Single nucleotide polymorphism (SNP) in the doublesex ( dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae

Background: Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex ( dsx) gene, involved in mosquitos’ somatic sexually dimorphic traits determination. However, the pathways that trigger the signal of dsx gene exon skipping alternative splicing mechanism in anopheline mosquitoes are not well characterized. This study aims to screen the An. gambiae dsx gene splice site sequences for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene ( Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs. Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

The widespread of insecticide resistance in natural vector populations has intensified researches on alternative malaria vectors control strategies. Alternative tools for vector control have included technologies such as cytoplasmic incompatibility with the use of natural Wolbachia bacteria infection 10,11 ; repressible dominant lethal systems in Aedes aegypti 12,13 ; Y-chromosome shredding gene drive (gene drive cassette that also incorporates a programmable endonuclease that shreds the Y chromosome, converting XY males into fertile XO females) 14 ; and the genetic sterilisation of Anopheles sp., known as Sterile Insect Techniques (SIT) 15 . The SIT technique, as firstly developed, is based on the repeated, high-density release of radio-sterilized males, through gamma radiation, into the environment in order to compete with wild males for mating with the native female anopheles mosquitoes, hindering the production of offspring 16,17 . Indeed, mated females will not produce viable offspring, resulting in reduced population numbers or even local elimination of the target species. However, instead of exposing males to a source of radiation, sterility could be induced by genetic modification of the mosquito genome and may improve the effectiveness of classical SIT-based approaches 15 .
In An. gambiae, one of the major malaria vectors, population suppression strategies are already under investigation by targeting the gender determination genes such as the doublesex (dsx) transcription factor gene 18,19 . Therefore, the Anopheles gambiae doublesex gene (Agdsx) represents a useful candidate gene for genetic manipulation and improvement of the alternative mosquito control technologies. Interest in this gene comes from the fact that it undergoes alternative splicing and results in female and male-specific transcripts necessary for gender determination in this species 20 . The use of transgenic tools in anopheline mosquitoes through targeting the dsx gene could improve the sterility induction and genetic sexing which are major requirements for genetic SIT technologies. However, the molecular mechanisms underlying gender determination are highly variable.
The only well-known model of the dsx splicing comes from the fly Drosophila melanogaster sex determination pathway 21 . The dsx gene acts as a transcription factor targeting several genes which have mostly sex-and tissue-specific functions that determine somatic sexual dimorphism traits in later stages of sexual development 22,23 . Transformer (TRA) and Transformer 2 (TRA2) are the key regulatory factors of the female-specific alternative splicing of dsx pre-mRNA (dsxF isoform) under the control of the Sex lethal gene (Sxl) product while the absence of TRA (non-productive form) leads to the male-specific splicing (dsxM isoform) in fruit fly 22 . Unfortunately, An. gambiae dsx gene (Agdsx) has different gene organization and regulatory elements positions suggesting that Agdsx gender-specific splicing event is caused by a mechanism different from that of the D. melanogaster dsx 20,24 . Recently, it was reported that in An. gambiae, femaleness gene (Fle) is necessary for the splicing of dsx into the female-specific mRNA

