Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystin synthetase (mcy) gene clusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers and TaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Effects of microcystin on protein profile in hepatopancreas of Litopenaeus vannamei.

For intensive aquaculture in freshwater ponds, microcystin (MC-LR) generated from cyanobacterial blooms is one of the bottlenecks for the healthy and sustainable development of shrimp aquaculture industry. In this study, we measured the MC-LR content in the hepatopancreas and muscles of Litopenaeus vannamei stressed by MC-LR, and analyzed protein expression in the hepatopancreas using DIA high-throughput proteomics technology. The results showed that MC-LR content in the hepatopancreas and muscles reached the highest at 1 h after MC-LR injection, which was (6.12±0.45) µg·kg-1 and (5.00±0.19) µg·kg-1, respectively. Then, it decreased gra-dually, with that in the hepatopancreas being significantly higher than in muscles. We identified 820 differential expressed proteins, including 586 up-regulated and 234 down-regulated ones. Results of bioinformatics analysis showed that MC-LR stress significantly affected immune-related pathways such as lysosome, RIG-Ⅰ receptor signals and interleukin-2. It also altered energy metabolisms including citrate cycle, metabolism of starch and sucrose, and interconversion of pentose and glucoronate, which in turn led to the disorder of carbohydrate metabolism. In addition, MC-LR significantly upregulated 19 cytoskeleton-related blood shadow proteins and damaged the hepatopancreas cytoskeleton. It was concluded that MC-LR mainly affected the physiological processes associated with immunity, energy metabolism, and cytoskeleton in the hepatopancreas of L. vannamei.

Integrated analysis of mRNA and microRNA expression profiles in hepatopancreas of Litopenaeus vannamei under acute exposure to MC-LR.

Intensive shrimp farming is often threatened by microcystins Hepatopancreas is the primary target organ of MCs in shrimp. To investigate the response of hepatopancreas to acute MC-LR exposure, the expression profiles of RNA-seq and miRNA-seq in the hepatopancreas of L. vannamei were determined, and data integration analysis was performed at 72 h after MC-LR injection. The expression of 5 DEGs and three DEMs were detected by Quantitative PCR (qPCR). The results showed that the cumulative mortality rate of shrimp in MC-LR treatment group was 41.1%. A total of 1229 differentially expressed genes (844 up- and 385 down-regulated) and 86 differentially expressed miRNAs (40 up- and 46 down-regulated) were identified after MC-LR exposure. Functional analysis indicated that DEGs is mainly involved in the oxidative activity process in molecular functional categories, and proteasome was the most enriched KEGG pathway for mRNAs profile. According to the functional annotation of target genes of DEMs, protein binding was the most important term in the GO category, and protein processing in endoplasmic reticulum (ER) was the most enriched KEGG pathway. The regulatory network of miRNAs and DEGs involved in the pathway related to protein degradation in endoplasmic reticulum was constructed, and miR-181-5p regulated many genes in this pathway. The results of qPCR showed that there were significant differences in the expression of five DEGs and three DEMs, which might play an important role in the toxicity and hepatopancreas detoxification of MC-LR in shrimp. The results revealed that MC-LR exposure affected the degradation pathway of misfolded protein in ER of L. vannamei hepatopancreas, and miR-181-5p might play an important role in the effect of MC-LR on the degradation pathway of misfolded protein.

The role of attached bacteria in the formation of Microcystis colony in Chentaizi River.

To further understand the role of attached bacteria in the formation of Microcystis colonies, we conducted a field investigation in Chentaizi River in Tianjin, China, which frequently suffers Microcystis blooms in summer. The results showed the average cell density of Microcystis was 2.31 × 107 cell/L from July 19 to July 27, 2021. Free-living and attached bacteria communities shared similar phylum diversity, but the abundance changed obviously. The colony size of Microcystis and attached bacterial number in the colony showed an increasing trend during the whole sampling period. There was a significant positive correlation between Microcystis colonial size and attached bacterial density (P < 0.01), indicating attached bacteria could contribute the colony formation of Microcystis. The genus composition in attached bacterial community varied in colonies of different sizes. The relative abundance of Acinetobacter, Cloacibacterium, Sphingobacterium, and Ralstonia in >90 µm colonies were significantly higher than those in 8-20 µm and 20-90 µm colonies (P < 0.05). These genera might have positive effects on the colony formation of Microcystis during sampling.

Light limitation inducing overcompensatory growth of cyanobacteria and function of serine/threonine kinase (STK) genes involved.

The rapid overcompensatory growth that appears when cyanobacteria are supplied with adequate resources after a period of resource deprivation might contribute to the occurrence of cyanobacterial blooms. We investigated the changing characteristics of overcompensatory growth and serine/threonine kinase (STK) genes expression of cyanobacterium Microcystis aeruginosa in response to light limitation. The results showed M. aeruginosa exhibited overcompensatory growth for 2 days after light recovery, during which the increase in growth was inversely related to light intensity. Expression of STK genes, such as spkD, was upregulated significantly at 0.5-4 h after light recovery (P < 0.05). To investigate the function of STK genes in the overcompensatory growth, M. aeruginosa spkD was heterologously expressed in Synechocystis. Transgenic Synechocystis exhibited greater and longer overcompensatory growth than wild-type Synechocystis after light recovery. Relative expression levels of STK genes in transgenic Synechocystis were significantly higher than those in wild-type Synechocystis at 24 h of light recovery (P < 0.05). Heterologous expression of Microcystis spkD might stimulate overcompensatory growth of Synechocystis by affecting its STK gene expression.

Comparing effects of berberine on the growth and photosynthetic activities of Microcystis aeruginosa and Chlorella pyrenoidosa.

