Controlling the development of the dengue vector Aedes aegypti using HR3 RNAi transgenic Chlamydomonas

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.

aegypti larvae. The growth and death of the larva and the observation of its anatomy and histomorphology were analyzed. Here is the report.

Algal strains and culture conditions
The C. reinhardtii strain CC425 (cw15 arg2) was grown in TAP medium supplemented with 250 μg mL -1 arginine [17,18]. Liquid cultures were grown under continuous light of 150 μmol m -2 s -1 at 25˚C under shaking conditions at 200 rpm. Strains on TAP-agar plates were incubated at a light intensity of 100 μmol m -2 s -1 at 22 C [19].

Bioinformatics analysis
The HR3 protein domains were determined from the Ae.aegypti HR3 protein sequence downloaded from The Aedes aegypti genomics database (https://www.vectorbase.org/organisms/ aedes-aegypti). The homology and otherlogy protein sequences of HR3 were analyzed for recognizable domains using the NCBI Batch conserved domain search tool (http://www.ncbi. nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Domains were also verified using the HMMERbased SMART website (http://smart.embl-heidelberg.de) and PFAM website (http://pfam. sanger.ac.uk/). A schematic diagram of the protein domain structures with functional domains was constructed using DOG 2.0 (Domain Graph, version 2.0) (http://dog.biocuckoo.org/) [20]. Protein alignment and phylogenetic analysis were aligned in the multiple sequence alignment tool CLUSTALX2.0 by using Gonnet Protein Weight Matrix with default parameters [21]. A phylogenetic tree was constructed by the Neighbor-Joining algorithm using MEGA 6.0. The bootstrap consensus tree was inferred from 1000 replicates.

Construction of pMaa7 IR/HR3IR, an RNAi expression vector of the HR3 gene
The cDNA from the total RNA of Ae.aegypti was used as the template, and the HR3 RNAi fragment was amplified using HR3RNAIF (ATTTGCGCTAACATGCTATCG) and HR3RNAIR (CAGCCATTTCAAGTTCACTACG) as primers. The amplified fragment was inserted into pMD18-T to obtain pMD-HR3. The plasmid was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. Then, the fragments were digested by HindIII/BamHIand XbaI/SalI and subsequently inserted into the corresponding cloning sites of plasmid T282 to acquire T282-HR3. Both RNAi vector of pMaa7 IR/XIR and intermediate vector of T282-HR3 were digested by EcoRI and then ligated to produce the RNAi recombinant pMaa7 IR/HR3IR [22].

Algae transformation
The cells used for transformation (C. reinhardtii strain CC425 [cw15 arg2]) were grown to a density of 1-2 × 10 6 cells mL -1 , and the constructs were introduced into the cells by using the glass bead method [23]. The cells were collected through centrifugation and then washed twice before their resuspension in the TAP medium without arginine to a cell density of approximately 1 × 10 8 cells mL -1 . Exactly 2 μg of plasmid DNA and 400 μL of cells were mixed with 100 μL of 20% polyethylene glycol and 300 mg of sterile glass beads. After the mixture was vortexed for 15 s, the cells were washed from the glass beads and subsequently plated on TAP agar with 1.5 mM L-tryptophan, 5 μg paromomycin ml-1, and 5 μM 5-fluoroindole [24]. To test the positive tranformants algea with HR3, we use the primers GATTTAAATGCCAGAAGGAG and CGCGGTGAGCACCGGAACGG for PCR amplification.