Introduction
Malaria is a vector-borne infectious disease caused by the protozoan parasite belonging to the Plasmodium genus 1 . The transmission occurs among humans through the bite of the female Anopheles mosquito. This disease is among the top ten causes of death in low-income countries (World Health Organization) 2 and continues to take a heavy toll on communities, especially in African regions. The malaria transmission cycle involves four major elements: the host (human), the parasite, the vector, and the environment 3 . In the absence of effective vaccine or sustainable treatment options, vector control is the cornerstone of malaria management and is based on the prevention of human-host contact and reduction in vector population density 1,4 . The traditional vector control strategies rely on longlasting insecticidal net (LLIN) distribution and indoor residual sprays (IRS) which have contributed to the decreasing malaria cases and mortality 5,6 . However, vector resistance (AgdsxF) 25 . However, Fle is not involved in the dsx splicing into the male-specific transcript (AgdsxM) 25 . Indeed, Yob1 gene (Y-linked) which is activated at earlier stage of zygotic transcription and expressed all throughout a male's life, regulates male-specific dsx splicing 26 .
Agdsx is located in the 17C band of the chromosome 2R (2R: 48703664 -48788460) on the reverse strand. The gene is 84.8 kb long and encodes AgdsxM and AgdsxF transcripts. AgdsxM transcript (6975 bp) is shorter than that of AgdsxF (8667 bp). The difference between the two gender-specific transcripts is due to the alternative splicing of exon 5. The latter is a cassette exon, which is retained in female and skipped in male transcript. The whole sequence of the female-specific exon 5 is included in the male intron 4 region and is spliced out. This gene structure causes a shift in intron/exon number in male. Thus, although male and female share the same exon/intron or intron/exon boundaries, they have common and specific splice sites ( Figure 1). Though it was demonstrated that Fle and Yob1 genes control respectively AgdsxF and AgdsxM specific splicing, the pathways triggering the signal of dsx gene exon skipping alternative splicing mechanism in An. gambiae are not well characterized.
The exon definition by the spliceosome requires interplays between splice sites on either side of the exon. Donor sites (5'-splice site) are defined by GT dinucleotide at the 5' end of exon-intron border, while AG dinucleotide defined acceptor sites (3'-splice site) at the 3' end of intron-exon border 27 . In mammalian cells, the presence of genetic variations such as single nucleotide polymorphisms (SNPs) within the donor and acceptor splice sites is susceptible to influence the splicing and might lead to changes in normal splicing pattern 28-30 . The presence of SNP at the acceptor splice site of several genes is reported in human and lead to the alternative splicing of the corresponding genes 28 . Indeed, in humans, splicing signals are a common point of mutations. Most of the splicing mutations analysed so far directly influence the conventional consensus splicing sequence, and consequently lead to skipping of the adjacent exon 29 . Lamba et al., revealed that a nonsynonymous SNP (15631G>T), which disrupted an exonic splicing enhancer (in exon 4), and a SNP (15582C>T) in an intron-3 branch  Taking together these observations in humans and animal models, we hypothesized that the same events could be possible in insects and that SNPs could occur in acceptor and/or donor splice sites in mosquitoes that might result in the splice variation. The current report seeks then to screen Anopheles gambiae doublesex gene (Agdsx) splice site sequences for single-nucleotide polymorphisms (SNPs) that could be associated with alternative splicing. Splice site sequences are given in 5'  3' direction on the reverse strand. Exonic coding sequences are shown in uppercase letters, and non-coding regions are in lowercase letters. The 12 bp preceding the 3' splice-acceptor site (NYag) is indicated, where Y = T or C and N = any nucleotide.

Sequence analysis and SNP identification
From the Agdsx reference sequence, the list of genomic positions of donor and acceptor sites was extracted. VCFtools version 0.1.15 (https://vcftools.github.io/index.html) 35 was used to extract the SNPs within the genomic region corresponding to the Agdsx sequence from the SNPs annotation file. The polymorphic nucleotides were then identified within the splice sequences, in comparison to the reference sequence. SNPs were then visualized using TASSEL version 5.2.63 software (https://tassel.bitbucket.io/) 36 . The genomic position of the acceptor sites was used to select SNPs in the last 12 nucleotides of an intron preceding the 3' splice pattern NYAG and in the first six nucleotides of an exon. In donor splice sites, SNPs were identified within in the last six nucleotides of an exon and the first 16 nucleotides in an intron. The average nucleotide diversity at the dsx locus between male and female was calculated using scikit-allel version 1.2.1 (https://scikitallel.readthedocs.io/en/stable/) 37 in order to determine whether SNPs density at the dsx locus differed between the two genders.

Results
Identification of An. gambiae dsx gene (Agdsx) donor and acceptor splice sites sequence Male and female mosquitoes share exon 1, 2, 3, 4 and 6 donor splice sites while exon 5 donor site is specific to female as it is only recognized by the spliceosome in females (Table 2). Similarly, both male and female share intron 1, 2, 3, and 6 acceptor sites. Male intron 4 and female intron 5 share the same 3' end as the female, and exon 5 is included in the male intron 4 sequence. However, females have the intron 4 specific acceptor site, as the cassette exon 5 definition is not established in males (Table 2).
SNPs in female-specific intron 4 acceptor and exon 5 donor splice sites Along the Agdsx gene, 17,196 polymorphic sites were identified. Wherever both male and female mosquitoes are present (in Burkina Faso, Cameroon and Mayotte), the nucleotide diversity is similar between both genders ( Figure 2). This was expected as male and female in each country make up a single population. In addition, no difference in the nucleotide diversity was observed between male populations from the three countries (Burkina Faso, Cameroon and Mayotte) ( Figure 2, top panel). The same trend was observed between female populations as well (Burkina Faso, Cameroon, Mayotte, Gabon, Ghana, Guinea, Equatorial Guinea and Uganda).
The potential splice-relevant SNPs that could trigger the female-specific exon 5 skipping should be in the intron 4 acceptor and exon 5 donor sites. However, there was no SNP in the acceptor sequence of female-specific intron 4 nor in the corresponding male region ( Figure 3). However in the female-specific exon 5 donor site, two SNPs (rs48712947, rs48712962) were found. Nevertheless, they were not specific to females as the rs48712947 was found in Cameroon female mosquitoes and in both males and females from Burkina Faso ( Figure 4). The rs48712962 was absent in the male mosquito population, while it was found only in females in Cameroon. The minor allele frequencies (MAF) of both SNPs identified were very low in each population. The MAF of rs48712947 and rs48712962 amounted to less than 1% in each female population, and only 2% of Burkina Faso male carried the rs48712947.

SNPs in other splice sites of Agdsx
The other splice sites were also examined for identification of gender-specific SNPs. No SNP was found in the shared exon 1 donor, introns 1. No splice-relevant SNP was found in the other donor ( Figure 5A, Figure 6, and Figure 7B) and acceptor ( Figure 5B, and Figure 7A) splice sites. The highest number of SNPs (7) was found in the common intron 3 acceptor site sequence (rs48715291, rs48715294, rs48715302, rs48715306, rs48715307, rs48715308, rs48715309) ( Figure 8). However, each of these SNPs occurred in a non-specific manner in both male and female populations, with variable minor allele frequencies.

Discussion
The An. gambiae doublesex (Agdsx) gene is a candidate gene of interest for genetic SIT strategy 18,19,25 . The translation and the success of using dsx in SIT methodology require a clearer understanding of the genetic bases of the gender determination pathway. This study screened the Agdsx donor and acceptor splice sites for identification of splice-relevant SNPs.
The alternative splicing of Agdsx gene is governed by exon 5 skipping in male mosquitoes 20 suggesting a silencing mechanism of the female-specific splice sites recognition (intron 4 acceptor and exon 5 donor sites) by the splicing machinery in males. Such silencing mechanism could be due to changes in splice site sequence. However, female-specific intron 4 acceptor site sequence is present within male intron 4 and no SNP was found in this sequence in both males and females. The SNPs rs48712947 and rs48712962 identified in female-specific exon 5 donor site were neither splice-relevant nor gender-specific. They appeared only is two mosquito populations (Burkina Faso and Cameroon) over the eight populations considered. In each population where these SNPs have been identified, they appeared in very few individuals, less than 1% in females and no more than 2% in males. These observations suggest that the Agdsx cassette exon 5 was not associated with changes in splice site patterns due to the presence of SNPs. The presence of SNPs in the other splice sites had also different distribution and were non-specific to the gender of the mosquitoes.
Another factor for exon skipping is the pyrimidine content of the polypyrimidine tract in acceptor splice sequence. Indeed a poor polypyrimidine tract causes a shift of the splicing machinery to the next acceptor site, leading to the skipping as the case of exon 4 skipping in male Drosophila 21 . In Anopheles gambiae the number of pyrimidine (8) in the 12bp preceding the acceptor site pattern (acag) ( It was known that the regulation of alternative splicing evolved tans-acting splicing factors, such as serine-arginine-rich (SR) family proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind to the auxiliary silencer and enhancer cis-element (ESE: exonic splicing enhancers; ESI: exonic splicing silencers; ISE: intronic splicing enhancers; ISI: intronic splicing silencers) 38-40 . Similar regulatory cis-elements were found in Drosophila melanogaster female-specific exon and putative homologs were identified in An. gambiae female-specific exon 5 20 . Therefore, further molecular analyses are needed toward characterizing these regulatory sequence and their binding trans-factors in order to underpin the somatic sex determination in An. gambiae.  Moreover, the epigenetic system was also reported to regulate the alternative splicing in mammalian and other insect cells. Indeed, it was demonstrated that changes in DNA cytosine methylation on the gene body in honey bees may lead to alternative splicing 41-43 . Also histone post-translational modifications (PTMs) such as lysine acetylation and methylation were associated to the alternative splicing event 44-46 . Consequently, similar mechanisms could happen in the malaria vector An. gambiae to regulate gene alternative splicing. However, no significant DNA methylation was reported in Diptera including An. gambiae 47,48 . Then, the only epigenetic modifications that could be linked to the alternative splicing in this species are histone PTMs. Indeed, the methylation and acetylation of lysines 4, 9 and 29 of histone H3 were reported in    An. gambiae 49,50 . Then, it will be interesting to evaluate whether such histone modifications enrichment in Agdsx between male and female mosquitoes could be critical for dsx alternative splicing.

Conclusion
Sustainable vector control strategies will rely on the integrated use of chemical and biological vector control. Given the potential of the Agdsx gene for SIT, the understanding of mechanisms of it regulation could help to improve the tools engineering targeting this locus. SNPs were identified within the Agdsx and their putative association with the dsx alternative splicing was analysed. No splice-relevant SNP was found in the specific male and female splice site. The SNPs were distributed in few proportion of individuals in the populations where they were identified. With the advances in genetic biotechnology, other mechanisms remain to be explored for providing solid background on somatic sexual fate determination in Anopheles gambiae. This will pave the way to find new biochemical and genetics target for malaria vector control. The description must be deleted.

Discussion
It looks fine.

Conclusion
Why the An coluzzi was not included in the study. The result of your study has opened some interesting questions to address these question, but you must include the An coluzzi for the next studies because it is one the major vector and widely distributed in western Africa

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed. There are some minor edits needed though: "Moreover, the epigenetic system was also reported to regulate the alternative splicing in mammalian and other insects cells." -> change to 'other insect cells'. ○ Some citations (e.g. World Health Organization fact sheet) seems like they should be in a format consistent with other references (hyper link text to numbers).

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

Version 1
Reviewer Report 08 July 2022 https://doi.org/10.21956/wellcomeopenres.19431.r51402 © 2022 Lee Y. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Yoosook Lee
Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, Vero Beach, FL, USA

Overall comment:
The authors should update the Introduction section to reflect the latest development of sex determination research on Anopheles gambiae and related species/genes and set reasonable expectations and hypotheses.
The detailed suggested edits are noted below: "The malaria transmission cycle involves, four major elements: the host (human), the parasite, the vector and the environment." Use oxford style commas. "… the vector, and the environment." ○ "In the last decade, scientific advances in additional tools for vector control have included technologies such as …" -> the papers cited for novel vector control tools are over 10 years old. There are newer publications in this realm and should be changed to newer citations or change wording to "In the last two decades, …" ○ "The latter technique, SIT, is based on …" -> construct of listing more than two things and referencing 'the latter' for the last element is a bit awkward.

○
The 2 nd paragraph of the introduction needs to be changed. As far as I am concerned there has not been radio-sterilized male SIT for Anopheles species although some people have attempted to test it in lab conditions and prepare for field trials. The paper 13 cited for SIT is introducing a genetic tool for inducing SIT not radiation. So explaining general radiationbased SIT method and citing paper 13 is not appropriate. It is especially concerning as this paper pertains to the Anopheles gambiae in Africa. Combined with more-than-a-decade-old citations for the alternative vector control strategies, it is somewhat questionable if the authors have due diligence in background research.
○ "The use of transgenic tools in anopheline mosquitoes through targeting the dsx gene could improve the sterility induction and genetic sexing which are major requirements for SIT technologies." -> add 'genetic' in front of 'SIT technologies' as radiation-based SIT does not require genetic sexing.

○
The manuscript has mixed use of gender and sex. I suggest using the same and consistent terminology throughout the manuscript.
○ "Unfortunately, An. gambiae dsx gene (Agdsx) has a different structure suggesting that Agdsx sexspecific splicing event is caused by a mechanism different from that of the D. melanogaster dsx" -> word 'structure' in this case is not clear to readers. Structure is a very generic term and ○ can mean very different things (e.g. RNA structure? Protein structure?). Clarification would be helpful for readers.
"Indeed, in humans, splicing signals are a common point of mutations…. Taking together these observations in humans and animal models, we hypothesized that the same events could be possible in insects and that SNPs could occur in acceptor and/or donor splice sites in mosquitoes that might result in the splice variation."-> There is some logical gap about expecting mutations in splicing regions determining sex locus. It seems like this is such a fundamental biological function that has huge implications in the downstream process so this region would be likely conserved. The authors should find examples of not just having mutations in splicing regions but splicing mutations in sex-determining genes to be relevant and set reasonable hypotheses/expectations. ○ Table 1 -Since the study examined the allele frequency between males and females, Table 1 should provide the number of male and female sequences used for the analysis. Some sites of the Ag1000 data only contain females so generalizing the sex-dependent patterns with skewed data can be misleading. ○ "The An. gambiae doublesex (Agdsx) gene is a candidate gene of interest for SIT, as a candidate for genetic modifications"-> candidate is repeated twice. Perhaps change it to "… is a candidate gene for genetic SIT strategy" or something to that effect? ○ Important paper on this topic like https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955153/ 1 is not cited in this paper. boundary (the region of study here). Therefore, a refocusing of the analysis approach used in the current report, but instead to the probability of variants occurring (and being tolerated) that might show positive selection (or not) in the face of a gene drive suppressing the population by targeting this region, would be a way to resolve the current limitations. This obviously would require a re-focusing of the manuscript and a tweaking of the analysis by the authors.
Another potential approach (unrelated) is to look at the conservation across the introns in general, and how (as I guess it is) this conservation is higher towards the donor and acceptor splice sites (since these are more functionally constrained). This analysis might reveal the extent and nature of the splicing sites/ ○ Minor points: the doublesex (dsx) gene, encoding somatic sexually dimorphic traits -I would tweak this language slightly: dsx does not encode these traits directly ○ It is not explicitly stated (yet probably should be) that alternative, sex-specific splicing of dsx is critical for the initiation of downstream transcriptional pathways that underpin sexual dimorphism.
○ "Y-chromosome shredding gene drive" could be read ambiguously -the gene drive is on the Y chromosome but shreds the X chromosome. "The SNPs association to the sex phenotype (male or female) was evaluated by running the association analysis using the general linear model (GLM) function in TASSEL." -see my earlier comment. I think this approach needs rethinking. ○ "The difference between the two sex-specific transcripts is due to the alternative splicing of exon 5. The latter is a cassette exon, which is retained in female and skipped in male transcript. The whole sequence of the female-specific exon 5 is included in the male intron 4 region and is spliced out. This gene structure causes a shift in intron/exon number in male. Thus, although male and female share the same exon/intron or intron/exon boundaries, they have common and specific splice sites." -this would surely benefit from a figure (even if it's been done before, it would help here in visualisation) " Agdsx gene, 17,196 polymorphic sites were identified." What is the definition for polymorphic here? A site where at least one individual in the sampled population that has a variant base compared to the published consensus genome? ○ "Wherever both male and female mosquitoes are present (in Burkina Faso, Cameroon and Mayotte), the nucleotide diversity is similar between both sexes (Figure 1). This was expected as male and female in each country make up a single population. In addition, no difference in the nucleotide diversity was observed between male populations from the three countries (Burkina Faso, Cameroon and Mayotte) (Figure 1, top panel). The same trend was observed between female populations as well (Burkina Faso, Cameroon, Mayotte, Gabon, Ghana, Guinea, Equatorial Guinea and Uganda)" again, see earlier comment about rationale for looking at sex-specific differences.
○ Figure 3 shows a heatmap of SNPs around exon 5 donor site, but the corresponding heatmap for the region around intrn4/exon 5 (for which a nice cartoon was made in Fig 2) is not shown.
○ Re: the 'absence' of transformer in An. gambiae -there are several competing hypotheses for this, and not all are mentioned here (e.g difficulty with finding it by homology-based approaches due to extremely rapid sequence divergence) ○ Throughout the article, it's never really made explicitly clear how this information on Dsx, and the splicing details specifically, would be relevant for genetic control, and SIT (which is mentioned frequently) in particular.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: vector control, gene drive, genetic control, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 03 Dec 2022 Oswald Djihinto #Reviewer 1. The report looks at variations around the splicing sites in the gene doublesex, a transcription factor essential in determining male and female sexual dimorphism. It mines data from the AG1000g initiative, which is sampling wild caught Anopheles malaria vectors and sequencing their genomes. The approach taken here, to mine the same data and look for insight on genetic variation that would be informative in genetic control approaches, is welcome. The methods and approach taken are generally well described and background information provided on doublesex gives good context. Unfortunately, though, I think there are some issues with the rationale applied and these currently, in my view, would preclude the indexing of this study. These issues are detailed below but I do think the methodology applied if given a different focus -for example in focusing the analysis on target sites around the female-specific splice junction that are used in a specific form of genetic control called gene drive. I'm struggling with the logic that SNPs in a splice site could definitively determine, and be sufficient for, sex-specific splicing. By extension, the fact that the SNPs are found in both sexes is taken to mean that these particular SNPs cannot be critical for alternative, sex-specific splicing. But all genes, and alleles thereof, must be found in males and females at some point unless linked to a sex determining chromosome or locus. Am I missing something? In short, I just don't see how gender-specific SNPs will be helpful or informative (or at all likely) in this instance. The good thing at least is that your results confirm this.

Reply:
The reviewer is correct. The presence of SNPs in both sexes means that these particular SNPs cannot be critical for alternative sex-specific splicing. However, this study was aiming to investigate any gender-specific SNP in the target splice site sequences that could be critical for exon 5 skipping splicing mechanism. This objective was driven by reports of SNPs in the splice site that lead to an alternative exon splicing. #Reviewer 1. Gene drives that target the doublesex gene, and specifically, the female-suggesting that Agdsx sex-specific splicing event is caused by a mechanism different from that of the D. melanogaster dsx" I am not quite sure what is meant by this statement -from what I know the overall structure in terms of sequence conservation and splicing structure, as well as last coding exon being sex-specific etc is pretty well conserved between Anopheles and Drosophila.