Comparative genomics identifies male accessory gland proteins in five species Glossina

Accessory gland proteins (ACPs) are important reproductive proteins produced by the male accessory glands (MAGs) of most insect species. These proteins are essential for male insect fertility, and are transferred alongside semen to females during copulation. ACPs are poorly characterized in   species Glossina (tsetse fly), the principal vector of the parasite that causes life-threatening Human African Trypanosomiasis and Animal trypanosomiasis in endemic regions in Africa. The tsetse fly has a peculiar reproductive cycle because of the absence of oviposition. Females mate once and store sperm in a spermathecal, and produce a single fully developed larva at a time that pupates within minutes of exiting their uterus. This slow reproductive cycle, compared to other insects, significantly restricts reproduction to only 3 to 6 larvae per female lifespan. This unique reproductive cycle is an attractive vector control strategy entry point. We exploit comparative genomics approaches to explore the diversity of ACPs in the recently available whole genome sequence data from five tsetse fly species ( Glossina morsitans, G. austeni, G. brevipalpis, G. and ). We used previously described ACPs in pallidipes G. fuscipes Drosophila and as reference sequences. We identified melanogaster Anopheles gambiae 36, 27, 31, 29 and 33 diverse ACP orthologous genes in G. austeni, G. and genomes respectively, brevipalpis, G. fuscipes, G. pallidipes G. morsitans which we classified into 21 functional classes. Our findings provide genetic evidence of MAG proteins in five recently sequenced genomes. It Glossina highlights new avenues for molecular studies that evaluate potential field control strategies of these important vectors of human and animal disease. 1,2 1,2 2


Introduction
Accessory gland proteins (ACPs) are important reproductive proteins produced by the male accessory glands (MAGs) of most insect species. These proteins are essential for male insect fertility, and are transferred alongside semen to females during copulation 1 . ACPs trigger significant physiological and behavioral changes in females after copulation, which include: egg laying, reduced sexual receptivity and refractoriness to subsequent inseminations, induce the expression of immune peptides and reduction of female lifespan [1][2][3][4][5] . ACPs are only resynthesized after transfer of seminal fluid to females, but topical application of juvenile hormone on the male's cuticles stimulates in vivo re-synthesis to pre-mating levels 2 . Female Anopheles gambiae mosquitoes copulated by males with degenerate testes and MAGs fail to oviposit and readily re-mate 1 . Conversely, those copulated by males with degenerate testes but fully developed MAGs lay unfertilized eggs and do not re-mate 1 . This underscores the relevance of ACPs as an entry point for vector borne disease control.
ACPs are poorly characterized in Glossina (tsetse fly), compared to Drosophila and Anopheles species 1,6 . The tsetse fly is the principal vector of the parasite that causes life-threatening human (sleeping sickness) and cattle (nagana) trypanosomiasis in endemic regions in Africa 7 . Over 60 million people and 80 million cattle are at risk of contracting disease 8 . Female tsetse flies only mate once during their lifespan and store the male ejaculate in their spermathecae, which they subsequently use to self-fertilize 9 . They have a peculiar reproductive cycle because of the absence of oviposition, with females producing a single fully developed larva at a time that pupates within minutes of exiting their uterus. This slow reproductive cycle, compared to other insects, significantly restricts reproduction to only 3 to 6 larvae per female lifespan 10 . This unique reproductive cycle is an attractive vector control target. An improved understanding of tsetse fly's reproductive biology, and specifically ACPs that are crucial determinants of successful reproduction in other insect species, may provide valuable possible vector control strategy entry points.
Comparisons between ACP gene orthologs in Drosophila simulans and D. melanogaster show they are rapidly evolving, relative to non-ACP genes 11-13 . However, there is strong ACP peptide structural homology between closely related species, which decreases as species phylogenetic distances increase 14 . This rapid rate of ACP genes evolution has made it challenging to reliably identify orthologs across insect species in the absence of genomic data 15,16 . The recently available whole genome sequence data from five tsetse fly species (Glossina morsitans, G. austeni, G. brevipalpis, G. pallidipes and G. fuscipes) has made it possible to revisit detailed examination of ACP gene distribution and genetic diversity in tsetse flies. We exploit comparative genomics approaches to interrogate these genomes, using previously described ACPs in D. melanogaster and A. gambiae as reference sequences. Determining the direction and extent of selection pressure The magnitude and direction of selection pressure on the ACP sequences was tested based on the ratio (ω = d N /d S ) of the average number of non-synonymous substitutions per non-synonymous site (d N ) to the average number of synonymous substitutions per synonymous site (d S ). If ω = 1, amino acid substitution is assumed to be under neutral selection, ω > 1 is indicative of positive selection whereas ω < 1 is evidence of negative or purifying selection. Sequence alignments of each of the ACP clusters

