
Larva M. mongolica (mL/ind·h)Adult M. mongolica (mL/ind·h)
15 °C20 °C25 °C30 °C35 °C15 °C20 °C25 °C30 °C35 °C
40.07 ± 0.010.05 ± 0.010.09 ± 0.000.10 ± 0.000.07 ± 0.000.07 ± 0.010.08 ± 0.010.15 ± 0.020.14 ± 0.000.08 ± 0.01
80.04 ± 0.000.04 ± 0.000.05 ± 0.000.02 ± 0.000.03 ± 0.000.03 ± 0.000.03 ± 0.000.07 ± 0.000.07 ± 0.000.04 ± 0.00
120.03 ± 0.000.04 ± 0.000.05 ± 0.000.02 ± 0.000.02 ± 0.000.01 ± 0.000.05 ± 0.000.06 ± 0.010.05 ± 0.000.05 ± 0.00
160.01 ± 0.000.01 ± 0.000.04 ± 0.000.02 ± 0.000.02 ± 0.000.01 ± 0.000.03 ± 0.000.05 ± 0.000.04 ± 0.000.04 ± 0.00
200.01 ± 0.000.00 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.03 ± 0.000.03 ± 0.000.04 ± 0.00
240.02 ± 0.000.00 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.03 ± 0.000.03 ± 0.00
Table
Table 3. Feeding rates of M. mongolica at two age stages under different light intensities (data are listed as the style of mean ± standard deviation, L and A represent larva and adult M. mongolica, respectively).
Table 3. Feeding rates of M. mongolica at two age stages under different light intensities (data are listed as the style of mean ± standard deviation, L and A represent larva and adult M. mongolica, respectively).
TimeM. mongolica (103 Cells/ind·h)
1000 lx3000 lx5000 lx7000 lx9000 lx
LALALALALA
478.90 ± 8.7897.58 ± 4.9085.88 ± 0.44143.15 ± 14.0722.29 ± 0.4763.13 ± 5.1343.37 ± 0.1550.58 ± 4.9755.35 ± 8.8436.91 ± 5.10
822.35 ± 0.0829.80 ± 2.5349.70 ± 2.7271.25 ± 6.7010.90 ± 0.0652.68 ± 5.0921.19 ± 0.0428.25 ± 2.5545.66 ± 2.6133.36 ± 2.39
1218.31 ± 2.8529.30 ± 2.6847.00 ± 3.2058.80 ± 5.5429.63 ± 4.4828.41 ± 2.7123.26 ± 3.5022.04 ± 5.2323.48 ± 1.5219.77 ± 1.55
Table
Table 4. Water filtration rates of M. mongolica at two age stages under different light intensities (data are listed as mean ± standard deviation).
Table 4. Water filtration rates of M. mongolica at two age stages under different light intensities (data are listed as mean ± standard deviation).
TimeM. mongolica
Larva M. mongolica (mL/ind·h)Adult M. mongolica (mL/ind·h)
1000 lx3000 lx5000 lx7000 lx9000 lx1000 lx3000 lx5000 lx7000 lx9000 lx
40.08 ± 0.010.09 ± 0.000.02 ± 0.000.04 ± 0.000.06 ± 0.000.10 ± 0.000.15 ± 0.000.07 ± 0.000.05 ± 0.000.04 ± 0.00
80.02 ± 0.000.05 ± 0.000.01 ± 0.000.02 ± 0.000.05 ± 0.000.03 ± 0.000.07 ± 0.010.05 ± 0.010.03 ± 0.000.03 ± 0.00
120.02 ± 0.000.05 ± 0.000.03 ± 0.000.02 ± 0.000.02 ± 0.000.03 ± 0.000.06 ± 0.010.03 ± 0.000.02 ± 0.010.02 ± 0.00
Table
Table 5. Feeding rates of M. mongolica at two age stages with different salinity conditions (data are listed as mean ± standard deviation).
Table 5. Feeding rates of M. mongolica at two age stages with different salinity conditions (data are listed as mean ± standard deviation).
TimeM. mongolica (103Cells/ind·h)
0‰2‰4‰6‰8‰10‰
LALALALALALA
488.18 ± 0.3284.49 ± 4.9560.01 ± 0.3064.00 ± 5.5062.92 ± 5.1751.81 ± 5.1948.26 ± 5.2059.31 ± 5.2451.90 ± 5.2444.48 ± 9.0814.83 ± 5.3118.52 ± 5.27