Breeding and biological detection of Ae.aegypti mosquitoes
Fresh water was added to a large Petri dish in an incubator at a temperature of 26˚C and a humidity of 75%, and a small amount of food was added. After 8 h, the residual feed that was not dissolved in water was sucked out. The filter paper containing the mosquito eggs was placed in water, and the hatched larvae were observed after 2 h of incubation. The oil film on the surface of the incubating water was scraped off on time every day. When the larvae became pupae, they were sucked out into a paper cup filled with clean water and then transferred to a mosquito cage. When the mosquito pupae developed into adult mosquitoes, they were fed sugar water.
The experimental design includes experimental groups and multiple control groups. Each group contains 10 larvae hatched from eggs. The larvae fed with the HR3 RNAi transgenic algal strains were set as the experimental groups, whereas the larvae fed with non-transgenic C. reinhardtii CC425 and fodder were used as controls. Trails were repeated three times. The mortality of the larvae was recorded every day. The surviving larvae were fed continuously until they became pupae and developed into mosquitoes. The length and pupation time of the larvae were recorded. Some larvae were embedded in paraffin, sectioned, stained, and then examined by light microscopy.
In the large-scale feeding experiment, 300 larvae hatched from eggs were selected for each treatment group and control group. The duration of the experiment is 30 days. The larvae fed with the transgenic strains HR3-C9 was set as the experimental group, whereas the larvae fed with non-transgenic C. reinhardtii CC425 and fodder were used as controls. The survival rate of larvae and adults mosquito were recorded.

Treatment of RNA-Seq samples of Larvae
C. reinhardtii CC425 and pMaa7IR/HR3IR transgenic strain HR3-C9 were inoculated into 50 mL of TAP liquid medium and shaken to the middle of the logarithmic growth phase. The same amount of algae was fed to Ae.aegypti larva every day. On the 4th and 6th days after feeding, the larvae were collected by centrifugation and frozen rapidly in liquid nitrogen. Total RNA of larvae was extracted, and the purity of the RNA was analyzed to meet the requirements of RNA-Seq sequencing. RNA-Seq was conducted by Illumina HiSeqTM 2500, and the results were plotted as heat maps by alignment statistics and gene differential expression analysis.

HR3 gene expression detction of Aedae larvae
The endogenous gene expression of HR3 levels was detected by isolating total RNA from 3rd instar larva that were collected 10-20, and performing Real time PCR. Expression levels of HR3 were determined as compared to the endogenous control, Ae.aegypti RPS17(Ribo ribosomal protein S17, GenBank accession no. AAEL004175 (KY000705)). LarvaRNA was isolated using the TRIzol Reagent (Takara). Single-strand cDNA was synthesized from total RNA using oligo-dT primers in Bio-Rad Iscript-selected cDNA synthesis kit. Real-time PCR was performed on a BioRad iCycler iQ Real-Time PCR Detection System using SYBR Green as a fluorescent dye. Primers for RPS17 amplification are AAGAAGTGGCCATCATTCCA and GGTCTCCGGGTCGACTTC. The primer sequences for HR3 gene quantification are ATTTGCGCTAACATGCTATCG and CAGCCATTTCAAGTTCACTACG. The amplification rate of each transcript (Ct) was calculated by the PCR baseline subtracted method performed in the iCycler software at a constant fluorescence level. Cts was determined over three repeats. Relative fold differences were calculated using the relative quantification analytical method (2 -

Statistical analyses
Data are presented as mean ± S.D. Duncan's multiple range tests and T-test were performed to examine significant differences between means. In all cases, differences showing P < 0.05 were considered significant.

Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of this paper.

Ethical approval
The laboratory research was conducted at the Chinese Academy of Tropical Agricultural Science. All methods met the ethical requirements of the institute, and followed guidelines of the Committee of Publication Ethics.

Research involving human participants and/or animals
This article does not involve any studies with human participants or vertebrate animals.

Bioinformatics analysis results
The open reading frame of the Ae.aegypti HR3 gene (GenBank accession number: AF230281, Ae.aegypti database: AAEL009588-PA) is 1407 bp in length, encoding 468 amino acid residues, and its 5 0 non-coding region is 54 bp in length. It contains two C4-type zinc fingers with orphan receptors and hormone receptor functions. Compared with HR3 homologous genes in other insects, as shown in Fig 1, the HR3 protein of Ae.aegypti contains a DNA-binding domain at the N-terminal end and a ligand-binding domain at the C-terminal end. Mega6 cluster analysis showed that the HR3 protein sequence of Ae.aegypti has the highest homology (99%) with A. albopictus (XP_019549827), followed by Anopheles sinensis (KFB44899), Papilio machaon (XP_014364014), and Papilio polytes (XP_013136657). However, the homology was the lowest (71%) with Zeugodacus cucurbitae (XP_011182274) (Fig 2).

