Microalgae are often used as nutritional supplements for aquatic animals and are widely used in the aquaculture industry, providing direct or indirect nutrients for many aquatic animals. Microalgae are abundant in nature, of high nutritional value, and some of them are non-toxic and rich in antioxidants so that they can be explored as a medicinal carrier for human or animals. Natural wild-type microalgae can be adopted as an immunostimulant to enhance non-specific immune response and improve growth performance, among which Haematococcus pluvialis, Arthrospira (Spirulina) platensis, and Chlorella spp. are commonly used. At present, there have been some successful cases of using microalgae to develop oral vaccines in the aquaculture industry. Researchers usually develop recombinant vaccines based on Chlamydomonas reinhardtii, Dunaliella salina, and cyanobacteria. Among them, in the genetic modification of eukaryotic microalgae, many examples are expressing antigen genes in chloroplasts. They are all used for the prevention and control of single infectious diseases and most of them are resistant to shrimp virus infection. However, there is still no effective strategy targeting polymicrobial infections and few commercial vaccines are available. Although several species of microalgae are widely developed in the aquaculture industry, many of them have not yet established an effective and mature genetic manipulation system. This article systematically analyzes and discusses the above problems to provide ideas for the future development of highly effective microalgae recombinant oral vaccines.

Background: Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine. Results: Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p<0.05). Among those, functional classifications were found in the Gene Ontology database for 3891 and in the Kyoto Encyclopedia of Genes and Genomes database for 3467. Several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of WED immunization. Further qPCR analysis confirmed that the genes encoding the factors involved in major histocompatibility complex (MHC)-I processing pathway were up-regulated, while those involved in MHC-II pathway were down-regulated. Conclusion: These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts.

Microalgae are cosmopolitan organisms in nature with short life cycles, playing a tremendous role in reducing the pressure of industrial carbon emissions. Besides, microalgae have the unique advantages of being photoautotrophic and harboring both prokaryotic and eukaryotic expression systems, becoming a popular host for recombinant proteins. Currently, numerous advanced molecular tools related to microalgal transgenesis have been explored and established, especially for the model species Chlamydomonas reinhardtii (C. reinhardtii hereafter). The development of genetic tools and the emergence of new strategies further increase the feasibility of developing C. reinhardtii chloroplasts as green factories, and the strong genetic operability of C. reinhardtii endows it with enormous potential as a synthetic biology platform. At present, C. reinhardtii chloroplasts could successfully produce plenty of recombinant proteins, including antigens, antibodies, antimicrobial peptides, protein hormones and enzymes. However, additional techniques and toolkits for chloroplasts need to be developed to achieve efficient and markerless editing of plastid genomes. Mining novel genetic elements and selectable markers will be more intensively studied in the future, and more factors affecting protein expression are urged to be explored. This review focuses on the latest technological progress of selectable markers for Chlamydomonas chloroplast genetic engineering and the factors that affect the efficiency of chloroplast protein expression. Furthermore, urgent challenges and prospects for future development are pointed out.