Integrated Analyses of miRNome and Transcriptome Reveal the Critical Role of miRNAs Toward Heat Stress Response in Isochrysis galbana.

Isochrysis galbana is widely used in aquaculture as a bait microalgal species. High temperature (HT) can severely impair the development of I. galbana, exerting adverse effects on its yield. MicroRNAs (miRNAs) play an essential role in modulating stress-responsive genes. However, the role of miRNAs in response to HT in microalgae remains largely unexplored. In the present study, we identified several conserved and novel miRNAs in I. galbana through miRNome sequencing. Among these identified miRNAs, 22 miRNAs were differentially expressed in response to heat stress, and their target genes were predicted accordingly. Moreover, a comprehensive and integrated analysis of miRNome and transcriptome was performed. We found that six potential reversely correlated differentially expressed miRNA (DEM) and differentially expressed gene (DEG) pairs were associated with heat stress response (HSR) in I. galbana. The expressions of DEMs and DEGs were further verified using real-time quantitative PCR (RT-qPCR). Integrated analyses showed that miRNAs played fundamental roles in the regulatory network of HSR in I. galbana mainly by regulating some heat-responsive genes, including heat shock proteins (HSPs), reactive oxygen species (ROS) signaling-related genes, and specific key genes in the ubiquitination pathway. Our current study identified the first set of heat-responsive miRNAs from I. galbana and helped elucidate the miRNA-mediated HSR and resistance mechanisms in I. galbana. This new knowledge could provide ways to enhance its heat stress tolerance.

RNA-seq Insights Into the Impact of Alteromonas macleodii on Isochrysis galbana.

Phycospheric bacteria may be the key biological factors affecting the growth of algae. However, the studies about interaction between Isochrysis galbana and its phycospheric bacteria are limited. Here, we show that a marine heterotrophic bacterium, Alteromonas macleodii, enhanced the growth of I. galbana, and inhibited non-photochemical quenching (NPQ) and superoxide dismutase (SOD) activities of this microalgae. Further, we explored this phenomenon via examining how the entire transcriptomes of I. galbana changed when it was co-cultured with A. macleodii. Notable increase was observed in transcripts related to photosynthesis, carbon fixation, oxidative phosphorylation, ribosomal proteins, biosynthetic enzymes, and transport processes of I. galbana in the presence of A. macleodii, suggesting the introduction of the bacterium might have introduced increased production and transport of carbon compounds and other types of biomolecules. Besides, the transcriptome changed largely corresponded to reduced stress conditions for I. galbana, as inferred from the depletion of transcripts encoding DNA repair enzymes, superoxide dismutase (SOD) and other stress-response proteins. Taken together, the presence of A. macleodii mainly enhanced photosynthesis and biosynthesis of I. galbana and protected it from stress, especially oxidative stress. Transfer of fixed organic carbon, but perhaps other types of biomolecules, between the autotroph and the heterotroph might happen in I. galbana-A. macleodii co-culture. The present work provides novel insights into the transcriptional consequences of I. galbana of mutualism with its heterotrophic bacterial partner, and mutually beneficial associations existing in I. galbana-A. macleodii might be explored to improve productivity and sustainability of aquaculture algal rearing systems.

Molecular method for rapid detection of the red tide dinoflagellate Karenia mikimotoi in the coastal region of Xiangshan Bay, China.

The species Karenia mikimotoi is a common nearshore red tide alga that can secrete hemolytic exotoxin and ichthyotoxin, which can induce the death of fish and shellfish, causing severe economic losses. In this study, loop-mediated isothermal amplification (LAMP) was employed in combination with the lateral flow dipstick (LFD) visual detection method to establish the LAMP-LFD rapid detection method for K. mikimotoi. The internal transcribed spacer ITS1-5.8S-ITS2 of K. mikimotoi was used as the target sequence and was amplified with specific primers designed in this study. The results indicated that the amplification optimal reaction conditions for LAMP in this paper were for 20 min at 65 °C. Moreover, LAMP had excellent specificity, showing negative results for other common red tide causing algal species. In field samples, we successfully reduced the total time, with only 23 min needed from LAMP amplification to LFD result display, which was shorter than that of conventional PCR. Consequently, LAMP-LFD should be useful for rapid field detection of low-density K. mikimotoi and for the early prevention of red tide induced by such algae.

[Effects of Different Concentrations of Ammonia Nitrogen on the Growth and Enzyme Activity of Four Common Algae Strains].

Treating swine wastewater with a high ammonia nitrogen content with microalgae cultures has proved difficult. In this paper, the strains Chlamydomonas 715, Botryococcus braunii 357, Porphyridium cruentum 806, and Scenedesmus obliquus 417 were tested. Ammonia nitrogen concentrations of 50 mg·L-1, 500 mg·L-1, and 2000 mg·L-1 applied to the media according to the concentrations of biogas slurry. This allowed the effect of different concentrations of ammonia nitrogen on the growth and cell enzyme activity of microalgae to be tested. The results showed that the growth of Chlamydomonas 715 and Scenedesmus obliquus 417 was inhibited at different concentrations of ammonia nitrogen, and the biomass and biomass productivities were lower than for the normal media. However, the biomass and biomass productivity of Porphyridium cruentum 806 in 50 mg·L-1 ammonia nitrogen were 1.78 g·L-1 and 0.16 g·(L·d)-1, respectively, which were higher than the values obtained using KOCK medium. Furthermore, the biomass and biomass productivity of Botryococcus braunii 357 in 500 mg·L-1 ammonia nitrogen were 1.95 g·L-1 and 0.18 g·(L·d)-1, respectively, which were higher than the values obtained using BG11 medium. The SOD, POD, and CAT of all algae species showed a decreasing tendency in response to an increase in the concentration of ammonia nitrogen, as did MDA. These results provide a theoretical basis for the treatment of swine wastewater with high ammonia nitrogen content using microalgae cultures.

