Background: Anopheles pharoensis has a major role in transmitting several human diseases, especially malaria, in Egypt?. Controlling Anopheles is considered as an effective strategy to eliminate the spread of malaria worldwide. Galaxaura rugosa is a species of red algae found in tropical to subtropical marine environments. The presence of G. rugosa is indicative of the ecosystem's overall health. The current work aims to investigate UPLC/ESI/MS profile of G. rugosa methanol and petroleum ether extracts and its activity against An. pharoensis and non-target organisms, Danio rerio and Daphnia magna. Methods: Galaxaura rugosa specimens have been identified using DNA barcoding for the COI gene and verified as G. rugosa. The UPLC/ESI/MS profiling of G. rugosa collected from Egypt was described. The larvicidal and repellent activities of G. rugosa methanol and petroleum ether extracts against An. pharoensis were evaluated, as well as the toxicity of tested extracts on non-target organisms, Dan. rerio and Dap. magna. Results: The UPLC/ESI/MS analysis of methanol and petroleum ether extracts led to the tentative identification of 57 compounds belonging to different phytochemical classes, including flavonoids, tannins, phenolic acids, phenyl propanoids. Larval mortality was recorded at 93.33% and 90.67% at 80 and 35 ppm of methanol and petroleum ether extracts, respectively, while pupal mortality recorded 44.44 and 22.48% at 35 and 30 ppm, respectively. Larval duration was recorded at 5.31 and 5.64 days by methanol and petroleum ether extracts at 80 and 35 ppm, respectively. A decrease in acetylcholinesterase (AChE) level and a promotion in Glutathione-S-transferase (GST) level of An. pharoensis 3rd instar larvae were recorded by tested extracts. The petroleum ether extract was more effective against An. pharoensis starved females than methanol extract. Also, tested extracts recorded LC₅₀ of 1988.8, 1365.1, and 11.65, 14.36 µg/mL against Dan. rerio, and Dap. magna, respectively. Conclusions: Using red algae derivatives in An. pharoensis control could reduce costs and environmental impact and be harmless to humans and other non-target organisms.

This study systematically evaluated the efficiency of rose bengal and methylene blue as photosensitizers against the immature aquatic stages of Anopheles pharoensis. Genetic identification using the COI partial sequence confirmed the species, and the obtained sequence was submitted to GenBank (Accession No. PQ346929). Both photosensitizers exhibited 100% mortality in larvae I within 24 h at their highest concentrations, demonstrating strong biocidal activity. LC₅₀ values for rose bengal increased from 1.50 ppm (24 h) and 1.34 ppm (48 h) in larvae I to 3.83 ppm (24 h) and 3.12 ppm (48 h) in pupae. Similarly, methylene blue showed LC₅₀ values rising from 1.14 ppm (24 h) and 0.90 ppm (48 h) in larvae I to 2.91 ppm (24 h) and 2.51 ppm (48 h) in pupae, indicating stage-dependent susceptibility. Enzymatic responses revealed a progressive increase in acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in the developmental stage, suggesting a physiological adaptation to the photosensitizers. Molecular docking against the AChE protein (PDB ID: 6xyu) confirmed insecticidal bioactivity, with methylene blue exhibiting superior binding affinity, aligning with the in-vitro larvicidal results. Furthermore, a Complex GAPI assessment confirmed the environmental sustainability of both photosensitizers, supporting their potential as eco-friendly alternatives for mosquito control. The use of Complex GAPI in assessing the environmental sustainability of photosensitizers in mosquito control represents a novel approach in the field of integrated pest management. This advancement not only aligns with the principles of green chemistry but also addresses the growing need for sustainable alternatives to traditional chemical insecticides. These findings highlight the feasibility of utilizing light-activated photosensitizers for sustainable vector management.