Amendments from Version 1
We have revised the Abstract, Introduction and Discussion sections to address the over-emphasis that ACPS play a crucial role in the tsetse fly life reproductive cycle, and thus vector control.
We have revised the Discussion section to show that this is a preliminary study using the initial release of the Glossina genomes, which are publicly available, and future studies using RNASeq/transcriptome datasets of the male accessory glands and testis may identify rapidly evolving Glossina-specific ACPs, which perhaps do not bear a well-known protein domains, and could escape in silico investigation dependent on a comparative genomic approach alone.
We have amended the Results and Discussion sections to reflect that we identified possible signatures of selection, since a wider analysis including more closely related taxa was not performed.
We used a different approach (outlined in the Methods section with references 17 & 18) to that used by Scolari et al., 2016. Our approach is able to distinguish functional redundancy (orthologs from paralogs) from BLASTp reciprocal hits; and resolve the many-to-many orthologous relationships inherent in comparisons across multiple genomes (reference 17). Only orthologs with a reciprocal BLASTP E-value cut-off 1e-5 and inflation index 2.5 (default settings) were considered for further analysis. We also provide annotated (gene name and exact genome location) sequences for each of the orthologous ACP clusters in the Supplementary materials section.
Further, we have examined transcriptome results from Scolari et al., 2016 (reference 37), which uses a different approach (reciprocal BLASTp hits). This analysis only identified 4 non-annotated ACPs (GMOY000024, GMOY007757, GMOY009744, GMOY012189) orthologous to D. melanogaster not detected in our analysis; and no orthologs to A. gambiae ACPs.

REVISED
containing A. gambiae, D. melanogaster, G. austeni, G. brevipalpis, G. fuscipes, G. morsitans and G. pallidipes were generated. Each alignment was then uploaded to the SNAP program 21 (www.hiv.lanl.gov), which calculates synonymous and nonsynonymous substitution rates to determine the magnitude of selection pressure.

Data visualization
A list of the relative abundance of ACPs with secretory signals identified within each ortholog cluster for Glossina, Anopheles and Drosophila species was generated (Supplementary Figure 1). The 21 ACPs clusters were visually presented in a single circular ideogram using CIRCOS software 22 .

Identification of ACP gene orthologs in Glossina species
We analyzed five recently sequenced Glossina genomes: Glossina morsitans, G. austeni, G. brevipalpis, G. pallidipes and G. fuscipes, to examine the presence of orthologs to ACP genes previously identified in Drosophila melanogaster (n=173) and Anopheles gambiae (n=57) 1,6 . First, we identified 41 ACP ortholog clusters that we broadly classified into 23 groups based on the encoded protein's functional class. These ACPs have a distinct species distribution with: 12 ortholog clusters common to Glossina, A. gambiae, and D. melanogaster species; and some clusters only present in A. gambiae and Glossina species (n=7) or D. melanogaster and Glossina species (n=5) (Supplementary Table 1). The remaining 17 clusters consist of ACP orthologs exclusive to either A. gambiae (n=7) or D. melanogaster (n=10). Next, we shortlisted genes that encode proteins carrying classical secretory signals 23 to distinguish the matched testes-specific secreted male accessory gland proteins from other insect peptides. We identified 36, 27, 31, 29 and 33 ACP orthologs with secretory signals in G. austeni, G. brevipalpis, G. fuscipes, G. pallidipes and Glossina morsitans genomes respectively (Figure 1), across 21 functional class groups. α2-macroglobulins (Group 1) and heat shock proteins (Group 17) are the most abundant ACP orthologs in Glossina species ( Figure 1). Interestingly, Glossina species lack orthologs to Acp70A (Group 18) and andropin (Group 19), which has antimicrobial properties and safeguards the male ejaculate, and stimulation of long-term post mating responses in females respectively 1,24,25 .