pMaa7IR/HR3IR transgenic algal strains are lethal to Ae.aegypti
Reverse transcription of the total RNA from Ae.aegypti was used as a template to amplify the HR3 RNAi interference fragment, and a band of about 362 bp was obtained. This fragment was cloned into pMD18-T to obtain pMD-HR3. Sequencing results showed that the cloned HR3 fragment shared 100% homology with the Ae.aegypti HR3 gene. After the recombinant RNAi vector of pMaa7IR/HR3IR was transformed into C. reinhardtii CC425, 38 transgenic algal strains were identified as positive and used for further studies.
When fed with the pMaa7IR/HR3IR transgenic algal strain, almost all the larvae bit their anal fistula in the early stage before death. Their bodies were floating on the water surface and trembling with active movements. In the late stage, their bodies were inactive and eventually sank into the water. The negative control group (larvae fed with non-transgenic C. reinhardtii CC425 and fodder) had stable growth with no biting and active movement (Fig 3).
The length of the 4th-instar larvae were tested. The treated group fed with pMaa7IR/HR3IR transgenic strain HR3-C7 was 4259 ± 66 μm. However, the controls including group fed with fodder was 7876±78 μm, fed with non-transgenic C. reinhardtii CC425 was 8689 ± 81 μm ( Fig  4). The larvae fed with pMaa7IR/HR3IR transgenic strain had a 51.1% reduction in body  length compared with the larvae fed with C. reinhardtii CC425. These results indicate that feeding the larvae with the HR3 RNAi transgenic algal strain largely affected their growth.
Under the same feeding conditions, compared with control groups (fed with water, fodder, and C. reinhardtii CC425), the larvae fed with pMaa7IR/HR3IR transgenic algae died as early as the 2nd day. All tested larvae fed with transgenic algal strain HR3-C9 or HR3-D8 died on the 3rd day. Except for HR3-C6, all the other larvae fed with pMaa7IR/HR3IR transgenic algae died on the 10th day at the latest. This result indicates that the pMaa7IR/HR3IR genetically engineered algae exert a serious lethal effect on Ae.aegypti (Fig 5).

HR3 mRNA levels in Ae.aegypti larvae fed with HR3 RNAi transgenic alage were significantly reduced
To detect the expression of the HR3 gene in Ae.aegypti larvae fed with transgenic engineered algae, we tested the cells by real-time PCR on the 6th day of larval incubation using Ae.aegypti fed with C. reinhardtii CC425 as a control. Compared with that of the control, the HR3 gene expression level of the Ae.aegypti larvae fed with the pMaa7IR/HR3IR transgenic engineered strain decreased significantly (Fig 6). The expression of HR3 in the larvae fed with HR3-B7 decreased by 99.7% compared with the control. The HR3 expression of the larvae fed with HR3-B6 and HR3-C9 decreased by 99.5% and 99.2%, respectively. The HR3 expression of the other larvae fed with HR3-B3, HR3-C3, and HR3-C7 decreased by 82.6%, 97.6%, and 91.1%, respectively. The above results show that the engineered algal strains can effectively silence the HR3 gene in Ae.aegypti.

Epidermis, muscle, and midgut tissues of larvae fed with HR3 RNAi transgenic algae were significantly damaged
To understand whether the tissues of Ae.aegypti larvae fed with HR3 RNAi transgenic algae show pathological changes that might cause subsequent death, we prepared paraffin sections of the larvae and performed microscopic observation. The anatomical structure of Ae.aegypti larvae consists of an epidermis of the head and body wall, a digestive system, a reproductive system, and a respiratory system. Tissue sections were observed under a stereomicroscope. Results showed that the larvae fed with non-transgenic C. reinhardtii CC425 had intact epidermis, thick muscles, uniform muscle distribution, and clear lines. Their midgut was normal and intestinal lumen was intact ( Fig  7A-7C). In contrast, The integumentary system of the larvae fed with HR3 RNAi transgenic algal strains was severely damaged and Their brush edge disappeared. Muscles of them were unevenly distributed, disordered, and some had even dissolved. The abnormal morphology of their midgut were characterized by disintegration of the intestinal cavity and vacuolation of some cells. Moreover, the striate edge was detached (Fig 7E and 7F).

