Revised. 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.

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 ^[@ref-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 ^[@ref-1]--\ [@ref-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 ^[@ref-2]^. Female *Anopheles gambiae* mosquitoes copulated by males with degenerate testes and MAGs fail to oviposit and readily re-mate ^[@ref-1]^. Conversely, those copulated by males with degenerate testes but fully developed MAGs lay unfertilized eggs and do not re-mate ^[@ref-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 ^[@ref-1],\ [@ref-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 ^[@ref-7]^. Over 60 million people and 80 million cattle are at risk of contracting disease ^[@ref-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 ^[@ref-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 ^[@ref-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 ^[@ref-11]--\ [@ref-13]^. However, there is strong ACP peptide structural homology between closely related species, which decreases as species phylogenetic distances increase ^[@ref-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 ^[@ref-15],\ [@ref-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.

Methods
=======

Identification of *Glossina, Anopheles* and *Drosophila* species ACP homologs
-----------------------------------------------------------------------------

The proteomes of *G. austeni, G. morsitans, G. pallidipes, G. fuscipes* and *G. brevipalpis* were retrieved manually from VectorBase ( [www.vectorbase.org](http://www.vectorbase.org)). The retrieved *Glossina* protein sequences alongside previously described ACP sequences from *A. gambiae* (n=57) and *D. melanogaster* (n=173) ( [Supplementary Table 1](#ST1){ref-type="other"}) ^[@ref-1]^, used as references, were assigned to homologous clusters using OrthoMCL with default settings (BLASTP E-value cut-off 1e-5 and inflation index 2.5) ^[@ref-17]^ ( [Supplementary File 1](#SF2){ref-type="other"}). Clusters with singletons were omitted from further processing. Mapped orthologs were subsequently processed using BMX ^[@ref-18]^.

Sequence alignment and phylogeny reconstruction
-----------------------------------------------

Multiple sequence alignments were performed using MUSCLE ^[@ref-19]^. Maximum likelihood (ML) phylogenetic analysis of the multiple aligned sequences with bootstrap values of 100 replicates was performed using PHYML version 3.5 ^[@ref-20]^.

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 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 ^[@ref-21]^ ( [www.hiv.lanl.gov](http://www.hiv.lanl.gov)), which calculates synonymous and non-synonymous 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](#SF1){ref-type="other"}). The 21 ACPs clusters were visually presented in a single circular ideogram using CIRCOS software ^[@ref-22]^.

Results
=======

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) ^[@ref-1],\ [@ref-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](#ST1){ref-type="other"}). 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 ^[@ref-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](#f1){ref-type="fig"}), 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](#f1){ref-type="fig"}). 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 ^[@ref-1],\ [@ref-24],\ [@ref-25]^.

![Identification of male accessory gland proteins (ACPs) in five *Glossina* species.\
( **A**) We identified 38 clusters of ACPs, which were classified into 21 functional classes. ( **B**) The identified *Glossina* species ACPs are orthologous to well-characterized ACP genes in *Anopheles gambiae* and *Drosophila melanogaster genomes*. ( **C**) 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~). A ratio greater than 1 indicates positive selection, and a ratio less than 1 indicates purifying selection. ( **D**) The relative abundance of ACPs identified per ortholog cluster. 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. ( **E**) Schematic presentation showing the relative abundance of each ACP functional class in *Glossina* species, *A. gambiae*, and *Drosophila melanogaster*. α2-macroglobulins \[A\] and, heat shock proteins \[Q\] are the most abundant ACPs in *Glossina* species.](wellcomeopenres-2-14370-g0000){#f1}

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](#f1){ref-type="fig"}). We found that α2-macroglobulins, which have been shown to be important in mosquito and *Drosophila* immunity ^[@ref-26],\ [@ref-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](#SF1){ref-type="other"}). 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](#f1){ref-type="fig"}). *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 ^[@ref-26]--\ [@ref-29]^. Our analysis did not detect β-defensin orthologs, which are antimicrobial peptides involved in immune responses ^[@ref-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* ^[@ref-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* ^[@ref-16]^. ACPs exhibit high evolutionary changes, thus displaying between species divergence and within species polymorphism ^[@ref-3],\ [@ref-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 ^[@ref-26],\ [@ref-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 ^[@ref-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 ^[@ref-1],\ [@ref-24],\ [@ref-25]^.

Acp70A or sex peptide stimulates long-term post mating behavior, resulting in non-receptivity to mating and increased oviposition ^[@ref-1],\ [@ref-25],\ [@ref-32]^. Andropin is an antimicrobial peptide transferred to the female during copulation, and defends the female reproductive tract against microbes ^[@ref-33]^. Andropin also protects the male ejaculate from Gram-positive and Gram-negative bacterial infections ^[@ref-1]^. Acp26Ab stimulates oviposition in *Drosophila melanogaster* females ^[@ref-34]^, and together with Acp26Ab protects the male ejaculate from microbial infections, and displacement by a second ejaculate ^[@ref-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 ^[@ref-35]^. Acp62F also protects sperm in the female reproductive tract from protease attack ^[@ref-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 ^[@ref-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.

Data availability
=================

The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Abry MF et al.

