Photoactivation Activity Of 2-Thiophenylfuranocoumarin Induces Midgut Damage And Apoptosis In Aedes Aegypti Larvae

Furanocoumarins are photoactive compounds derived from secondary plant metabolites.They possess many bioactivities,including antioxidative,anticancer,insecticidal,and bactericidal activities.Here,we designed a new scheme for synthesizing2-arylfuranocoumarin derivatives by condensation,esterification,bromination,and Wittig reaction.It was found that 2-thiophenylfuranocoumarin（Iy）had excellent photosensitive activity.Therefore,in this study,the fourth instar larvae of Aedes aegypti were treated with Iy,and the effects of Iy on the development parameters of Aedes aegypti larvae at sublethal concentrations were evaluated through previous studies on the virulence of Iy.Low,medium and high concentrations(LC₂₅,LC₅₀,LC₇₅)were set,and ROS detection,enzyme activity determination,paraffin section,ultrastructural analysis,detection of intestinal permeability,high-throughput sequencing and apoptosis staining were used.The photoactivation activity of Iy to Aedes aegypti larvae was studied from the aspects of mitochondrial dysfunction,oxidative stress,intestinal barrier dysfunction,intestinal microbiota and apoptosis.The specific research contents are as follows:1.After being treated under ultraviolet light for 48 h,the LC₂₅,LC₅₀and LC₇₅of Iy to larvae were 54,65 and 78 mg/m L,respectively.At the same time,the symptoms of poisoning were observed.Aedes aegypti larvae were exposed to different Iy concentrations in the dark.After the light treatment,the Iy-exposed groups displayed different degrees of toxic reactions,and the activity of the larvae was increased during the initial period,especially at the medium and high concentration of Iy.After 48 h observation,the exposed larvae were weakened,and they swam up and down spasmodically after being touched by the needle.Most dead larvae were in a rigid state with a black abdomen,and their color was deepened at the higher Iy concentrations.The larvae which were treated with different sublethal concentrations of Iy(LC₁₀,LC₂₀,LC₃₀)for 48 h could be washed with dechlorinated water and fed normally,which could affect the growth and development of larvae,and had obvious post lethal effect.2.Under ultraviolet light,Iy can produce ¹O₂,which can further induce the body to produce ROS.Through the qualitative observation and quantitative determination of ROS,it was found that ROS burst out after 3 h of light treatment,and obvious green fluorescence could be observed in the total digestive organs including the gastric caeca,midgut,and Malpighian tubules.3.The origin of ROS is related to mitochondria.Ultrastructural analysis showed that the structure of the control group mitochondria was intact,and their membrane and cristae were clearly visible.In contrast,the membrane and cristae of the Iy treatment group mitochondria were fuzzy,and there were vacuoles.The enzyme activities of ETC and TCA cycle were promoted at low concentration and inhibited at high concentration.The activity of complexⅠandⅢ,as the main producing sites of ROS,was significantly inhibited at high concentration,and the enzyme activity decreased by 35.19%and 59.57%respectively compared with the control group.At the same time,mitochondrial membrane potential（MMP）decreased significantly（p<0.05）.The content of ATP decreased significantly（p<0.05）.The function of mitochondria was impaired and excessive production of ROS was induced.4.Excessive ROS will consume and destroy the enzyme and non-enzyme antioxidant defense system,resulting in oxidative stress.The results showed that the enzyme activities of SOD and CAT were promoted at low concentration,but significantly inhibited at medium and high concentration（p<0.05）,GR enzyme activity was promoted at low concentration,but significantly inhibited at high concentration（p<0.05）,and GPx enzyme activity was significantly inhibited（p<0.05）.The ratio of GSH to GSSG decreased from0.48 to 0.09,indicating that the midgut cells were in a state of high oxidative stress.The lipid peroxidation product MDA and protein oxidation product protein carbonyl increased significantly（p<0.05）,indicating that the oxidative damage of midgut cells was aggravated.Histopathological changes of midgut were observed by paraffin section.In the control group,the midgut epithelial cells were intact and orderly arranged,the nucleocytoplasmic staining generated strong signals,the striated border and peritrophic membrane were not broken.In contrast,under Iy treatment,the midgut tissues were damaged at varying degrees,the striated border was diminished,the cells were swollen,and there were instances of vacuolation,cell disintegration,associated with the appearance of intercellular space and midgut disruption.Ultrastructurally,microvilli were destroyed,and no histological changes were observed in the control group.The results showed that there were pathological changes in midgut.5.The tissue structure of midgut was destroyed to explore the effect of Iy on intestinal barrier function.Intestinal permeability test showed that,when the peritrophic membrane was destroyed,（FITC）-dextran will exude from the intestine.Iy treatment was associated with increased fluorescence intensity and intestinal permeability,compared with those in the control group.Chitin is the main component of the perineal membrane.Compared with the control group,the content of chitin in the treatment group decreased by 12.74%,24.25%and 27.21%respectively（p<0.05）,and the perineal membrane was damaged,resulting in intestinal barrier dysfunction.6.Excessive ROS can affect the composition of intestinal microbiota.After Iy treatment,compared with the control group,the OTU of the treatment group decreased,the composition of intestinal microbiota of larvae changed,the proportion of Chryseobacterium increased,and the proportion of Tessaracoccus decreased,which changed the balance of intestinal microbiota.7.When ROS was eliminated and blocked,it accumulates continuously in the cell,induces and activates apoptosis-related genes such as Ae Dronc,Ae Caspase7 and Ae Caspase8,and induces cell apoptosis.The Hoechst 33342 stain densely labeled the nuclei of midgut cells treated with Iy.The TUNEL staining detected broken genomic DNA in the midgut cells.The terminal deoxynucleotidyl transferase（TDT）added FITC-conjugated d UTP to exposed 3’-OH groups,which clearly labeled apoptotic spots.The TEM analysis indicated that Iy treatment was associated with karyopyknosis.Overall,these results suggested that Iy induced apoptosis in midgut cells.