Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton

Abstract

Background: More than half of the world's population suffers from epidemic diseases that are spread by mosquitoes. The primary strategy used to stop the spread of mosquito-borne diseases is vector control. Interference RNA (RNAi) is a powerful tool for controlling insect populations and may be less susceptible to insect resistance than other strategies. However, public concerns have been raised because of the transfer of antibiotic resistance marker genes to environmental microorganisms after integration into the recipient genome, thus allowing the pathogen to acquire resistance. Therefore, in the present study, we modified the 3-hydroxykynurenine transaminase (3hkt) and hormone receptor 3 (hr3) RNAi vectors to remove antibiotic resistance marker genes and retain the expression cassette of the inverse repeat sequence of the 3hkt/hr3 target gene. This recombinant microalgal marker-free RNAi insecticide was subsequently added to the suburban water in a simulated-field trial to test its ability to control mosquito population.

Methods: The expression cassette of the 3hkt/hr3 inverted repeat sequence and a DNA fragment of the argininosuccinate lyase gene without the ampicillin resistance gene were obtained using restriction enzyme digestion and recovery. After the cotransformation of Chlamydomonas, the recombinant algae was then employed to feed Aedes albopictus larvae. Ten and 300 larvae were used in small- and large-scale laboratory Ae.albopictus feeding trials, respectively. Simulated field trials were conducted using Meishe River water that was complemented with recombinant Chlamydomonas. Moreover, the impact of recombinant microalgae on phytoplankton and zooplankton in the released water was explored via high-throughput sequencing.

Results: The marker-free RNAi-recombinant Chlamydomonas effectively silenced the 3hkt/hr3 target gene, resulting in the inhibition of Ae. albopictus development and also in the high rate of Ae. albopictus larvae mortality in the laboratory and simulated field trials. In addition, the results confirmed that the effect of recombinant Chlamydomonas on plankton in the released water was similar to that of the nontransgenic Chlamydomonas, which could reduce the abundance and species of plankton.

Conclusions: The marker-free RNAi-recombinant Chlamydomonas are highly lethal to the Ae. albopictus mosquito, and their effect on plankton in released water is similar to that of the nontransgenic algal strains, which reduces the abundance and species of plankton. Thus, marker-free recombinant Chlamydomonas can be used for mosquito biorational control and mosquito-borne disease prevention.

Keywords: Aedes albopictus; Chlamydomonas; High-throughput sequencing; Marker-free RNA interference; Plankton.

Fig. 1

Schematic diagram of co-transformation strategy. pMaa7 IR/HR3IR and pMaa7 IR/3HKTIR were digested with XhoI, and the expression cassettes containing RbcS Promoter and HR3/3HKT inverted repeats were recovered by agarose gel electrophoresis. The plasmid pUC-Arg7-lox-B containing the genomic ARG7.8 gene was digested with EcoRV, and the fragment containing the argininosuccinate lyase (asl) gene was recovered. Through co-transformation, Chlamydomonas cells were plated on TAP agar without arginine until the algal colonies appeared 5–10 days later

Fig. 2

Aedes albopictus mortality (A, B), pupation (C, D) and eclosion rate (E, F) when fed recombinant Chlamydomonas. Water: water is fed to the larvae; CC48: larvae fed C. reinhardtii CC48; fodder: larvae fed fodder; 3HKT-1 to 3HKT-4: larvae fed with 3HKT RNAi expression cassette co-transformation Chlamydomonas strains 3HKT-1 to 3HKT-4; HR3-1 to HR3-4: larvae fed with HR3 RNAi expression cassette co-transformation Chlamydomonas strains HR3-1 to HR3-4. HR3-D1 and 3HKT-D3: larvae fed with inactive dry powder of recombinant Chlamydomonas HR3-1 and 3HKT-3, respectively. The experiment was done three times, and the average values are presented. Each treated and control group contained ten Aedes larvae. Time frame: 15 days