Bacterial Community Diversity and Screening of Growth-Affecting Bacteria From Isochrysis galbana Following Antibiotic Treatment.

Algal cultures are generally co-cultures of algae and bacteria, especially when considering outdoor cultivation. However, the effects of associated bacteria on algal growth remain largely unexplored, particularly in the context of Isochrysis galbana. In the present study, we investigated the effects of antibiotic on the growth of I. galbana and its associated bacterial community. We found advantageous responses of I. galbana to antibiotic exposure, evidenced by the increased growth, and the maximal photochemical efficiency of PSII (Fv/Fm). Since antibiotics can cause major disturbances within bacterial community, we further conducted 16S rDNA amplicon sequencing to determine the changes of bacterial community diversity following antibiotic treatment. We found that antibiotic treatment considerably and negatively affected the abundance and diversity of bacterial community, and 17 significantly decreased bacterial species in the antibiotic-treated medium, including Pseudomonas stutzeri, were identified. Further co-culture experiments revealed that P. stutzeri inhibited the growth of I. galbana, and the inhibitory activity was retained in the cell-free bacterial filtrate. These results indicated that the negative effect of bacteria was not exclusively transmitted through contact with I. galbana but could be also mediated via secretory compounds. Taken together, our findings not only fully characterized the bacterial community associated with I. galbana and how the bacterial community changed in response to antibiotic perturbations, but also provided a valuable information about the interactions between I. galbana and its associated bacteria, which might help improve the yield, and quality of I. galbana during its cultivation processes.

Distribution of Karlodinium veneficum in the coastal region of Xiangshan Bay in the East China Sea, as detected by a real-time quantitative PCR assay of ribosomal ITS sequence.

Athecate dinoflagellate Karlodinium veneficum is a universal toxic species possessing karlotoxins recognized especially as ichthyotoxic as well as cytotoxic and hemolytic. Blooms of K. veneficum, both single-species or accompanied with other species, occurred more frequently worldwide in recent years, including the coastal region of China. Normally, K. veneficum present in relatively low abundance in phytoplankton communities in estuary regions. Being small and difficult to identify with light microscopy, it has been ignored for a long time till its blooming and toxins being confirmed. How it presents in background level and what is its relationship with critical geological and hydrological environment factors are basically not clear. In this study, the paper reports the application of a real-time quantitative PCR (qPCR) method to investigate the abundance and distribution of K. veneficum in the coastal waters of Xiangshan Bay in the East China Sea (ECS), a typical bay area of harmful algae blooms and heavily affected by anthropogenic activities. The real-time qPCR assay came out being an efficient method at detecting even low cell densities of K. veneficum of different genotypes. A total of 38 field samples of surface (0.5 m) and bottom water (9-100 m in depth) were analyzed and 12 samples were found positive for K. veneficum. At least 3 genotypes of K. veneficum present in this region. Temperatures in sites of K. veneficum positive ranged from 21.7 to 23.4 °C, and salinity levels were between 21.1 and 26.3. The K. veneficum distributed quite extensively in the waters of Xiangshan Bay, cell abundance varied from a low of 4 cells/L to a maximum of 170 cells/L. Most of the samples containing K. veneficum were collected from bottom water in different sites. At three of the 19 sampling sites, K. veneficum was detected in both surface and bottom water samples. Especially at sampling site near Beilun port, where the water is typically muddy with low transparency, relative high cell numbers of K. veneficum were found in both surface and bottom waters. Mixotrophy and vertical migration of K. veneficum could be important eco-physiological factors to consider in terms of understanding these distribution characteristics. The ideal conditions for K. veneficum growth and aggregation in this area still needs further study.

Detection of Skeletonema costatum based on loop-mediated isothermal amplification combined with lateral flow dipstick.

We developed a new assay method, which combines loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) for the rapid and special detection of the diatom Skeletonema costatum. Four groups of LAMP primers were derived from a conserved DNA sequence unique to S. costatum. The amplifications were carried out at 61, 63, and 65 °C for 60 min in various combinations by the quantitative PCR thermal cycler to confirm optimal primers and reaction temperature. The LAMP-LFD detection limit was 0.94 pg/µL of S. costatum genomic DNA and was 100 times more sensitive than conventional PCR. The LAMP-LFD method had high specificity and accurately identified S. costatum algal isolates, but not other algal isolates. The new LAMP-LFD assay can be used as a reliable and easy method to detect S. costatum.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Karlodinium veneficum.

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4pg/µL (approximately 6.5cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assay's accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum.

[Model study on CO2 removal by photobioreactor].

The key point of study on CO2 removal by microalgae cultured in a photobioreactor is to improve CO2 removal capability. In this paper, a model of air-lift photobioreactor was developed by combination of conditions including the velocity of flow, the degree of mixing, the gas-liquid mass transfer and the rate of photosynthesis, and two corresponding simplified methods, such as time discretization and lumped parameters were put forward. Using a method of lumped parameters, the model for simulation of time course of DO, pH in the column air-lift photobioreactor and prediction of CO2, O2 concentrations in the outlet gas under different CO2 concentration in the aeration gas was thoroughly discussed. Experimental data were also used to verify the model which could potentially be applied to rational design of the photobioreactor, high-density culture of microalgae and efficient removal of CO2.