By David McKalip, M.D.*
Risk: The OX513A bugs could establish a new subspecies and actually survive in nature.
Implications: The FDA thinks the risk of this is low, but bases their conclusions on some assumptions that are not entirely accurate. However if the survival rate, percentage of females released or genetic assumptions are wrong, then the GMO mosquito could establish itself permanently.
Introduction to Part II.
The FDA has approved a genetic experiment on humanity and the ecosystem in Key West Florida. They propose to release genetically Modified Organisms (GMO), mosquitos, into the wild to see if they can control the Aedes mosquito population and decrease transmission of disease. The Sunbeam Times continues its series on the GMO Mosquito, known as OX513A by evaluating the risk of the new mosquito establishing itself in nature and the potential consequences. The FDA believes this risk is low, but they have based it on faulty assumptions and some deceptive statistics used by Oxitec to obtain FDA approval. In Particular, Oxitec claims that the GMO mosquito offspring will die quickly, when in fact a large percent can live weeks and breed more than once.
The Genetics of the GMO Mosquito – Ox 513A
The Oxitec Mosquito was made in a lab and modified the genes of the Aedes subspecies it created so that the offspring of the mosquito would die quickly. The Aedes aegyptii mosquito transmits multiple diseases (Zika, Dengue, Yellow Fever and Chikungunya) and the Zika scare is renewing dramatic efforts to kill the mosquitos. Oxitec owns the OX513A Mosquito that has been modified in two major ways using multiple genes. First, the GMO mosquito has a red fluorescence gene inserted in it so it can be identified under special lights in the laboratory for further study after recovery from the wild. Secondly it contains a so-called “lethal trait”, which is a gene for a protein called a TAV (Transcription Activator Variant). The DNA inserted into the mosquito will cause the offspring to produce the TAV protein which shuts off reading of the DNA by the cells of the mosquitos. That means the mosquito will not produce the other proteins it needs to develop and will die. The TAV gene was cut out of a Herpes Simplex Virus by scientists in 1992 (a gene called VP16 – Virion Protein 16). Transcription activators are normally occurring proteins that allow our DNA to be decoded (transcribed) into RNA which is then read by the cell (translated) to make proteins. However the TAV in the Oxitec mosquito is designed to overproduce itself and take up all the other sites for transcription of proteins so that the needed proteins are not produced, leading to death. To keep the mosquitoes alive in the lab so that they can be released into the wild, the gene also includes a tetracycline sequence. Thus Oxitec can feed tetracycline to the mosquitoes during production in the lab and keep them alive long enough to be introduced into the wild. Thus the lethal trait is called tTAV (Tetracycline Responsive Transcription Activator Variant).
The genetic engineering was done by injecting the mosquito embryos under a microscope with two different tools used to alter DNA in living organisms. Known as plasmids, these tools are circles of DNA that contain sequences allowing them to integrate into the native DNA of organisms. The embryos are injected very soon after fertilization in what is called the pre-blastoderm phase, as shown in this video. One plasmid contains a very powerful set of modifiers called Transposons, specifically known as “piggyBac”. Transposons allow genetic material to insert – often randomly – into genomes and can cause massive damage to the DNA. That means most injected embryos will die before producing a successfully transformed organism, in this case the OX513A. This sort of transposon can be very dangerous if artificially inserted into nature without control, which is why the OX513A uses what are called “non-autonomous” transposons. The piggyback transposon requires a helper protein – called transposase – which is contained in the second plasmid injected into the embryos and not present on the plasmid containing the lethal trait called “tTAV”. Theoretically, that should prevent any runaway activity of these transposons which now exist in the GMO mosquito. However, nature and genetic evolution being what they are, there is always a chance that over hundreds, thousands or more generations the GMO mosquitos or their offspring could change. That means these transposons could somehow become autonomous or find another organism with transposase that will allow them to leap all over a gene causing major man-made mutations in nature that would not happen naturally.
In summary, Oxitec intends to release male GMO mosquitos into the wild that contain a fluorescence gene and a lethal trait designed to kill the offspring that will occur between the OX513A and wild type (WT) mosquitoes. The idea will be that eggs from the pairing (GMO/WT) will die due to interference in the protein production of the embryos, leading to early death before they emerge from the water. The GMO mosquito will contain new genes not present in the rapidly and voluminously breeding mosquito population that also contain other elements designed to cause integration and mutation of their DNA if man-made control mechanisms fail. The mosquitos are also intended to survive if they access tetracycline as a food source, which can happen (see below).
Genetic Mutation and Unexpected Genetic Impact in Nature from the OX513A mosquito
The risk to the ecosystem comes from uncontrolled survival of the GMO mosquito in many ways. The mosquito could displace other organisms, interfere with food supply or in a worst case scenario cause unintended genetic alterations in the population or ecosystem at accelerated, man-made rates. This would be a sort of “genetic pollution”.