835.87 ± 2.5726.90 ± 0.0515.80 ± 2.7623.71 ± 0.1126.44 ± 0.0921.15 ± 0.0737.75 ± 4.2748.56 ± 4.5728.77 ± 2.4521.58 ± 0.075.35 ± 0.0114.25 ± 2.51
1224.16 ± 2.748.05 ± 1.599.00 ± 1.8019.29 ± 3.0816.83 ± 0.0324.68 ± 4.2230.52 ± 2.6841.83 ± 1.7140.70 ± 2.8941.82 ± 1.608.13 ± 1.629.29 ± 1.64
1623.51 ± 0.0712.19 ± 1.2316.60 ± 1.4411.71 ± 0.0515.22 ± 0.0414.37 ± 1.1619.48 ± 4.4828.12 ± 2.5421.97 ± 1.2923.72 ± 0.099.65 ± 1.227.90 ± 0.03
2015.81 ± 2.678.86 ± 1.3919.55 ± 1.578.52 ± 0.9210.99 ± 2.376.80 ± 1.3715.11 ± 0.8119.76 ± 1.8211.52 ± 0.8712.73 ± 0.0214.52 ± 1.848.27 ± 0.96
2413.77 ± 1.364.02 ± 0.819.58 ± 1.609.57 ± 1.536.76 ± 1.346.76 ± 0.0410.18 ± 1.3816.53 ± 0.7022.04 ± 1.3823.73 ± 1.395.66 ± 0.783.96 ± 0.79
Table
Table 6. Water filtration rates of M. mongolica at two age stages with different salinity conditions (data are listed as mean ± standard deviation).
Table 6. Water filtration rates of M. mongolica at two age stages with different salinity conditions (data are listed as mean ± standard deviation).
TimeM. mongolica
Larva M. mongolica (mL/ind·h)Adult M. mongolica (mL/ind·h)
0‰2‰4‰6‰8‰10‰0‰2‰4‰6‰8‰10‰
40.09 ± 0.000.07 ± 0.000.06 ± 0.010.05 ± 0.010.05 ± 0.010.02 ± 0.010.09 ± 0.000.07 ± 0.010.05 ± 0.010.06 ± 0.010.05 ± 0.010.02 ± 0.01
80.04 ± 0.000.02 ± 0.000.03 ± 0.000.04 ± 0.000.03 ± 0.000.00 ± 0.000.03 ± 0.000.03 ± 0.000.02 ± 0.000.05 ± 0.000.02 ± 0.000.01 ± 0.00
120.02 ± 0.000.01 ± 0.000.02 ± 0.000.03 ± 0.000.04 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.02 ± 0.000.04 ± 0.000.04 ± 0.000.01 ± 0.00
160.02 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.02 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.03 ± 0.000.02 ± 0.000.01 ± 0.00
200.02 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.02 ± 0.000.01 ± 0.00
240.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.00 ± 0.000.01 ± 0.000.01 ± 0.000.02 ± 0.000.02 ± 0.000.00 ± 0.00
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Xing, H.; Jiang, R.; Chen, T.; Liu, H.; Yin, Y.; Zhang, J.; He, W.; Tang, C.; He, P. Effects of Different Environmental Variables on the Ingestion of Microcystis aeruginosa by Moina mongolica. J. Mar. Sci. Eng. 2023, 11, 570. https://doi.org/10.3390/jmse11030570

AMA Style

Xing H, Jiang R, Chen T, Liu H, Yin Y, Zhang J, He W, Tang C, He P. Effects of Different Environmental Variables on the Ingestion of Microcystis aeruginosa by Moina mongolica. Journal of Marine Science and Engineering. 2023; 11(3):570. https://doi.org/10.3390/jmse11030570

Chicago/Turabian Style

Xing, Hao, Ruitong Jiang, Taoying Chen, Hongtao Liu, Yusu Yin, Jianheng Zhang, Wenhui He, Chunyu Tang, and Peimin He. 2023. "Effects of Different Environmental Variables on the Ingestion of Microcystis aeruginosa by Moina mongolica" Journal of Marine Science and Engineering 11, no. 3: 570. https://doi.org/10.3390/jmse11030570

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