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New biocontrol strategies are urgently needed to combat vector-borne infectious diseases. This study presents a low-cost method to produce a potential mosquito insecticide that utilizes the microalgae released into suburban water sources to control mosquito populations. Chlorella microalgae are ubiquitous in local waters, which were chosen as the host for genetic transfection. This species facilitated the recombinant algae to adapt to the prevailing environmental conditions with rapid growth and high relative abundance. The procedure involved microalgae RNAi-based insecticides developed using short hairpin RNAs targeting the Aedes aegypti chitin synthase A (chsa) gene in Chlorella. These insecticides effectively silenced the chsa gene, inhibiting Aedes metamorphosis in the laboratory and simulated-field trials. This study explored the impact of recombinant microalgae on the phytoplankton and zooplankton in suburban waters. High-throughput sequencing revealed that rapid reproduction of recombinant Chlorella indirectly caused the disappearance of some phytoplankton and reduced the protozoan species. This study demonstrated that a recombinant microalgae-based insecticide could effectively reduce the population of Aedes mosquitoes in the laboratory and simulated field trials. However, the impact of this technology on the environment and ecology requires further investigation.

The Aedes aegypti mosquito plays an important role in the spread of diseases, including epidemic ones, such as dengue fever, Zika virus disease, yellow fever, and chikungunya disease. To control the population of Ae.aegypti, we transferred an HR3 RNAi fragment into the microalgae Chlamydomonas, which serves as food for Ae.aegypti larvae. Results showed that the HR3 RNAi transgenic algal strains were lethal to Ae.aegypti. The integumentary system of larvae fed with HR3 RNAi transgenic algal strains was severely damaged. Muscles of the larvae were unevenly distributed and disordered, and their midgut showed disintegration of the intestinal cavity. RNA-Seq results demonstrated that on the 4th day of inoculation with the transgenic algae, the abundance of early expressed genes in the 20E signal transduction pathway of larvae fed with the HR3 RNAi transgenic algal strain significantly reduced. These genes include E74, E75, E93, and 20E receptor complex EcR/USP and FTZ-F1 gene regulated by HR3. In later experiments, a scale test of 300 Ae.aegypti eggs per group was carried out for 30 days, and the survival rate of Ae.aegypti fed with the HR3 RNAi transgenic strain was only 1.3%. These results indicate that the HR3 RNAi transgenic strain exerts obvious insecticidal effect.

E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.

Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.