Dr. Ricarda A. Steinbrecher, a published molecular geneticist and co-director of “Eco-Nexus” has expressed serious concern about the unexpected genetic impact from introduction of the OX513A mosquito. She (on behalf of over 70 other organizations) warned the Malaysian government against proceeding with Oxitec’s field tests there. However these began without approval of local citizens and apparently without their knowledge. She indicated there was a real risk for unintended escape of the GMO mosquitoes artificial genes into other organism in the ecosystem. She specifically warns that unexpected mutations may occur in surviving mosquito populations and that these genes could also transfer to other mosquito species while in nature. In fact, species within the Aedes genus have been shown to be able to mate between each other in a 2011 study of mosquitos in Florida, although with less effective results for producing offspring. This was also shown in a 1949 Hawaii study showing inter-species mating for Aedes.
The OX513A mosquito can therefore cause unexpected genetic outcomes in the ecosystem by surviving longer than expected in the wild or breeding with other Aedes mosquitos. It is unknown if the genes could somehow reach other mosquito types like Anophiles or Culex which are responsible for Malaria and West Nile Virus respectively. It is unknown if these genes could somehow be picked up by recombining with viruses or other genetic material in the blood the mosquito drinks for egg production purposes. While these outcomes are theoretically less likely to happen, and no scientific evidence yet exists to show this can happen, the nature of evolution over thousands and millions of years should cause concern.
Oxitec Deception on GMO mosquito survival and reproduction.
For unexpected and negative genetic impacts to occur as a result of man’s genetic interference in the ecosystem, the Oxitec GMO mosquitos would need to survive at higher rates than expected and breed enough to produce offspring. This is entirely possible and likely. Oxitec has been deceptive regarding the chance of these mosquitoes surviving in nature.
Actual Life Expectancy of GMO Offspring. The FDA, in their study leading to the approval of release of these mosquitoes, repeatedly points to Oxitec’s claim that the mosquitoes survive “a median of two days” compared to the 68 day survival of wild type mosquitoes. Section 13.2.3. in the FDA report “Environmental Assessment for Investigational Use of Aedes aegypti OX513A”:
“…data demonstrating hemizygous females reared without tetracycline have a median lifespan of two days relative to a wild-type median lifespan of 68 days indicate a further reduction in the likelihood of survival of OX513A mosquitoes and their progeny. FDA therefore concludes it is highly unlikely that OX513A mosquitoes and their progeny would be able to establish at the proposed trial site. Nevertheless, Oxitec would monitor ovitraps for a period of time that covers a full mosquito season after the conclusion of the trial in order to detect any persistence of OX513A mosquitoes.”
The FDA refers to Appendix F of their evaluation to prove this. Appendix F shows an internal Oxitec study on the longevity and reproductive capacity of the mosquitoes that led to the claim of a median two day survival of the GMO/WT offspring. This is also known as the F1 population – the first generation of hybrids from two different parental sources (in this case the GMO (OX513A) and the wild type (WT) mosquitos found in nature). A basic review of statistics is in order. Referring to a “MEDIAN” survival means the “middle” of numbers if the all the numbers are placed into orders. So if 100 tests were done and 80 of the mosquitoes survived for 2 days, but 20% survived longer, then the median is likely to be 2. However, this should give little comfort in a natural system where mosquitoes can breed quickly and produce dozens of eggs each in the process.
The data from Appendix F shows the source of the Oxitec deception. They graphed the likelihood of survival of the F1 hybrids (offspring of GMO and Wild mosquitoes). They found that about 20% survived for long enough to “take two blood meals and some produced two clutches of eggs” (page 6 of 9 of appendix F). The graph they supplied tells the story.
This graphs of the Oxitec data shows that a substantial number (20%) of the F1 offspring of these GMO mosquitos will survive after they are produced from mating with wild mosquitoes. This means there is a markedly higher chance of survival of the GMO mosquito in the wild than Oxitec and the FDA admits. In fact, when these OX513A mosquitoes were first produced, they had about a 3-4% survival rate in 2007. These new Oxitec mosquitos have been aggressively interbred over at least 100 generations and now have about a 20% survival rate, raising concern that other changes have occurred to these GMO mosquitoes that has improved their hardiness. These longer surviving OX513A mosquitoes can live long enough to take blood meals and reproduce.
Egg Clutch Sizes and hatch rates are higher for GMO mosquitoes! As it turns out, the Oxitec GMO mosquitoes actually produce more eggs than those in nature and more of them hatch than those in nature. In the Appendix F experiment, the OX513A mosquitoes had average clutch sizes of 69.9 eggs versus only 54.8 for wild type Aedes mosquitoes. In addition, a higher percentage of the GMO mosquitoes hatch (92%) versus the wild Aedes (82%). Oxitec attributed this as possibly due to the intensive inbreeding of the GMO mosquitoes that was used to produce them.
