Team:CIDEB-UANL Mexico/Safety-ProjectDocument

SAFETY ESSAY
The organism used in the project is E. Coli, which is part of the risk group with a biosafety level of one. This means that it’s unlikely to cause human or animal disease and, therefore, it is safe to use. The protein that is going to be synthetized is derived for beauveria bassiana and it also belongs to the risk group one. Like any micro-organism, B. bassiana has the potential to act as an opportunistic pathogen, but as the literary study demonstrates, Beauveria infections are extremely rare events. Anyway, there is no need to work directly with, B. bassiana because it is just an organisms from which the insecticidal part is derived.

There are also some potential risks for the team members while they are working in the laboratory. The laboratory used is a basic biosafety level one only used for basic teaching and research; it is an open bench work. The only risk in this laboratory is that the team members do not follow the security rules for every laboratory causing an accident. If the project result as it is supposed to do, it must not be of danger for any public, as long as the product is not ingested. If it is ingested, E. Coli may cause diarrhea and a little fever depending the patient.

About the environmental impact, the bacteria is modified in order to cause the death of plague insects, this means that the population of the plague will be reduced, affecting in one or another way, a change in the environment. The insecticide may also affect some species of the same family but not all, it is difficult to predict its impact and danger because of the lack of information about this new insecticide.

The risk of an accident inside the laboratory depends of the level of care that the team members have during the practices. The biological parts and organisms represent a risk if they enter in contact with a body. We are working with E. coli strains that are specifically weakened for laboratory use. On the other hand, there are some equipment and chemicals that represent a danger if they are not used correctly. Some chemicals used in laboratory represent a safety risk so all members of the team were told to handle them with extreme care. The same goes for the lab equipment; some cannot be used without authorization.

We are addressing these issues in our project design and lab work by taking precautions and using safety equipment in order to prevent an accident. Every member of the team is aware of the risk that means work in the laboratory. Each one know about the security rules during a laboratory practice because we have had biosafety training before the real practices even started. On it we learned about the correct procedures to carry out any experiment; and also about some security precautions referring to the material that we were going to use. When using ethidium bromide (risks include: irritant to eyes, skin, mucus, and respiratory tract) we make sure the designated research zone is properly closed and we use the necessary protection. When observing stained DNA, through the use of UV rays, we use special protection for the eyes. While managing electrical devices and circuits, all members of the team were told to handle them with extreme care. This training was in order to prevent accidents and for the success of the project.

Environment Safety of the VIP3
Vip3 proteins are original from the bacteria Bacillus thuringiensis. Vip proteins have pesticide properties against some plagues from the family of the lepidopteron. This document seeks to make a collection of information and data relevant to the overall assessment of the environmental hazard of the protein Vip3Ca3 when it is produced in genetically modified organisms as E.Coli and the GM corn plant.

Bacillus thuringiensis is a soil bacterium although is found throughout the environment. The protein pesticides produced by B. thuringiensis showing a great variety with regard to the mode of action, the specificity of the objective and the mechanism of expression. Pesticide proteins expressed by the strains of B. thuringiensis include anti-fungal compounds, δ-exotoxinas,3 and δ-endotoxins, which include the protein Cry and Cyt, that are not structurally related but come from the same bacterium. Cry proteins are so named because it is stored as parasporals crystals during the formation of the spore, unlike the new protein that was produced by B. thuringiensis during their vegetative phase of growth, in addition to during the stage of sporulation, so that was called vegetative insecticidal protein (Vip). In addition, while the Cry proteins are isolated as crystals, the Vip proteins are secreted by bacteria and can be isolated directly from the culture medium.

It has been determined that, in reality, there are several variants of Vip which are classified into three classes according to the similarity of the aminoacid sequence: Vip1, Vip2 and Vip3. Of these, the group whose insecticidal properties are more efficient is the Vip3. The members of the Vip3 family characterized to date exhibit activity against lepidopterans, and several of them do not compete with Cry proteins for binding sites. They are classified into two subfamilies (Vip3A and Vip3B), and some are especially toxic for species with little susceptibility to several Cry proteins. All of these features have made Vips a research target for broadening the host-range of B. thuringiensis-based biopesticides and for the management of insect resistance to B. thuringiensis proteins.

Relation between Vip3A and Vip3C
The taxonomic group of proteins that Vip is currently used in, for the production of GM crop plants resistant to insects, is Vip3Aa. Vip3Aa1 may vary in size from 62 to 66 kDa among different Vip3 proteins and is occasionally known as the “trypsin-resistant core". Also, Vip3C proteins maintained only one of the three residues of the C terminus stabilizing domain described for Vip3Aa1 (5). This higher divergence toward the C terminus might indicate lower functional constraints and, consequently, more permissibility to non-synonymous substitutions.

For a preliminary screening of the insecticidal properties of Vip3Ca (host range and toxicity), Vip3Ca3 was picked because of its high yields, expressed in Escherichia coli, column purified, quantified by densitometry, and used to challenge larvae of 10 different lepidopteran species. Bioassays were conducted with neonate larvae that were placed over a surface-contaminated artificial diet. It has been given considerable attention to the possible impacts of the proteins of B. thuringiensis in the Monarch Butterfly (Danaus plexippus), which is not a lepidopter pest in North America. Studies have shown that the proteins Vip3Aa are not toxic to this specie of butterfly. However, the differences between the Vip3A and the Vip3C are such that it is not possible to determine exactly the toxicity of the pesticide for the butterfly without making tests.

