Development of Entomotoxic Maize for Biological Control of Mosquitoes

Executive Summary: BSF - 2007037 Development of Entomotoxic Maize for Biological Control of Mosquitoes Robert Shatters, Charles Powel and Dov Borovsky (USDA, Fort Pierce, FL, USA), Arieh Zaritsky and Eitan Ben-Dov (BGU, Be’er-Sheva, Israel) A. Synergy between Cry toxins and TMOF Starved 1st instar Aedes aegypti larvae are 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis (Bti) toxins than fed larvae. TMOF (Trypsin Modulating Oostatic Factor) is synergistic to Bti Cry and Cyt toxins: larval death was enhanced when fed on a mixture of Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for Ae. Aegypti’s TMOF) with Escherichia coli cells expressing cry4Aa, cry11Aa, cyt1Aa and p20.

Codon-optimized synthetic genes, cry11-tmfA and gst-cry11-tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae, caused 50% and 87.5% mortality in 8 and 12 days respectively. GST (glutathione-S-transferase) enhances the activity of Cry11A-TMOF and protects it from heat denaturation. Cell free extracts of recombinant P. pastoris cells killed 40% of tested 4th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa and TMOF. The combined efforts of our laboratories demonstrate for the first time that Cry toxins and TMOF are synergistic to larval Ae. aegypti.

B. Trasnsformation of maize and biological testing Zea maize plantlets were transformed with the following gene combinations: cry11Aa-tmfA, cry4Aa-tmfA and cyt1Aa-tmfA. The genes were incorporated as single, double or triple genes. Pollen of mature plants were used to self-pollinate in order to select for homogeneous plants. newly hatched Ae. Aegypti larvae were fed singly by the pollen at different concentrations. Seeds of plants with pollen that caused less than 25% mortality were stored. Self-pollinated plants that caused high larval mortalities (60-80%) were propagated. Plant numbers 13-1, 29-1, 76-1 and 24-3, causing 60% - 80% mortalities (Table 1) are currently grown in the green house.

Expression of cry11Aa, cry4Aa, cytAa and tmfA was quantified in leaf DNA and tassel mRNA. These genes are driven by a pollen specific promoter that expresses proteins only in the pollen. RT-qPCR showed that several plants that had been transformed with 3 genes (detected in the leaves) expressed double or single transcripts of these genes in the tassels.

After 4 years of active, intense research our funds ran out. Seeds of several promising plants that efficiently kill mosquito larvae were stored. More work is needed to finish and conclude this important project and we hope to secure more funds from other sources to be able to (a) select homogeneous lines that express robust transcript of the 3 Bti and TMOF transcripts in the pollen, (b) cross plants that produce a single or two genes so that all the useful plants will express all the 3 Bti genes and TMOF together, (c) analyze the pollen protein by mass spectrometry for the expected genes spectra, and (d) assay the plants for larval mortality in the green house.

During the grant period, several delays happened due to the fact that Professor Borovsky retired from the University of Florida and could not start working at the USDA for a year.

C. Publications * Borovsky D, Khasdan V, Nauwelaers S, Theunis C, Bertier L, Ben-Dov E, Zaritsky A. 2010. Synergy between Bacillus thuringiensis subsp. israelensis Crystal Proteins and Aedes aegypti Trypsin Modulating Oostatic Factor. The Open Toxinol J 3: 141-150 * Zaritsky A, Ben-Dov E, Borovsky D, Boussiba S, Einav M, Gindin G, Horowitz R, Kollot M, Melnikov O, Mendel Z, Yagil E. 2010. Transgenic Organisms Expressing Genes from Bacillus thuringiensis to Combat Insect Pests. Bioeng Bugs 1: 341-344 * Shatters RG Jr, Borovsky D, Zaritsky A, Powell CA, Ben-Dov E. 2013. Maize Pollen to Control Mosquitoes. Ecological Impact of Genetically Modified Organisms. IOBC WPRS Bull, in press * Shatters RG Jr, Borovsky D, Zaritsky A, Powell CA, Ben-Dov E, et al. 2013/14. Pollen of transgenic Zea Mays expressing combination of genes toxic to larvae of Aedes aegypti. in preparation * Several additional articles were published that acknowledge the grant, but authored by the Israeli team only, they will therefore not be fully recorded here.

D. Inventions disclosures: * "Control of Mosquito Larvae with Bti Toxins and TMOF". Inventors: Dov Borovsky, University of Florida, USA; Arieh Zaritsky and Eitan Ben-Dov, Ben-Gurion University of the Negev, Israel. Submitted (number UF.778XPCT) by the University of Florida to the USA patent office.

* "Control of Insects with Entomotoxic Pollen". Inventors: Dov Borovsky, University of Florida, USA; Andrew Spielman and Richard Pollack, Harvard University, USA; Arieh Zaritsky and Eitan Ben-Dov, Ben-Gurion University of the Negev, Israel. Disclosure is in preparation by the University of Florida patent lawyer for submission to the USA patent office.

E. Conference / Meetings / Lectures: 1. Borovsky, D. Second International Conference on Biotechnology for the Wellness Industry. "Potential Control of Malaria using Bioengineered Maize Pollen Expressing TMOF and Bti Toxins; A Simple Holistic Approach". July 23rd-26th, 2009.

2. Borovsky, D. Malaysian Ministry of Health by invitation by the deputy secretary of Health. Control of mosquitoes and dengue with a combined Bti-TMOF yeast cells. July 30th, 2009, Kuala Lumpur, Malaysia.

3. Zaritsky, A. The first, plenary lecture at the 7th Pacific rim Conference on the Biotechnology of Bacillus thuringieneis and its Environmental Impact, held in New Delhi, November 25-28, 2009. Lecture Title: "Transgenic Organisms Expressing Genes from Bacillus thuringiensis to Combat Insect Pests".

4. Zaritsky, A. Transgenic Organisms Expressing Genes from Bacillus thuringiensis to Combat Insect Pests. Invited Lecture, the Instituto de Biotecnologia UNAM, Cuernavaca Morelos, Mexico, November 22, 2010

5. Zaritsky et al., Two Posters, presented at the FEMS Conference, Geneva, Switzerland, June, 2011

6. Zaritsky et al., A Lecture: Maize Pollen to Control Mosquitoes. Presented at the “Ecological Impact of Genetically Modified Organisms”. IOBC WPRS, Berlin, June 2013