Female GMO mosquitoes are released and survive. In addition, there are female mosquitoes released with the GMO population. The lab goes through a process of mechanically sorting out the mosquitoes into male and female and kills the females while releasing the males. However the process is not perfect and about 0.2% of mosquitoes released are actually females that will bite humans and animals and can then reproduce (see paragraph 11.3.3 of the FDA report). When one considers that at least 14 million mosquitoes are planned for release over two years, that means that the release of 28,704 reproducing mosquitoes. Oxitec and the FDA attempted to minimize this by stating there would be 62 female mosquitoes per person over the 104 week period, or 2.4 per person per week. There are only about 1,000 residents and 475 homes in all of Key Haven and less than half of the area will be treated. However there is very little else in the area and it is likely humans will be the main source of blood meals for the OX513A mosquitoes. The local citizens certain have every reason to be concerned that they will be subject to the bites of experimental mosquitoes in a larger public health experiment.
Enhanced survival of the GMO mosquito. As discussed, the OX513A mosquito from Oxitec is designed to survive when fed the antibiotic tetracycline. The presence of this antibiotic in the mosquito food supply could cause extended survival of the mosquitoes. In fact, in Brazil, the OX513A mosquitoes have been shown to have higher survival rates when they were able to access tetracycline in livestock that are routinely given this antibiotic as part of local agriculture. As these GMO mosquitoes hop rides on local Fed Ex and mail trucks and spread into Florida, there is every chance they can also find some tetracycline-fed livestock on ranches and have enhanced survival.
The Math – Over 14 million Mosquitoes to be released – Over 92 million GMO’s to feed on humans over two years.
The FDA and Oxitec anticipate releasing at a “minimum” 14,352,000 mosquitoes over two years. With about a 20% survival of the GMO mosquito offspring through two blood feedings, there are likely to be a high number of mutant mosquitoes that remain in nature. Here is the math.
- 924,268,800 new GMO offspring mosquitoes produced. Of the 14,352,000 mosquitoes, each is capable of resulting in about 70 eggs per fertilized wild type female. Of these, about 92% hatch (more than wild type). That means there could be 1004640000 eggs resulting in 924,268,800 over two years. (Number released X Number of Eggs each X hatch rate of eggs).
- 92,426,880 new GMO mosquitoes feeding on humans twice for two years. Of the nearly 1 billion newborn GMO offspring mosquitoes, about 20% can survive for two blood feedings. That means 184,853,760 mosquitoes produced with an assumption of half being female for a total of 92,426,880 (Offspring x 20% survivors x 50% as females)
- 2,221 GMO mosquitoes feeding on Key Haven Test Humans every week. Over 92 million new GMO feeding (female) offspring will exist over two years meaning that there will be 888,720 GMO offspring mosquito feeding per week. If one assumes about 400 people in the Testing area (without any migration of mosquitoes out to the rest of the keys), there will be 2,221 GMO mosquitos available to feed on each person per week for two years. (Female offspring ÷ 104 weeks ÷ 400 estimated test humans in Key Haven)
There will clearly be an enormous population of new GMO offspring created in the study area of Key Haven over two years. According to calculations based on Oxitec’s own data, over 92 million of the GMO/WT offspring (F1 Hybrids) could survive and would be available to feed. Another 92 million male GMO hybrids will exist to mate and create more surviving offspring. There will of course be a die off of the local Aedes population but they can be replaced by other mosquitoes or other pests in the void (and nature abhors a void). What will be the impact on fish, birds, and other animals that rely on the mosquitoes for their food and the downstream effect on the ecosystem?
These new OX513A hybrid mosquitoes will then be able to feed on the very dense population of humans on Key Haven to survive. The potential for genetic mutation in 184 million offspring Oxitec mosquitoes over two years is enormous. The potential for crossbreeding with other Aedes species and subsequent mutations cannot be ignored with 184 million new Oxitec hybrid mosquitoes in play. The potential short and long term impact on the ecosystem and nature itself appears entirely unpredictable and could be catastrophic. Based only on this, there should be no approval to release these Oxitec mosquitoes into Key Haven as part of a corporate/Government genetic experiment on humans and our ecosystem.
*Dr. McKalip is a private practice neurological surgeon with firsthand experience in genetically engineering. While a researcher and academic neurosurgeon, he ran a laboratory that created viruses to alter the DNA of brain and spinal cord cells to produce proteins to promote regrowth of neurons after spinal cord injury. Dr. McKalip was awarded an NIH grant to pursue this research. Dr. McKalip is also a clinical researcher familiar with the science of clinical trials and statistical analysis of results.