Preliminary bioassays of larvae from 10 different lepidopteron species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 g/cm2. How the few changes in the Vip3Ca proteolytic processing sites may modify either the protein activity or the host range is uncertain, but different Vip3 proteins have shown very distinct insecticidal properties. For example, two of the species tested showed very low susceptibility to Vip3Ca3. One of them was Ostrinia nubilalis, for which no toxic effects of any of the Vip3Aa proteins assayed so far have been reported. The other one was L. botrana, which has been challenged with Vip3 for the first time.

Mechanism of the insecticidal activity of Vip3
A prerequisite for the insecticidal activity of Vip3Aa is an enzymatic proteolysis that is found in the intestine of the insect. Curiously, the Vip3 proteins are processed by the fluids in the intestine of the insect and arrive to the active form regardless of if the insect is susceptible to the toxin. Once the Vip3 protein is found in its active form, it joins the vesicular membrane of the mucosa of the midget of susceptible species. The site of union is not the same site in the different types of Vip3 and connection to the intestine is correlated with toxicity.

After the union, Vip3 causes paralysis of the bowel, followed by the lysis of the epithelial cells of the intestine, allegedly because of an interference of the transmembrane potential, resulting in the death of cells. The activated protein can form transmembrane pores, and it is considered that these pores contribute to lysis and death of the epithelial cells of the medium intestine.

Probes in non-target organism (NTO)
There have been probes of some Vip3 proteins to determine their environmental damage, specifically to see if they are a risk against not pest organisms. Only the Vip3Aa have been used in GM plants and based on the information recovered, it is possible to make inferences about the general effects of Vip3, but remembering that the properties change in different branches of Vip. The proteins Vip3Aa have insecticidal properties against lepidopteran insect, and when used in genetically modified crops, the proteins are aimed at lepidopterans pests of insects to reduce the damage food. They are called non-target organism (NTO) to the agencies of the environment, directly or indirectly exposed to proteins Vip, which are not pests in the agricultural system.

Direct exposure occurs when the braids are fed of tissues of live cultures or crop wastes that are on the ground or on the floor. The indirect exposure occurs when an organism feeds on another agency that has consumed tissues of plants that contain proteins Vip3. Have been submitted the data to show that the much exposed to the protein Vip3Aa in the environment, either directly or indirectly, they do not suffer damage. Regulatory decisions have been derived from the broad history of the use of insecticides for microbes formulations of B. thuringiensis, as well as the collected data from field tests of GM crops that produce one of the proteins Vip3Aa. These data have established that the proteins Vip3Aa are specifically active against the subgroup of Lepidoptera pests that consume the crop and that they are safe to vertebrate species and other NTO. However, because the experiment was realized with with Vip3Aa is could be a different behavior of Vip3Ca3.

Environmental exposure pathways
Regulatory decisions, generally, take into account three main routes of exposure in addition to the direct contact with the plant or bacteria that expresses one of the Vip3 protein: exposure to pollen contains Vip3, exposure to the Vip3 deposited on the ground by the material object in decomposition, and exposure tritrofic through herbivores that feed on the plant or bacteria with the protein.

In some countries like the United States, require data related to the longevity of the Bt proteins in the soil, and the data suggest that Vip3Aa degrades quickly once freed of the plant tissue in decomposition, which is not likely to persist or accumulate in the soil environment.Regulatory authorities have considered the potential impact of the protein Vip3Aa in natural populations of braids and determined that the adverse effects on are unlikely in NTO for various reasons. At first, the proteins Vip3Aa have a narrow spectrum of pesticide activity but there is not information that confirms the same occur with all Vip3 proteins. Second, in studies of level I has been also demonstrated that proteins Vip3Aa have no observable effect on aquatic vertebrate species nor representative. Fourth, the levels of Vip3Aa used in these tests of level I were much higher than those measured in the production a plants and bacteria.

In addition when compared with the control of insects through Vip3, the usual insect control, which uses chemical pesticides, alters the diversity of species in a meaningful way and damage species NTO. But it is not prudent to forget the large time effects that Vip3 can have in immediately depredators of the plagues.

Conclusion
Vegetative insecticidal proteins (Vip) are secret able proteins from Bacillus thuringiensis which do not share sequence homology with known Cry proteins and display insecticidal activity against a wide variety of lepidopterans and coleopterans and some sap-sucking insect pests. Field studies suggest that the cultivation of plants and other producers that express Vip3 does not affect the abundance of non-target arthropods, with the possible exception of the specific predators of pests. Together, these findings indicate that it is unlikely that the proteins Vip3Aa will have adverse effects in natural populations of organisms, except for the crop pest lepidopterans, and probably there would be similar results in general for similar Vip3 proteins.

1.Revisión de la seguridad ambiental de Vip3Aa. Center for Environmental Risk Assessment, ILSI Research Foundation. July 15th, 2012.
2.Vip3, a novel class of vegetative insecticidal proteins from Bacillus Thuringiensis. Instituto de Agrobiotacnología CSIC-UPNA, Spain, 2012.
3.Ministerio de agricultura, alimentación y medioambiente.
4.http://www.glfc.forestry.ca/bacillus/BtSearch.cfm(The Bacillus thuringiensis toxin specificity database)
5.http://cfs.nrcan.gc.na/projects/119/2(The Bacillus thuringiensis toxin project)