Most ACP genes are under positive selection
We inferred the direction and magnitude of selection pressure on the identified ACP orthologs using dN/dS ratios. We observed possible signatures of positive selection in all genes except five are evolving under positive selection ( Figure 1). We found that α2-macroglobulins, which have been shown to be important in mosquito and Drosophila immunity 26,27 , display signatures of purifying selection suggesting they are critical for successful reproduction and all deleterious variations are purged. Our analysis was restricted to reference genes present in A. gambiae and D. melanogaster, and future studies that integrate data from more closely related taxa will highlight evolutionary changes associated with ACPs in more detail. We reconstructed the phylogeny of ACP orthologs within each cluster (Supplementary Figure 1). We failed to identify any pattern associated with the diverse ecological niche and unique reproductive style in Glossina species in our analysis.

ACP distribution in Glossina species
Distribution of ACP orthologs varies widely between species (Figure 1). Glossina species have a disproportionately large number of α2-macroglobublin and heat shock proteins, which are important in immunity in A. gambiae and Glossina species 26-29 . Our analysis did not detect β-defensin orthologs, which are antimicrobial peptides involved in immune responses 30 , in the G. pallidipes and G. brevipalpis genomes. We also did not detect Acp29AB, Acp70A, and andropin orthologs in this comprehensive catalogue of Glossina genes. This raises the possibility that these genes were lost by tsetse flies after evolutionary radiation of insects into multiple taxa, and alternative species-specific proteins might compensate for the same roles.

Discussion
We performed comparative genomics analysis to detect the presence of male accessory gland proteins (ACPs) orthologs previously identified in A. gambiae and Drosophila 1 . The motivation here was to improve knowledge on the biology of Glossina species ACPs given the importance of reproductive molecules in strategic designs of vector control. We identified 21 functional classes of ACP orthologs with secretory signals in five Glossina species genomes. We observed genetic signatures of a high rate of ACP protein divergence, supporting similar findings on male reproduction-related genes in Drosophila 16 . ACPs exhibit high evolutionary changes, thus displaying between species divergence and within species polymorphism 3,31 . We restricted analysis to reference genes present in A. gambiae and D. melanogaster, and future studies integrating datasets from more closely related taxa will be useful to understand evolutionary changes associated with ACPs in more detail. α2-macroglobulins and heat shock proteins are the most abundant ACP orthologs in Glossina species. α2-macroglobulins are important in mosquito and Drosophila immunity 26,27 , and display signatures of purifying selection, suggesting they are critical for successful reproduction and all deleterious variations are purged. α2-macroglobulin over-representation, and the absence of other ACP orthologs implicated in immunity in the Glossina genomes points to their critical role in ensuring successful tsetse fly reproduction. Heat shock protein silencing in A. gambiae down-regulates up to 50% of male accessory gland proteins, half of which are male reproductive tract specific and encode the homologs of 13 known Drosophila ACPs that include Acp70A 1 . Interestingly, Glossina species lack orthologs to Acp70A, andropin, Acp26Ab, Acp29AB, and Acp62F, which play critical roles in successful reproduction in Anopheles and Drosophila species 1,24,25 .
Acp70A or sex peptide stimulates long-term post mating behavior, resulting in non-receptivity to mating and increased oviposition 1,25,32 . Andropin is an antimicrobial peptide transferred to the female during copulation, and defends the female reproductive tract against microbes 33 . Andropin also protects the male ejaculate from Gram-positive and Gram-negative bacterial infections 1 . Acp26Ab stimulates oviposition in Drosophila melanogaster females 34 , and together with Acp26Ab protects the male ejaculate from microbial infections, and displacement  Click here to access the data.

Supplementary
Supplementary Figure 1: Reconstructed phylogenies of the ACP orthologs within each cluster.
Click here to access the data.
by a second ejaculate 1 . Drosophila Acp29AB and Acp62F up-regulate genes for egg production and muscle development, although Acp29AB or Acp62F null males do not show a reproduction impairment phenotype 35 . Acp62F also protects sperm in the female reproductive tract from protease attack 36 .
A limitation in our study was the absence of transcriptome data to measure ACP differential gene expression. Analysis of transcriptome data available to others using a different approach (reciprocal BLASTp hits) identified only 4 non-annotated ACPs (GMOY000024, GMOY007757, GMOY009744, GMOY012189) orthologous to D. melanogaster not detected in our analysis; and no orthologs to A. gambiae ACPs 37 . Future studies focused on transcriptome datasets of male accessory glands and testis may identify rapidly evolving Glossina-specific ACPs, which perhaps do not bear a well-known protein domains, and could escape in silico investigation dependent on a comparative genomic approach alone.
Our analysis detected orthologous Glossina, Anopheles and Drosophila ACPs belonging to the same functional classes, suggesting a conserved role for these proteins across all three genera. However, some ACPs may represent lineage-specific ACPs that may have evolved to perform species-specific reproductive functions. Our findings support evolutionary adaptation to different reproductive styles. Tsetse fly females produce a single fully developed larva at a time that pupates within minutes of exiting their uterus, and may have lost non-essential ACP genes after adaptation.

