# Comparative Genomics of Glossina palpalis gambiensis and G. morsitans morsitans to Reveal Gene Orthologs Involved in Infection by Trypanosoma brucei gambiense

Illiassou Hamidou Soumana, Bernadette Tchicaya, Stéphanie Rialle, Hugues Parrinello, Anne Geiger

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Illiassou Hamidou Soumana, Bernadette Tchicaya, Stéphanie Rialle, Hugues Parrinello, Anne Geiger. Comparative Genomics of Glossina palpalis gambiensis and G. morsitans morsitans to Reveal Gene Orthologs Involved in Infection by Trypanosoma brucei gambiense. Frontiers in Microbiology, 2017, 8, pp.540. 10.3389/fmicb.2017.00540. hal-02506528

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# Comparative Genomics of *Glossina palpalis gambiensis* and *G. morsitans morsitans* to Reveal Gene Orthologs Involved in Infection by *Trypanosoma brucei* gambiense

Illiassou Hamidou Soumana<fnref n="1" />, Bernadette Tchicaya<fnref n="1" />, Stéphanie Rialle²,³,⁴,⁵,
Hugues Parrinello²,³,⁴,⁵ and Anne Geiger<fnref n="1" />*

OPEN ACCESS

**Edited by:**
Alexandre Morrot,
Federal University of Rio de Janeiro,
Brazil

**Reviewed by:**
Cello Geraldo Freire De Lima,
Federal University of Rio de Janeiro,
Brazil
Elisangela Oliveira De Freitas,
University of Oxford, UK
Geneviève Milon,
Institut Pasteur (INSERM), France

*Correspondence:
Anne Geiger
anne.geiger@ird.fr

**Specialty section:**
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Microbiology

Received: 01 December 2016

Accepted: 14 March 2017

Published: 03 April 2017

**Citation:**
Hamidou Soumana I, Tchicaya B, Rialle S, Parrinello H and Geiger A (2017) Comparative Genomics of Glossina palpalis gambiensis and G. morsitans morsitans to Reveal Gene Orthologs Involved in Infection by Trypanosoma brucei gambiense. *Front. Microbiol.* 8:540. doi: 10.3389/fmicb.2017.00540

Blood-feeding *Glossina palpalis gambiense* (Gpg) fly transmits the single-celled eukaryotic parasite *Trypanosoma brucei gambiense* (Tbg), the second *Glossina* fly African trypanosome pair being *Glossina morsitans/T.brucei* rhodesiense. Whatever the *T. brucei* subspecies, whereas the onset of their developmental program in the zoo-anthropophilic blood feeding flies does unfold in the fly midgut, its completion is taking place in the fly salivary gland where does emerge a low size metacyclic trypomastigote population displaying features that account for its establishment in mammals-human individuals included. Considering that the two *Glossina*—*T. brucei* pairs introduced above share similarity with respect to the developmental program of this African parasite, we were curious to map on the *Glossina morsitans morsitans* (Gmm), the Differentially Expressed Genes (DEGs) we listed in a previous study. Briefly, using the gut samples collected at days 3, 10, and 20 from Gpg that were fed or not at day 0 on Tbg—hosting mice, these DGE lists were obtained from RNA seq—based approaches. Here, post the mapping on the quality controlled DEGs on the Gmm genome, the identified ortholog genes were further annotated, the resulting datasets being compared. Around 50% of the Gpg DEGs were shown to have orthologs in the Gmm genome. Under one of the three *Glossina* midgut sampling conditions, the number of DEGs was even higher when mapping on the Gmm genome than initially recorded. Many Gmm genes annotated as “Hypothetical” were mapped and annotated on many distinct databases allowing some of them to be properly identified. We identify *Glossina* fly candidate genes encoding (a) a broad panel of proteases as well as (b) chitin-binding proteins, (c) antimicrobial peptide production—Pro3 protein, transferrin, mucin, atttacin, cecropin, etc—to further select in functional studies, the objectives being to probe and validated fly genome manipulation that prevents the onset of the developmental program of one or the other *T. brucei* spp. stumpy form sampled by one of the other bloodfeeding *Glossina* subspecies.

