| Mosquitoes are one of the most important health pests in the world by transmitting many serious medical infectious diseases through the bloodsucking such as malaria,yellow fever,and dengue fever etc.Humans have a long history of mosquito control,including physical,chemical and biological methods.Currently,the most effective way to combat the mosquitoes is still to apply chemical insecticide.However,arsing issues of insecticide resistance caused by extensive continus usage of pesticides,environmental safety and killing of non-targets lead to seek altermative biological control agents.With the development of comprehensive pest management,a large number of biological agents have been widely used.The Pythium guiyangense has many advantages,such as good vitality,easy to cultivate in the laboratory,rapid growth,etc.In this article,we observed the infection process of Pythium guiyangense in the larvae of mosquito Culex pipiens pallens via two different routes:Exterior cuticle penetration by germination of zoospores and toxicate midguts through ingestion of myceliium and zoospores.We also analyze the biological function of one member of Protein Kinase A in Pythium guiyangense by constructing genetic tranformants.1.Infection of Pythium guiyangense to mosquitoScanning electron microscopy and paraffin section were used to observe and study the infection process of Pythium guiyangense to mosquito larvae.1):External cuticle invasion:Initially,zoospores produced by the mycelium of Pythium guiyangense could gathered around the whole body of msoquitoes incuding head,thorax,abodomen and siphone by unknown meachnisms.At about 2h of post infection,the zoospores began to produce mastoid structures and gradually germinated and extended to form germ tube around 4h-8h.It appears that the tip of the germ tube to be the only area of the spore in intimate contact with the cuticle.About 12h,slender penetration hyphae of P.guiyangense extended through the cuticle until it reached the haemocoel of the insect body.Additioanlly,the paraffin section results also showsuggested that the cortical cells of the lanvae were also destroyed during the penetrating process.Around 16 hours,mycelium began to colonize within the whole body of mosquoto and form a large amount of mycelium.About post 24h,the mycelium circulated and filled the body cavity and penetrated the outer cutile.In about 48h,mycelium protruded out of the cutile and produced sporangium to strat a new round of infection cycle.2):Digestive tract invasion:the larvae of Culex pipiens pallens actively and voluntarily ingested a large number of hyphae and zoospores of P.guiyangense.After feeding 8h,the cross-cutting mosquito segment was investigated using electron microscopy,and results revealed that the gut of larvae was filled with mycelium.DNA extraction of mycelium from the gut and feces after treantment and PCR amplification further confirmed that infection was caused by P.guiyangense.We also obsenved the change of melanization in the gut within 2h,4h,12h and 24h,and found that melanization intensified over time.Paraffin section additionally proved that the cells of midgut wall were progressively destroyed between treatment of 4h and 12h.The ultrastructural damages observed in larvae tissue in 24h were characteristic of cell lysis and rupture of the midgut epithelial.The myceliun in the larval body mass grew,accumulated and filled the whole body of larvae eventually in about 36h.2.Functional analysis of PKA gene in Pythium guiyangensePKA(Protein kinase A)is a centre enzyme in the activating signal way,and it can play a important role in the phytopathogen and insect pathogen.So,it is a significant importance to research the meanings of Pythium guiyangense killing the mosquito.In this research,the function of PKA03242 with a conserved protein of Pythium guiyangense was studied by RNAi strategy.The growth rates of the silenced#1 and#2 were dramatically reduced in comparison with WT and CK,also displayed visible morphological changes.We examined the expression levels of PgAGC1 at developmental stages of fungi and in infected mosquitoes using Realtime RT-PCR(qPCR).Analysis results showed that if the expression level is 1 in mycelia,no significant varies were detected in zoospore and cytospore,however,was significantly up-regulated during the early stage of infection(10 hours)and reached the maximum level at 20 hpi,then dropped down at the late stage of infection.We have used infection rates and mortality data to evaluate the pathogenicity of PgAGC1 in mosquito vector,Culex Pipiens pallens and the silenced reduced the pathogenicity of P.guiyangense.In this study,we incubated in a plate containing H2O2 Two PgAGC1-deficiency mutants in Py.guiyangense#1 and#2,resulted in reduced growth rated under the 100mM CaCI2?0.5M Sorbitol,2.5M H2O2 and 5MH2O2 respectively compared with WT and CK... |
PDF Full Text Request