RNA-Seq detection of metabolic pathway changes in Ae.aegypti larvae fed with HR3 RNAi transgenic algae
The larval RNA on the 4th and 6th days after fed with the transgenic algal strain was extracted and subjected to RNA-Seq. On day 4, the larvae fed with the pMaa7IR/HR3IR strain expressed 988 upregulated genes, 2282 downregulated genes, and 9696 genes with no significant difference. On day 6, 775 genes were upregulated, 709 genes were downregulated, and 11,831 genes showed no significant difference. The expression of some genes involved in the metabolism of xenobiotics by the cytochrome P450 pathway, oxidative phosphorylation pathway, DNA replication, and RNA degradation pathway increased significantly. These genes include ① GST, GSTK1, HPGDS, and UGT in the metabolism of xenobiotics by the cytochrome P450 pathway; ② PCNC of DNA polymerase B, ε1 subunit of DNA polymerase ε and Mcm2, 3, 4, 5, 7, RFA2, 4, and RPA3 of the MCM complex in DNA replication; and ③ Ndufa1, Ndufb4, Ndufb11, Ndufb12, and Ndufs4 of DADH dehydrogenase and COX5A, COX6A, COX6B, COX7C, and COX11 of cytochrome C oxidase in the oxidative phosphorylation pathway. This result may be attributed to the toxic effect of the transgenic algal strain, resulting in a significant increase in gene expression in toxic degradation, DNA replication, and energy synthesis after the larvae were fed with the engineered algae.
After the HR3 gene in the larvae was silenced by feeding with the pMaa7IR/HR3IR transgenic engineered algal strain (Fig 6), HR3 lost its regulation of downstream genes, which affected the metamorphic growth and formation of larvae, thus causing their death. The results of RNA-Seq showed that on the 4th day of incubation, the abundance of early expressed genes in the 20E signal transduction pathway of larvae fed with the pMaa7IR/HR3IR transgenic algal strain significantly reduced, including E74, E75, E93, and 20E receptor complex EcR/USP and FTZ-F1 gene regulated by HR3. In addition, the expression of late-stage genes, such as HR4 and KR-H1, decreased. On the 6th day of incubation, the mRNA abundance of E74, E75, EcR/ USP, HR4, FTZ-F1, and KR-H1 decreased (Fig 8). We speculate that these genes are positively correlated with HR3 silencing.
Silencing of the HR3 gene in Ae.aegypti larvae also affects the anabolism of ecdysone. RNA-Seq results showed that the expression levels of genes associated with ecdysone E20 synthesis, such as CYP306A1, CYP302A1, and CYP315A1, increased significantly on the 4 th day of incubation. However, the downstream gene HR3 in the 20E signal transduction pathway was blocked. Thus, despite the high accumulation of 20E in vivo, it could not regulate the downstream functional gene expression by normal signal transduction, which resulted in delayed development and normal pupation. The normal pupation time of Ae.aegypti larvae is 6-8 days. Feeding with the pMaa7IR/HR3IR transgenic engineered algae can delay the larvae pupation by period up to 30 days, resulting in the majority not being pupated.
Given that silencing HR3 blocks the delivery of 20E, the synthesis of the 20E precursor, cholesterol, and its derivatives significantly increased in the steroid biosynthesis pathway. Fig 8 shows that the expression of key genes for the synthesis of cholesterol and its derivatives in the steroid biosynthesis pathway, such as SC5DL, ERG3, STE1, and DWF1, increased significantly on day 4.