**Keywords:** human African Trypanosomiasis, *Glossina palpalis gambiensis*, *Glossina morsitans morsitans*, *Trypanosoma brucei gambiense*, differentially expressed genes, heterologous genes

<footnote n="1">¹ UMR 177, Institut de Recherche pour le Développement-CIRAD, CIRAD TA A-17/G, Montpellier, France, ² Centre National de la Recherche Scientifique Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Montpellier, France,
³ Institut National de la Santé Et de la Recherche Médicale U661, Montpellier, France, ⁴ Universités de Montpellier 1 and 2,
UMR 5203, Montpellier, France, ⁵ Montpellier GenomiX, c/o Institut de Génomique Fonctionnelle, Montpellier, France</footnote>
# INTRODUCTION

Trypanosomes causing either Human African Trypanosomiasis (HAT, i.e., sleeping sickness) or Animal African Trypanosomiasis (AAT, i.e., Nagana) are transmitted by Glossina spp. (tsetse flies). These hematophagous flies acquire their parasite during a blood meal on an infected host, and transmit the mature form of the parasite to another host during a subsequent blood meal. Two forms of HAT have been reported: a chronic and an acute form (Hoare, 1972; Aksoy et al., 2014; Beschin et al., 2014). The chronic form, spread throughout 24 sub-Saharan countries of West Africa, is caused by *Trypanosoma brucei gambiense* (Tbg) and is transmitted by *Glossina palpalis*; this form represents over 90% of all sleeping sickness cases (Welburn et al., 2009). The acute form, endemic to 12 East African countries, is caused by *Trypanosoma brucei rhodesiense* (Tbr), and is transmitted by *Glossina morsitans morsitans* (Gmm). Currently the disease persists in sub-Saharan countries (Louis et al., 2002), where more than 60 million people are exposed to the trypanosomiasis risk. Progress in deciphering the mechanisms of host-parasite interactions involves identifying the genes encoding the factors that govern tsetse fly vector competence (Vickerman et al., 1988; Maudlin and Welburn, 1994; Van den Abbeele et al., 1999), which may promote the development of anti-vector strategies that are alternative or complementary to current strategies.

Using a microarray approach, we recently investigated the effect of trypanosome ingestion by *G. palpalis gambiensis* (Gpg) flies on the transcriptome signatures of *Sodalis glossinidius* (Farikou et al., 2010; Hamidou Soumana et al., 2014a) and *Wigglesworthia glossinidia* (Hamidou Soumana et al., 2014b), two symbionts of tsetse flies (Aksoy et al., 2014). The aim of this previous work was to identify the genes that are differentially expressed in trypanosome infected vs. non-infected or self-cured (refractory) flies and that, consequently, can be suspected to positively or negatively control fly infection. Similarly, using the RNA-seq *de novo* assembly approach, we investigated the differential expression of *G. p. gambiensis* genes in flies challenged or not with trypanosomes (Hamidou Soumana et al., 2015). Furthermore, transcriptome profiling of *T. b. brucei* development in Gmm has recently been reported (Savage et al., 2016).

Since the acute form of HAT is caused by the Gmm/Tbr vector/parasite “couple,” the identification of molecular targets common to both Gpg and Gmm (i.e., orthologous genes) deserves further consideration. Indeed, identification of these targets would allow the development of common approaches to fight both forms of HAT. As Gpg and Gmm are two separate *Glossina* species, their genomes should display some differences between each other. Furthermore, the Gmm genome and the sequences of the Gpg RNA-seq *de novo* assembled genes have been annotated with reference to two distinct database sets: the first set comprises *Drosophila melanogaster*, *Aedes aegypti*, *Anopheles gambiae*, *Culex quinquefasciatus*, and *Phlebotomus papatasi* (International Glossina Genome Initiative, 2014), whereas the second set comprises *Ceratitis capitata*, *Drosophila melanogaster*, *D. willistoni*, *D. virilis*, *D. mojavensis*, *Acyrthosiphon pisum*, *Hydra magnipapillata*, *Anopheles* sp., *Bombyx* sp., *Aedes* sp., and *Glossina morsitans* (data that were available before the publication of the whole genome sequence;

