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Validation of reliable reference genes for comparison of gene expression across species in the Anopheles Hyrcanus Group

March 16, 2025

Primer set design and efficiency test

Primer sets for 10 of these candidate genes (excluding RPS18) were designed from conserved regions across the six Anopheles Hyrcanus Group species (Table 1). Although the RPS18 gene was used in a previous study with An. sinensis, it was excluded from our study because no suitable primers that met our criteria (a length of 20–21 bp, a GC content of 45%–60%, and a product size ranging from 90 to 180 bp) could be found within the conserved regions. After efficiency testing, the RPL49 and α-tub primers, which exhibited lower average amplification efficiencies (89.1 and 92.1%, respectively) than the other reference genes, were excluded from further analysis. The remaining primers demonstrated robust performance, with average efficiencies ranging from 95.8 to 103.1%. Notably, their species-specific efficiencies ranged from 90.7 to 106%, with an R² ≥ 0.99, thus satisfying the criteria for reference genes.

Table 1 Primers used for qPCR.

qPCR for C
_q
values and comparative delta C
_t

qPCR was performed to compare the transcript abundance and stability of the candidate genes. C_q values for the eight remaining candidate reference genes across the five developmental stages and six species were determined using LightCycler 96 software (Roche, Mannheim, Germany). The average C_q values for these eight genes ranged from 14.77 (EF1a) to 16.95 (GAPDH), thus indicating their robust expression levels (Fig. 1). Among the eight candidate reference genes, EF1α exhibited the highest abundance across the six species, with average C_q values ranging from 14.48 in An. lesteri to 15.07 in An. kleini.

The gene with the lowest SD was determined to be the most stable in the delta C_t analysis, which varied across species: RPL8 in An. belenrae (SD = 0.63) and An. kleini (SD = 0.74); RPS7 in An. lesteri (SD = 0.52); RPL32 in An. pullus (SD = 0.79); and RPS17 in An. sinensis (SD = 0.76) and An. sineroides (SD = 0.63) (Fig. 2, Table 2). In the 4L and ovi-A stages, RPL13a was found to be the most stable gene (SD = 0.42 and SD = 0.25, respectively), while RPL32 was the most stable gene in the other developmental stages (SD = 0.30–0.75) (Table 3).

Table 2 Ranking of gene stability across the six species was determined using various algorithms.

Table 3 Ranking of gene stability across the five developmental stages determined using various algorithms.

geNorm

In each species, the use of two genes consistently exhibited the least pairwise variation when compared to using three or four genes. However, an exception was observed in An. lesteri and An. sineroides, where lower pairwise variation values were estimated for three genes (Fig. 3). When testing the different stages across six species, three genes were found to be superior to two genes, as indicated by their lower pairwise variations. However, it is worth noting that two genes were also acceptable, as their V_2/3 values were < 0.1. When assessing the M values of the eight candidate genes, the combination of RPL32, RPL8, RPL13a, and RPS17 consistently exhibited lower values, indicating greater stability across all of the sample groups (Tables 2, 3, Fig. 4). Furthermore, the most stable genes with the lowest M values varied across species: RPL32 and RPS17 in An. belenrae (M = 0.1), An. pullus (M = 0.16), and An. sineroides (M = 0.16); RPL32 and RPL8 in An. kleini (M = 0.15) and An. sinensis (M = 0.1); and RPL13a and RPS7 in An. lesteri (M = 0.08).

BestKeeper and NormFinder

BestKeeper analysis, based on the SD and CV values using pairwise correlation, identified that RPS17 was the most stable gene for An. belenrae, An. lesteri, and An. pullus, while RPS7 was found to be the most stable gene for An. kleini, An. sinenesis, and An. sineroides (Table 2). In addition, RPS17 emerged as the best reference gene for the 4L and ovi-A stages across the six species. RPL8 was determined to be the best reference gene for the 24 h-A and pup stages, while RPL32 exhibited the highest stability as the reference gene for the 72 h-A in the Anopheles Hyrcanus Group (Table 3).

By NormFinder algorithm, the most stable genes for each species with the lowest SV, were determined as follows: RPL8 in An. belenrae and An. kleini (SV = 0.31 and 0.26, respectively); RPS17 in An. lesteri, An. pullus, and An. sineroides (SV = 0.26, 0.38, and 0.17, respectively); and RPS7 in An. sinensis (SV = 0.33) (Table 2). When testing across the different developmental stages, results similar to those of the delta C_t method were obtained, where RPL13a emerged as the most stable gene in the 4L and ovi-A stages (SV = 0.23 and 0.11, respectively), while RPL32 was the most stable gene in the 24 h-A and 72 h-A stages (SV = 0.07 and 0.24, respectively) (Table 3).

RefFinder

Using the results obtained from the four normalized methods described above, the overall ranking of gene stability was determined using RefFinder²³. For An. belenrae, An. pullus, An. sinensis, and An. sineroides, RPS17 was identified as the most stable reference gene, while RPL8 and RPS7 were identified as the most stable genes for An. kleini and An. lesteri, respectively. When using two reference genes, the following combinations were recommended: RPS17 + RPL8 for An. belenrae and An. sinensis; RPL8 + RPL32 for An. kleini; RPS17 + RPS7 for An. lesteri; and RPS17 + RPL32 for An. pullus and An. sineroides. When comparing the expression levels between species, RPL32 was identified as the most stable gene for the pup and adult stages, while RPL13a was found to be the most stable gene for the 4L stage. For normalization with two reference genes, RPL13a + RPL8 was recommended for the 4L stage, RPL32 + RPL8 for the pup and 72 h-A stages, and RPL32 + RPS17 or RPL32 + RPL13a for the 24 h-A and ovi-A stages.

Validation of reference genes

Chitin synthase 2 (CHS2) and vitellogenin 1 (Vg1) genes are known to exhibit distinct expression patterns throughout the mosquito life cycle^27,28. To validate the selected reference genes, qPCR was performed, and the transcription levels of CHS2 and Vg1 were normalized using either the two most stable genes or the least stable gene and compared. When using RPS7 and RPS17, which were identified as the most stable genes for An. lesteri, as references, the expression level of CHS2 was determined to be highest in the 24 h-A stage (Fig. 5A). However, when normalized using the least stable gene, actin, a different pattern of CHS2 emerged during the adult stages, with a 4.9-fold higher expression level in the ovi-A stage compared to the 24 h-A stage. The expression pattern of Vg1 across stages varied depending on whether it was normalized to the most or least stable gene (Fig. 5B).

When comparing the expression levels of CHS2 in the 24 h-A stage across the six species, An. pullus and An. sinensis exhibited the lowest expression levels when the stable reference genes (RPL32 and RPL13a) were used for normalization. In contrast, An. belenrae showed the lowest expression level when an unstable gene (EF1α) was used as the reference (Fig. 5C). The expression level of Vg1 also varied among the six species, but the order of expression level for each species was more consistent when using the RPL32 and RPL13a genes (An. pullus < An. kleini < An. sinensis < An. belenrae < An. sineroides < An. lesteri) compared to using EF1α (An. belenrae < An. pullus < An. sineroides < An. sinensis < An. kleini < An. lesteri) (Fig. 5D). These results emphasize the importance of appropriate reference gene selection and confirm the reliability of the reference genes identified in this study.