The HR3 RNAi transgenic algae exerts obvious insecticidal effect
A scale test of 300 Ae.aegypti eggs per group was carried out for 30 days, and the data of biological observations were statistically analyzed. The data of larval hatching are as follows. In the control group fed with fodder, 292 eggs hatched. The larvae began to pupate on the 4th day, and all larvae completely pupated on the 8th day. In another control group fed with non-GM algae CC425, 295 eggs hatched. The larvae began to pupate on the 4th day, and all larvae pupated completely on the 13th day. In the experimental group fed with the transgenic pMaa7IR/HR3IR strain, 268 eggs hatched. The larvae began to pupate on the 4th day, and all larvae pupated completely on the 28th day ( Fig 9A). The survival rate of adult mosquitoes was as follows. The larvae fed with fodder completely developed into adults on the 12th day, and 244 adult mosquitoes survived on the 30th day, with a rate of 81.3%. On the 16th day, the larvae fed with CC425, a non-GM algal strain, completely developed into adults. On the 30th day, 270 mosquitoes survived, with a rate of 90%. The larvae fed with the transgenic pMaa7IR/HR3IR strain were completely developed into adults on the 24th day, and four mosquitoes survived on the 30th day. The survival rate was 1.3%, accompanied by uninterrupted death of larvae and adults. These results suggest that the transgenic pMaa7IR/HR3IR strain exerts obvious insecticidal effect (Fig 9B).

Discussion
The use of biotechnology to control mosquitoes instead of traditional chemical pesticides is the future direction of vector mosquito control. At present, the use of Wolbachia infection and gene splicing male infertility techniques are relatively successful cases [26][27][28][29][30][31][32][33]. However, their disadvantage is that wild female mosquitoes prefer to mate with wild male mosquitoes in comparison to a Wolbachia bacterial infection male Aedes or a male Aedes that has been transferred with a lethal gene. The transgenic male sterile Aedes, code-named OX513A, which transgenic offspring have a survival rate of 15%-18% in outdoor after eating animal food, dog food, and cat food containing tetracycline residues [34][35][36]. This phenomenon undoubtedly poses a potential safety hazard for genetically modified organisms. Moreover, the obstacle to using Wolbachia's mosquito-control technology is that Wolbachia has mutual transmission (horizontal transmission) in other insects, although reports of Wolbachia bacteria infecting mammals and humans are lacking [37][38][39]. Wolbachia not only spreads among the individuals of Trichogramma but also enables cross-species transmission between parasitic wasps and Drosophila. Whether Wolbachia can be transmitted to other organisms in the environment through mosquitoes, including predators of mosquitoes, is not conclusive.
Microalgae are a natural food of Ae.aegypti larvae and widely found in nature. At present, microalgae such as Chlorella, Spirulina, and Chlamydomonas can be industrialized and produced on a large scale. Compared with transgenic Aedes and Aedes infected by Wolbachia, the cost of production of transgenic microalgae is low and the technology is well-developed. It can be placed in a closed area for a long time to form dominant algae species, thereby gradually eliminating Aedes in corresponding areas. This method provides a new concept and method for using bio-insecticides to block the prevalence of malignant infectious diseases, such as dengue fever, Zika virus disease, and yellow fever.
In this study, 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 algae was lethal to Ae.aegypti larvae. The epidermis of larvae was severely damaged. Their muscles of the larvae were unevenly distributed and disordered, and their midgut showed disintegrated. HR3 is located in the ecdysone 20E signal transduction pathway. By regulating FTZ-F1, it can regulate E74 and E75, thereby regulating the pupation of Aedes mosquitoes (Fig 10). If the expression of HR3 is blocked, it will affect the expression of FTZ-F1, E74, E75 genes, which will affect the pupation of larvae and cause the death of larvae. The RNA-Seq test results showed that the expression of E74, E75, FTZ-F1 genes was greatly reduced due to the silence of HR3. The results of a large-scale experiment of 300 larvae in this study showed that 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. This result indicates that HR3 RNAi transgenic Algae strains have commercial application prospects. In the future, we will conduct in-depth research on the survival of algae strains in environmental waters, the impact on ecology and other organisms, and genetic safety.