Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

Although pesticide resistance is common in insect vectors of human diseases, the evolution of resistance might be delayed if management practices are adopted that limit selection of resistance alleles. Outbreaks of dengue fever have occurred in Queensland, Australia, since the late 1800s, leading to ongoing attempts to control the mosquito vector, Aedes aegypti (L.). Since the 1990s, pyrethroid insecticides have been used for this purpose, but have been applied in a strategic manner with a variety of delivery methods including indoor residual spraying, lethal ovitraps, and use of insect growth regulators as larvicides. Separate selection experiments on mosquitoes from Queensland using Type I and Type II pyrethroids did not produce resistant lines of Ae. aegypti, and bioassays of field material from Queensland showed only weak tolerance in comparison with a susceptible line. There was no evidence of knockdown resistance (kdr) mutations in Ae. aegypti from Queensland, in stark contrast to the situation in nearby southeast Asia. We suspect that careful management of pyrethroid insecticide use combined with surveillance and interception of exotic incursions has helped to maintain pyrethroid (and particularly kdr-based) susceptibility in Ae. aegypti in Australia.

Background: In the inner city of Yogyakarta, Indonesia, insecticide resistance is expected in the main dengue vector, Aedes aegypti, because of the intensive local application of pyrethroid insecticides. However, detailed information about the nature of resistance in this species is required to assist the release of Wolbachia mosquitoes in a dengue control program, so that we can ensure that insecticide resistance in the strain of Ae. aegypti being released matches that of the background population. Methods: High-resolution melt genotyping was used to screen for kdr mutations associated with pyrethroid resistance in the voltage-sensitive sodium channel (V SSC) gene in Ae. aegypti of some areas in the inner city of Yogyakarta. Results: The results show that the V1016G mutation predominated, with individuals homozygous for the 1016G allele at a frequency of 82.1% and the mutant allele G at a frequency of 92%. Two patterns of co-occurrence of mutations were detected in this study, homozygous individuals V1016G/S989P; and heterozygous individuals V1016G/ F1534C/S989P. We found the simultaneous occurrence of kdr mutations V1016G and F1534C at all collection sites, but not within individual mosquitoes. Homozygous mutants at locus 1016 were homozygous wild-type at locus 1534 and vice versa, and heterozygous V1016G were also heterozygous for F1534C. The most common tri-locus genotype co-occurrences were homozygous mutant 1016GG and homozygous wild-type FF1534, combined with homozygous mutant 989PP (GG/FF/PP) at a frequency of 38.28%. Conclusions: Given the relatively small differences in frequency of resistance alleles across the city area, locality variations in resistance should have minor implications for the success of Wolbachia mosquito trials being undertaken in the Yogyakarta area.

Background: In the inner city of Yogyakarta, Indonesia, insecticide resistance is expected in the main dengue vector, Aedes aegypti, because of the intensive local application of pyrethroid insecticides. However, detailed information about the nature of resistance in this species is required to assist the release of Wolbachia mosquitoes in a dengue control program so that we can ensure that insecticide resistance in the strain of A. aegypti being released matches that of the background population.Methods: High-resolution melt genotyping was used to screen for kdr mutations associated with pyrethroid resistance in the voltage-sensitive sodium channel (VSSC) gene in A. aegypti of some areas in the inner city of Yogyakarta.Results: The results show that the V1016G mutation predominated, with individuals homozygous for the 1016G allele at a frequency of 82.1% and the mutant allele G at a frequency of 92%. Two patterns of co-occurrence of mutations were detected in this study, homozygous individuals V1016G/S989P; and heterozygous individuals V1016G/F1534C/S989P. We found the simultaneous occurrence of kdr mutations V1016G and F1534C at all collection sites, but not within individual mosquitoes. Homozygous mutants at locus 1016 were homozygous wildtype at locus 1534 and vice versa, and heterozygous V1016G were also heterozygous for F1534C. The most common tri-locus genotype co-occurrences were homozygous mutant 1016GG and homozygous wild-type FF1534, combined with homozygous mutant 989PP (GG/FF/PP) at a frequency of 38.28%.Conclusions: Given the relatively small differences in frequency of resistance alleles across the city area, locality variations in resistance should have minor implications for the success of Wolbachia mosquito trials being undertaken in the Yogyakarta area.

Abstract. Mulia DS, Raicha R, Lunggani CP, Erina SN, Wuliandari JR, Purbomartono C, Isnansetyo A. 2023. Antibacterial activity of ethanol extract of banana, cassava, and pineapple peels against a fish pathogen Aeromonas hydrophila. Biodiversitas 24: 481-485. Aeromonas hydrophila is the most virulent bacteria for almost all freshwater fish species. One method to control pathogenic bacteria in fish should be safe and environmentally friendly, including utilizing natural wastes such as banana peels (Musa paradisiaca), cassava peels (Manihot esculenta), and pineapple peels (Ananas comosus). These natural wastes contain bioactive compounds. Their utilization increases the waste value. This study aims to determine the potential of banana peel, cassava peel, and pineapple peel waste as an antibacterial against A. hydrophila, a causing agent of Motile Aeromonas Septicemia (MAS) in freshwater fish. Parameters observed included phytochemical compounds, MIC, and MBC values of banana, cassava, and pineapple peels. Phytochemical screening was performed using thin-layer chromatography (TLC) and foam test. The microdilution method carried out the antibacterial activity of banana peel, cassava peel, and pineapple peel against A. hydrophila. It determines the minimum inhibitory concentration (MIC); and the minimum bactericidal concentration (MBC) values. Phytochemical screening results showed that banana, cassava, and pineapple peel extracts contained flavonoids, alkaloids, terpenoids, tannins, and saponins. The MIC values of banana peel, cassava peel, and pineapple peel extract were 600, 400, and 200 µg/mL, respectively. The MBC values of banana peel, cassava peel, and pineapple peels were 4 × MIC, 2 × MIC, and 1 × MIC, respectively. Extract of pineapple peel exhibited better antibacterial activity than banana and cassava peels. However, the three waste extracts indicated the antibacterial potential to be developed as natural bactericides to control A. hydrophila bacterial disease.