Hamidou Soumana et al., 2015). This indicates that only the *D. melanogaster* database was common to the two database sets used to annotate the differentially expressed Gpg genes and the Gmm genome, respectively. Thus, for the present study, it was necessary to map the sequences of the Gpg RNA-seq *de novo* assembled genes on the Gmm genome and annotate them on the corresponding database. This has been achieved, and the Gpg genes that were previously shown to be differentially expressed (i.e., stimulated vs. non-stimulated flies, and infected vs. non-infected flies; Hamidou Soumana et al., 2015) were annotated on the Gmm database. Finally, the data resulting from the best hits annotation, which provide a translation product for each gene (and thus its potential biological function and physiological role), were compared with data resulting from the previous annotation of the same genes on the set of above-mentioned databases. The overall results provide a data platform that can be applied for further identification of candidate genes involved in the vector competence of both fly species. Importantly, these data could represent promising targets in the development of new anti-vector strategies in the fight against the chronic or acute forms of sleeping sickness.

# MATERIALS AND METHODS

## Ethical Statement

All animal experiments in this report were conducted according to internationally recognized guidelines. The experimental protocols were approved by the Ethics Committee on Animal Experiments and the Veterinary Department of the Centre International de Recherche Agronomique pour le Développement (CIRAD; Montpellier, France).

## Sample Processing, RNA-Seq Library Preparation, and Sequencing

Samples for this study were previously used to identify the differentially expressed genes (DEGs) in Gpg. The different steps are described in the corresponding report (Hamidou Soumana et al., 2015), as well as pro parte in reports related to the differential expression of *S. glossinidius* and *W. glossinidia* genes (Hamidou Soumana et al., 2014a,b). Sample processing is summarized in Figure 1.

## Preparation and Sequencing of the RNA-Seq Libraries

The sequential steps consisted of: RNA extraction from the pooled midguts of each biological replicate, resuspension of RNA pellets in nuclease-free water, concentration, RNA quantification, and quality control (to confirm the absence of any DNA contamination).

## Generation of RNA-Seq Libraries

RNA-seq libraries were generated using the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina; San Diego, USA). The sequential steps consisted of: mRNA purification from 4 µg total RNA using poly-T oligo-linked magnetic beads; fragmentation of RNA using divalent cations under elevated temperature (Illumina fragmentation buffer); first-strand cDNA
<figure><img src="image_4.png" /><figcaption>**FIGURE 1 | Samples processing.** (*) at day 10 the rate of infected flies was low, thus only 12 infected flies ("I10") could be sampled (instead of 28 at day 20). Self-cured flies are flies that have ingested trypanosomes (while they have taken a blood meal on an infected mouse), but the anal drops of which were trypanosome negative.</figcaption></figure>

synthesis using random oligonucleotides and SuperScript II; second-strand cDNA synthesis using DNA Polymerase I and RNase H; conversion of remaining overhangs into blunt ends via exonuclease/polymerase activities and enzyme removal; and adenylation of 3' ends of cDNA fragments, with ligation of Illumina PE adapter oligonucleotides for further hybridization. Finally, cDNA fragments were selected (preferably 200 bp in length) in which fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail, and the products were purified and quantified using the Agilent DNA assay on the Agilent Bioanalyzer 2100 system.

## Brief Summary of the Pipeline for Generating Quality-Controlled Reads

A total of 12 RNA-seq libraries were prepared, sequenced, and compared, including two biological replicates for each of the NS3, S3, I10, NI10, I20, and I20 samples. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit-cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000 platform, and 100-bp paired-end reads were generated. Image analyses and base calling were performed using the Illumina HiSeq Control Software and Real-Time Analysis component. Demultiplexing was performed using CASAVA 1.8.2. The quality of the raw data was assessed using FastQC (Babraham Institute) and the Illumina software SAV (Sequencing Analysis Viewer). Raw sequencing reads from this

study were exported in the FASTQ format and were deposited at the NCBI Short Read Archive (SRA) with the accession number SRP046074; aligned BAM files are available on request.

