Genetic Modification Of Pythium Guiyangnese Using Arthropod Toxins For Mosquito Control

Mosquitoes are essential medical vectors that transmit several deadly infectious diseases to humans worldwide.However,there is no treatment for most of these diseases,and vector control with synthetic chemical pesticides is still the primary method.The traditional insecticide-based strategies have their limitations attributed to insecticide resistance development as well as adverse impacts on the environment and non-target organisms.A previous study illustrated that Pythium guiyangense,a filamentous oomycete,from plant pathogens is an effective biological control agent against mosquitoes.P.guiyangense reproduces both sexually and asexually,adapts to a diverse natural environment firmly,and can easily achieve mass production.Recent studies found that this oomycete infects its host via two pathways;invasion through the exocuticle and the digestive tract.These two pathways give it an advantage over other entomopathogens that infect via cuticle penetration only.The studies also found that P.guiyangense attracts the mosquito larvae,which like feeding on the oomycete mycelia.Additionally,since it is a water mold,it has a greater probability of contacting the early stages of mosquitoes,unlike Beauveria and Metarhizium spp.that are soil-borne.Like other entomopathogenic fungi,infection via the cuticle is initiated by direct penetration of the external cuticle.Upon breaching the insect cuticle,this oomycete enters the insect hemocoel,where the hyphal cells absorb nutrients,produce toxins,destroy host cells,and eventually kill the host.On the other hand,infection via the digestive system is initiated by mosquito larvae feeding on this oomycete,followed by filling the internal organs with mycelia that eventually destroy the host’s internal organs,causing death within 48 hpi.Transmission is then achieved by sporulation from the cadaver.As an entomopathogenic fungus,it has an advantage over other biocontrol agents such as toxin-producing bacteria,protozoa,and viruses because:of its route of pathogenicity（direct contact of zoospores with the cuticle of the host is necessary to initiate the process of infection）,easy delivery,formulation improvement,and easy engineering techniques.P.guiyangense as an entomopathogen provides an alternative biocontrol to chemical insecticides and can help to reduce the reliance on the current insecticide-based mosquito control strategies.However,its low virulence（slow kill and high inoculum load needed）is a major setback to its commercial use compared to its counterpart cheap and fast-acting chemical insecticides.Genetic engineering has made it feasible to greatly improve entomopathogenic fungi virulence and their tolerance to abiotic stress.Modifying it genetically using insect-killing toxins provides a better alternative since it improves its efficacy and efficiency in controlling mosquitoes.Predatory arthropods inject venom-containing toxins into victims to kills or paralyze them.These predators have a natural ability to infiltrate insects,and thus entomopathogenic fungi are used to deliver the toxins to the prey,enhancing their virulence.Hybrid,together with Aa IT have successfully been used in the past with Metarhizium spp.and Beauveria spp.Based on this,we constructed a transgenic strain from the genome of P.guiyangense using insect neurotoxins Aa IT（a sodium channel blocker）,Hybrid（a potassium and calcium channels blocker）,and a combination of Hybrid and Aa IT:First,we synthesized the sequences of toxins and incorporated them into the genome of P.guiyangense using PEG-mediated transformation.This was followed by screening the successful transformants and verifying them with semi-QPCR and western blot.Secondly,we examined whether the insect toxins affected the phenotypes of P.guiyangense.There was no significant difference in the growth rate of transgenic strains on V8 medium and wild type.Similarly,the transgenic strains exhibited similar mycelial morphology and zoospores production as the wild-type strain.Secondly,we evaluated the ability and response of transgenic strains to withstand different stress inhibitors,namely:Ca Cl₂,sorbitol,and hydrogen peroxide（H₂O₂）.The results showed no significant difference in the growth rate of transgenic strains under abiotic stresses Ca Cl₂and sorbitol;however,there was a decrease in the growth rate of strains as H₂O₂concentration increased.Hybrid showed a significant difference in terms of inhibition rate at 5 and 10 m M H₂O₂.All these studies showed that the strains had no growth defects.Lastly,we evaluated the virulence of the strains by carrying out pathogenicity assays with 3 day old second instar mosquito larvae.The transgenic lines killed more mosquitoes than the wild type,and combining two toxins had a synergistic benefit and killed more mosquitoes than when the toxins were used individually.In conclusion,we successfully genetically engineered P.guiyangense using arthropod toxins to control mosquitoes.The inclusion of P.guiyangense in already existing control methods would efficiently reduce the mosquito population and thus help reduce the disease burden transmitted by mosquitoes.