Fig. 3

Length of larvae. The length of L3 larvae from each treatment was measured. Water: water is fed to the larvae; CC48: larvae fed C. reinhardtii CC48; fodder: larvae fed fodder; 3HKT-1 to 3HKT-4: larvae fed with 3HKT RNAi expression cassette co-transformation Chlamydomonas strains 3HKT-1 to 3HKT-4; HR3-1 to HR3-4: larvae fed with HR3 RNAi expression cassette co-transformation Chlamydomonas strains HR3-1 to HR3-4. HR3-D1 and 3HKT-D3: larvae fed with inactive dry powder of recombinant Chlamydomonas HR3-1 and 3HKT-3, respectively. Data are expressed as mean ± SD (n = 3), and significant differences (P < 0.05, Duncan’s multiple range tests) are shown by different letters

Fig. 4

Mortality (A), pupation (B) and eclosion rate (C) of Ae. albopictus fed with the recombinant Chlamydomonas and the relative 3HK/HR3 mRNA levels in Aedes L4 larvae fed with recombinant Chlamydomonas (D). Water: water is fed to the larvae; CC48: larvae fed C. reinhardtii CC48; fodder: larvae fed fodder; 3HKT-3: larvae fed with 3HKT RNAi expression cassette co-transformation Chlamydomonas strains 3HKT-3; HR3-1: larvae fed with HR3 RNAi expression cassette co-transformation Chlamydomonas strains HR3-1. Re-Chlamy: larvae fed with recombinant Chlamydomonas

Fig. 5

Simulated field tests in a Haikou neighborhood. A and B Meishe River was used for the test water. C 700 l Meishe River water and 100 l Chlamydomonas were combined in the barrel. D and E Each cage contained about 1000 pupae, and every week the number of adult mosquitoes was counted. F Aedes albopictus population and survival rates in MSH, CC48, 3HKT and HR3 treatments. MSH: During this treatment, mosquitoes exclusively drank water from the Meishe River. 3HKT and HR3: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with recombinant Chlamydomonas 3HKT-3 and HR3-1, respectively. CC48: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with C. reinhardtii CC48

Fig. 6

Variation in the class level of phytoplankton (A) and zooplankton (B) relative abundance in test waters from MSH, CC48, 3HKT and HR3. MSH: During this treatment, mosquitoes exclusively drank water from the Meishe River. 3HKT and HR3: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with recombinant Chlamydomonas 3HKT-3 and HR3-1, respectively. CC48: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with C. reinhardtii CC48

Fig. 7

Variations in the relative abundance of phytoplankton (A) and zooplankton (B) species at the genus level in test waters from MSH, CC48, 3HKT and HR3. MSH: During this treatment, mosquitoes exclusively drank water from the Meishe River. 3HKT and HR3: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with recombinant Chlamydomonas 3HKT-3 and HR3-1, respectively. CC48: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with C. reinhardtii CC48

Fig. 8

In test waters from MSH, CC48, 3HKT and HR3, phytoplankton (A) and zooplankton (B) were analyzed using a heat map. Sample names are indicated in the heat map’s horizontal ordinate at the bottom, and distinct phytoplankton or zooplankton classes are indicated in the ordinate on the right side. When values fall below the mean, the heat map’s hue is negative; when they rise, it is positive. The standard score (Z-values), which is represented by the color scale in the top right corner, is equal to (x − µ)/σ, where x represents the relative abundance of a particular plankton group. The average relative abundance of all plankton groups is known as μ. The standard deviation of relative abundance for all plankton groups is σ. For hierarchical clustering, the Bray-Curtis distance was determined using R software. MSH: During this treatment, mosquitoes exclusively drank water from the Meishe River. 3HKT and HR3: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with recombinant Chlamydomonas 3HKT-3 and HR3-1, respectively. CC48: In this treatment, mosquitoes were kept in water from the Meishe River that had been supplemented with C. reinhardtii CC48