*Anopheles gambiae* and *Drosophila melanogaster* ACPs were obtained from: DOI, [10.1073/pnas.0703904104](http://dx.doi.org/10.1073/pnas.0703904104) ^[@ref-1]^.

*Glossina* species genome sequences were obtained from [VectorBase](https://www.vectorbase.org/taxonomy/glossina).

We would like to thank Abraham Parwos, Anne Owiti, Edwin Rono, and Dr. George Obiero, and support from the University of Nairobi's Centre for Biotechnology and Bioinformatics, and ICIPE.

This work is published with permission from the University of Nairobi and ICIPE.

Supplementary material {#SM1}
======================

**Supplementary Table 1:** (A) *Anopheles gambiae* (n=57) and *Drosophila melanogaster* (n=173) ACP orthologs. (B) ACP ortholog clusters identified in *Glossina*, *A. gambiae*, and *D. melanogaster* species prior to signal-peptide filtering.

Click here for additional data file.

**Supplementary File 1:** Amino acid sequences of the ACP ortholog clusters.

Click here for additional data file.

**Supplementary Figure 1:** Reconstructed phylogenies of the ACP orthologs within each cluster.

Click here for additional data file.

10.21956/wellcomeopenres.14370.r28295

Referee response for version 2

Ram

Kristipati Ravi

1

Referee

Embryotoxicology Laboratory, Environmental Toxicology Group, Council of Scientific & Industrial Research (CSIR)- Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India

**Competing interests:**No competing interests were disclosed.

6

12

2017

Version 2

Authors have adequately addressed the concerns raised on the previous version.

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.

10.21956/wellcomeopenres.13476.r26501

Referee response for version 1

Ram

Kristipati Ravi

1

Referee

Embryotoxicology Laboratory, Environmental Toxicology Group, Council of Scientific & Industrial Research (CSIR)- Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India

**Competing interests:**No competing interests were disclosed.

23

10

2017

Version 1

The paper by Abry *et al*., is aimed at identification of accessory gland proteins (Acps) in five *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 *G. morsitans* has already been reported earlier (Scolari *et al.* 2016, Scientific Reports). The 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 *G. morsitans* (Scolari *et al.*, 2016) to assess if the identified genes are enriched/expressed in MAGs or form the part of male contributions to the spermatophore.

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.

Kulohoma

Benard

**Competing interests:**None declared

20

11

2017

We thank the reviewers for the comments. Our study is a preliminary *in silico* exploration of the MAG repertoire of *Glossina* species, and provides initial findings about genome annotations that 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 *Drosophila melanogaster* and *Anopheles gambiae*, given the importance of 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 *in silico* investigation dependent on a comparative genomic 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.

10.21956/wellcomeopenres.13476.r25531

Referee response for version 1

Mancini

Emiliano

1

Referee

Department of Science, Roma Tre University, Rome, Italy

**Competing interests:**No competing interests were disclosed.

4

9

2017

Version 1

The paper by Abry *et al.* presents a comparative genomics analysis to identify male accessory gland proteins in *Glossina*species. The paper is an \" *in silico*\" exploration of the MAG repertoire of *Glossina*, and has the merit to open the road to further investigation on these important reproductive proteins and their functions. However I think that the authors should give the right relevance to their results, which are, in a sense, quite preliminary, but presented, in my opinion, with too much emphasis.

First of all the authors have to clarify the aim of the work, especially in the context of vector control. Although it is true that a deep understanding of ACPS in *Glossina* can be useful for limit fertility of *Glossina* species and thus control their spread (these proteins have important roles in female post-mating behaviour, such as single mating, storing and sustaining viability of sperms etc.), I think that ACPS do not necessarily have a realistic connection with the peculiar post-mating characteristic of the reproductive cycle of *Glossina*(e.g. absence of eggs deposition). Since this point is recurrently discussed and emphasized in the paper, I suggest the author either to better explain how the ACPS can have a role on the origin of this particular life cycle, or to eliminate this point which, otherwise, remains quite speculative.

It is important that authors stress the importance that their work should be completed in the future by RNAseq of male accessory glands and testis of *Glossina*. This because some rapidly evolving ACPS could escape *in silico*investigation, as they could be *Glossina*-specific and not corresponding to any ortholog, or simply because they do not bear a well-known protein domain. Species-specific proteins can not be obtained by a comparative genomic approach alone, so that RNA analysis is required to reveal (probably) the most important, peculiar ACPS in this important vector.

Finally, I would be very cautious in assigning positive selection to genes with dN/dS\>1, since a wider analysis including more closely related taxa (and data on their intra-specific variability) should be performed. However, based on the dN/dS estimation here provided, I think that the authors should just argue about \"possible signatures of positive selection\" on these genes.

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.

Kulohoma

Benard

**Competing interests:**None declared

20

11

2017

We thank the reviewers for the comments. Our study is a preliminary *in silico* exploration of the MAG repertoire of *Glossina* species, and provides initial findings about genome annotations that 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 *Drosophila melanogaster* and *Anopheles gambiae*, given the importance of 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 *in silico*investigation dependent on a comparative genomic 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.

[^1]: **Competing interests:**No competing interests were disclosed.