Conclusions
Our findings provide genetic evidence of male accessory glad proteins in five recently sequenced Glossina genomes. It provides new avenues for molecular studies that evaluate potential field control strategies of these important vectors of human and animal disease.
Glossina species genome sequences were obtained from VectorBase.

Competing interests
No competing interests were disclosed. The paper by Abry ., is aimed at identification of accessory gland proteins (Acps) in five et al Glossina species through bioinformatic approaches. Given the importance of reproductive molecules in strategic designs of vector control, the reported data are of significant value. However, the study is quite preliminary, and hence it feels that authors have gone overboard with the interpretation of these preliminary results. Primarily, authors are required to be clear about the rationale of their study given that the testicular/MAG contributions towards spermatophore formation through transcriptomic and proteomic approaches in has already been reported earlier (Scolari 2016, Scientific Reports). The G. morsitans et al. inclusion of data on reciprocal hits would increase the reliability of predicted/putative Acps. Further, RT-PCR based analysis of the enrichment/expression of at least a couple of identified putative Acp orthologs in male accessory glands (MAGs) from any of the Glossina species would add excellent value to the data and support to the conclusions drawn. Alternatively, authors may compare their datasets with RNAseq/proteomic data of (Scolari , 2016) to assess if the identified genes are G. morsitans et al. enriched/expressed in MAGs or form the part of male contributions to the spermatophore.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
I have read this submission. I 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.

Benard Kulohoma
We thank the reviewers for the comments. Our study is a preliminary exploration of the in silico MAG repertoire of species, and provides initial findings about genome annotations that Glossina identifies ACPs orthologs. Our aim was to establish whether there are male accessory gland (ACPs) orthologs across the Glossina species genomes, by comparing them to well characterised ACP orthologs from and , given the importance of Drosophila melanogaster Anopheles gambiae reproductive molecules in strategic designs of vector control.
We have revised the manuscript to address the over-emphasis that ACPS have a play crucial role in the tsetse fly life reproductive cycle, and thus vector control. Lines 45, 73, 163, and 164.
We have revised the manuscript to show that this is a preliminary study using the initial release of the Glossina genomes, which are publicly available, and future studies using RNASeq/transcriptome datasets of the male accessory glands and testis may identify rapidly evolving Glossina-specific ACPs, which perhaps do not bear a well-known protein domains, and could escape investigation dependent on a comparative genomic in silico approach alone. Lines 194 -201.
We have amended the manuscript to reflect that we identified possible signatures of selection, since a wider analysis including more closely related taxa was not performed.

Benard Kulohoma
We thank the reviewers for the comments. Our study is a preliminary exploration of the in silico MAG repertoire of species, and provides initial findings about genome annotations that Glossina identifies ACPs orthologs. Our aim was to establish whether there are male accessory gland (ACPs) orthologs across the Glossina species genomes, by comparing them to well characterised ACP orthologs from and , given the importance of Drosophila melanogaster Anopheles gambiae reproductive molecules in strategic designs of vector control.
We have revised the manuscript to address the over-emphasis that ACPS have a play crucial role in the tsetse fly life reproductive cycle, and thus vector control. Lines 45, 73, 163, and 164.
We have revised the manuscript to show that this is a preliminary study using the initial release of the Glossina genomes, which are publicly available, and future studies using RNASeq/transcriptome datasets of the male accessory glands and testis may identify rapidly evolving Glossina-specific ACPs, which perhaps do not bear a well-known protein domains, and could escape investigation dependent on a comparative genomic in silico approach alone. Lines 194 -201.
We have amended the manuscript to reflect that we identified possible signatures of selection, since a wider analysis including more closely related taxa was not performed. Lines 137, 140-143, and 165-166.

None declared
Competing Interests: