The Anopheles Hyrcanus Group, comprising various mosquito species known for their vector capabilities, poses significant challenges for researchers aiming to understand malaria transmission dynamics. A recent study aimed to clarify the discrepancies among these closely related species by identifying reliable reference genes for gene expression studies.
Published on March 16, 2025, the research was conducted by Lee, D., Lee, S., and Kim, J. over three years, from 2021 to 2023. The researchers collected adult mosquitoes from three sites across Gyeonggi Province, South Korea, focusing on six species within the Anopheles Hyrcanus Group: Anopheles belenrae, Anopheles kleini, Anopheles lesteri, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides.
Gene expression profiling is pivotal for exploring distinguishing characteristics among these species, particularly their varying vector competencies. The study sought to identify reference genes whose stable expression levels would provide sound normalization for comparative gene expression analysis through quantitative polymerase chain reaction (qPCR).
Eight candidate reference genes were initially selected based on prior transcriptional studies and included ribosomal proteins and other housekeeping genes. The gene stability was evaluated across five developmental stages: the 4th instar larva (4L), pupa (pup), 24-hour-old adult female (24 h-A), 72-hour-old adult female (72 h-A), and mixed-age oviposited female (ovi-A).
The findings revealed ribosomal protein L8 (RPL8) and ribosomal protein L13a (RPL13a) as the most stable at the larval stage. For the adult stages, ribosomal protein L32 (RPL32) and ribosomal protein S17 (RPS17) demonstrated high stability. Among these, RPS17 emerged as the preferred reference gene for Anopheles belenrae, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides, whereas RPL8 and ribosomal protein S7 (RPS7) were deemed suitable for Anopheles kleini and Anopheles lesteri.
Conducted through structured methodologies, the study utilized advanced data analysis programs, including geNorm, BestKeeper, and NormFinder, to ascertain gene stability metrics. The research highlighted the significance of RPS17 and RPL8, which are key for accurate genetic analysis among species with similar morphologies and ecological niches.
Interestingly, previous studies faced challenges analyzing gene expression due to the overlapping habitats of these species and difficulties associated with their identification. This research serves to establish foundational knowledge on gene interactions defining vector competence and insecticide susceptibility.
Lee's team indicates, "RPL8 and RPL13a transcription were the most stable at the larval stage," underscoring the necessity of selecting genes with consistent expression levels across different developmental contexts. The identification of RPS17 as reliable, as stated, "RPS17 emerged as a reliable reference gene for Anopheles belenrae, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides," enhances the comparative studies within the Anopheles Hyrcanus Group.
Implications stemming from these findings can directly influence how researchers approach gene expression studies across various species of mosquitoes, providing insights beneficial for vector control and public health strategies. The stability identified within RPL32 and RPS17 across developmental stages makes them ideal candidates for future research endeavors focusing on malaria transmission pathways.
While establishing reliable reference genes is instrumental, Lee and colleagues note the need for additional validation to examine their applicability under varying biological and environmental conditions. The team suggests these genes can facilitate future investigations exploring detoxification mechanisms against larvicidal agents and immune responses to pathogens.
Conclusively, recognizing the stability of RPL8, RPL13a, RPS17, and RPS7 as dependable markers heralds new opportunities for scientific inquiry within the Anopheles Hyrcanus Group, potentially leading to enhanced malaria vector control measures and comprehensive understandings of mosquito biology.