CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE

Abstract

The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh.

Toward mosquito control with a green alga: expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas

“…Whether or not the RNAi will effect non-target species, or if the mosquitoes will develop resistance to RNAi are not known. A marine chlorophyte, Chlorella dessicata , was stably engineered to produce a regulatory peptide (trypsin modulating oostatic factor) that represses trypsin in the larval gut (Borovsky et al 2016). The transgenic Chlorella strains were lethal to A. aegypti larvae; however, the assays had extraordinarily high algal cell numbers (3 × 10 8 cells mL -1 ).…”

Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control

“…The procedure was repeated until no radioactivity was detected. The hybridization procedure and the washings were repeated as mentioned above and the X-ray film was exposed for 24 to 72 h at −80 • C (Borovsky et al, 2016).…”

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas

“…However, the elucidation of the Bti toxin at the molecular level has enabled an approach whereby the toxin could be delivered intracellularly (for protection) in a microbial host that grows in larval habitats [ 12 , 13 , 14 , 15 , 16 ]. Although such an organism has not yet been widely employed, there is still considerable interest in this approach [ 17 , 18 , 19 ].…”

“…Many microalgae are known to be important food sources for mosquito larvae [ 20 , 21 , 22 ], and a few cyanobacteria ( Agmenellum , Anabaena , Synechococcus ) and two eukaryotic green algae ( Chlamydomonas and Chlorella ) have been targeted for genetic modification to make them useful for larval control [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 23 ]. Our approach is to develop strains of Chlamydomonas reinhardtii ( Chlamydomonas ) that express genes in the chloroplast that are based on the Bti toxin.…”