Berberine is a potent algicidal allelochemical of Microcystis aeruginosa. To optimize its application in the control of Microcystis blooms, the effects of berberine on the growth and photosynthetic activities of M. aeruginosa and a non-target green alga, Chlorella pyrenoidosa, were compared. The results showed that the algicidal activity of berberine on M. aeruginosa was light dependent. Berberine had no algicidal effects on C. pyrenoidosa with or without light exposure. Under light-dark conditions, berberine significantly decreased the chlorophyll fluorescence parameters in M. aeruginosa while no significant berberine-induced changes were observed under constant darkness. Significant reductions of photosystem II (PSII) and whole chain electron transport activities in M. aeruginosa exposed to berberine suggested that PSII was the important target site attacked by berberine. Contrary to M. aeruginosa, no berberine-induced inhibition in photosynthesis activities were observed in C. pyrenoidosa. The differences in photosynthetic apparatuses of these two algae might be responsible for their different sensitivities to berberine.

Microcystins distribution, bioaccumulation, and Microcystis genotype succession in a fish culture pond.

In freshwater aquaculture ponds, cyanobacterial blooms and microcystins (MCs) pollution have attracted considerable attention due to their toxic effects. To provide an insight into cyanobacterial problems in aquaculture ponds, MCs distribution, bioaccumulation, and Microcystis genotype succession in a fishpond were investigated from May 2017 to November 2017. The distribution of MCs in filtered water, seston, and sediment varied considerably among months. MCs concentrations in filtered water, seston, and sediment ranged from 1.16 to 3.66 µg/L, 0.64 to 13.98 µg/g DW, and 1.34 to 5.90 µg/g DW, respectively. In addition, chemical oxygen demand was positively correlated with sestonic MCs concentrations. MCs concentrations accumulated in different tissues of market-size fish were in the order of liver > kidney > intestine > muscle. MCs content in muscle was 4.3 times higher than the WHO recommended tolerable daily intake level. Twenty-four ITS genotypes of Microcystis were identified from a total of 653 sequences. During the survey period, considerable genotype variation and rapid genotype succession were observed and dominant genotype was absent. A redundancy analysis revealed that Microcystis genotypes could significantly influence the variations in the proportions of the potentially toxic Microcystis, which could in turn influence the MCs concentrations in seston.

Effects of Bacillus subtilis on the growth, colony maintenance, and attached bacterial community composition of colonial cyanobacteria.

In freshwater aquaculture ponds, application of algicidal Bacillus is a promising way in the control of cyanobacterial blooms. To best understand Bacillus algicidal characters and mechanisms in the field, different-sized colonial cyanobacteria were isolated from an aquaculture pond, and the effects of B. subtilis on their growth, colony maintenance, and colony-attached bacterial community composition were investigated. The results showed that B. subtilis could inhibit the growth of colonial cyanobacteria. Bigger-sized colonies isolated from the field could spontaneously disintegrate into smaller-sized colonies in the laboratory. Algicidal B. subtilis could accelerate the disintegration of colonies and decrease colony size. B. subtilis not only decreased the colony-attached bacterial community diversity but also changed its composition. B. subtilis increased the relative abundances of some attached bacterial genera, including Pseudomonas, Shewanella, Bacillus, Shinella, Rhizobium, and Ensifer. These bacteria with algicidal, microcystin-degrading, and flocculating activities might be an important contributor to algicidal effects of B. subtilis on colonial cyanobacteria.

Effects of toxic Microcystis genotypes on natural colony formation and mechanism involved.

The cyanobacterium Microcystis occurs as colonies of different sizes with varying abundance of toxic genotypes versus non-toxic genotypes under natural conditions. To investigate the effects of toxic Microcystis genotypes on natural colony formation, samples collected from the mainstream of Haihe River from July to October 2015 were sieved into four colony classes with sizes of <8 µm, 8-20 µm, 20-90 µm and >90 µm. Each colony size class was analyzed for the proportion of toxic Microcystis genotypes, and microcystins (MCs) cellular production of toxic genotypes. The results showed the smallest size class of Microcystis colonies (<8 µm) showed the lowest proportion of toxic genotypes and the highest MC-RR and MC-YR cellular production. With the increasing colony sizes, the proportion of toxic Microcystis genotypes increased but the MC-RR and MC-YR cellular production decreased. A negative correlation between the MCs cellular production and the proportion of toxic genotypes was observed in all four colony size classes, suggesting that the less there were toxic Microcystis cells able to produce MCs, the more each toxic cell needed to produce that molecule. Toxic Microcystis played an important role in the colony formation in natural waters via producing MCs.

Effect of environmental factors on allelopathic inhibition of Microcystis aeruginosa by berberine.

To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.

Biochemical responses of the copepod Centropages tenuiremis to CO(2)-driven acidified seawater.

An ecophysiological experiment was conducted to examine the biochemical effects of acidified seawater containing elevated concentration of CO(2) (C(CO2) 0.08, 0.20, 0.50 and 1.00%) on the copepod Centropages tenuiremis. AchE, ATPase, SOD, GPx, GST, GSH level and GSH/GSSG ratio of the copepod were analyzed. The results showed that elevated C(CO2) and the duration of culture time significantly influenced several biochemical indices in C. tenuiremis (ATPase, GPx, GST, GSH and SOD). Furthermore, the principal component analysis results indicated that 72.32% of the overall variance was explained by the first three principal components (GPx, SOD and GSH). Changes in GPx and GSH levels may play a significant role in the antioxidant defense of copepods against seawater acidification. The long-term response of copepods to seawater acidification and the synergistic effects of acidification with other environmental factors, such as temperature, salinity and trace metal need further investigation.