## Identification of DEGs Once the Reads Generated from the 12 Gpg Fly Gut RNA Seq Libraries Were Mapped and Annotated on a Panel of Non-insect and Insect Genome Databases, One of Them Being Gmm

The RNA-seq reads that satisfied the quality control (i.e., removal of ambiguous nucleotides, low-quality sequences with quality scores <20, and sequences <15 bp in length) were mapped on the *G. m. morsitans* genome (13,807 scaffolds; International Glossina Genome Initiative, 2014) from VectorBase (www vectorbase.org) and GenBank (accession no. CCAG010000000). This was achieved via the splice junction mapper TopHat 2.0.13 (Kim et al., 2013) using Bowtie 2.1.0 (Langmead and Salzberg, 2012), to align RNA-seq reads to the *Glossina morsitans* genome (GmorY1 assembly, release date: January 2014). Final read alignments with more than 12 mismatches were discarded.

Gene counting (number of reads aligned on each gene) was performed before statistical analysis, using HTSeq count 0.5.3p9 (union mode; Anders et al., 2014). Genes with <10 reads (cumulating all analyzed samples) were filtered and removed. We used the Bioconductor (Gentleman et al., 2004) software package EdgeR (Robinson et al., 2010) 3.6.7. to identify genes
displaying a modified expression profile as a result of fly infection by trypanosomes. Data were normalized using the upper quartile normalization factors, using the quartiles method (Bullard et al., 2010). Genes with an adjusted *p* < 5% according to the False Discovery Rate (FDR) method from Benjamini and Hochberg (1995) were declared differentially expressed.

## Bio Informatics-Based Approaches Aimed to Identify Molecular DEGs in Both Gmm and Gpg Once the Latter are Subverted as T. brucei spp Hosts Per se

Tsetse fly gene orthologs were tentatively identified using BLAST searches (Mount, 2007) with annotation against the NCBI non-redundant (Nr) sequence database, using an *E*-value cut-off of $10^{-5}$ (*E* < 0.00001), according to the best hits against known sequences. This was performed to retrieve orthologous genes with the highest sequence similarity to the given unigenes along with putative functional annotations. The official gene symbols of tsetse fly gene orthologs were used for functional annotation. Along with Nr annotations, the “Database for Annotation, Visualization and Integrated Discovery” (DAVID; Dennis et al., 2003) was used to obtain GO annotations of unigenes. The KEGG pathway annotations of tsetse fly gene orthologs were performed using the BLASTX software against the KEGG database (Wixon and Kell, 2000).

Analyzing the two annotation processes of the Gpg DEGs consisted in comparing the list of the “best hits” resulting from the Gpg DEG annotation on the Gmm database with the list resulting from the Gpg DEG annotation previously performed on a set of other databases (Ceratitis capitata, Drosophila melanogaster, D. willistoni, D. virilis, D. mojavensis, Acyrthosiphon pisum, Hydra magnipapillata, Anopheles sp., Bombyx sp., Aedes sp., and Glossina morsitans; Hamidou Soumana et al., 2015). The first step consisted in mixing the DEGs identified at the three experimental times (3, 10, and 20 days) and removing the duplicates, so as to take into account all recorded DEGs except for one of each. The second step consisted in removing the DEGs in which the annotation (best hit) resulted in “hypothetical” or “uncharacterized” proteins, as well as those identified with a numerical identifier, in order to only consider identified and named proteins. Finally, the names of the proteins (best hits) were standardized and alphabetically classified. This process was performed separately for the DEGs annotated with reference to the Gmm database, as well as those previously annotated on the above-characterized set of other databases. The two final listings were then combined (Microsoft Excel software), and their content was arranged according to the alphabetical order of protein names. This procedure facilitated the detection of the best hits that are common to both annotation processes and their corresponding genes.

# RESULTS

## Mapping of PolyA+ mRNA

A total of 459,555,846 clusters were generated from the 12 RNA-seq libraries. Quality controls were performed to ensure the

reliability of the libraries after removal of ambiguous nucleotides, low-quality sequences (quality scores < 20), and sequences <15 bp in length. Finally, 436,979,101 clean clusters were obtained (Table 1). Clean reads had Phred-like quality scores at the Q20 level (i.e., a sequencing error probability of 0.01). These clean sequenced reads with no strand-specificity were mapped to the Gmm reference genome using TopHat (with Bowtie 2) software in order to identify exon-exon splice junctions and to ensure enough sensitivity in mapping reads with polymorphisms.

Filtering and removing any genes with <10 mapped reads allowed mapping 8,286 (stimulated vs. non-stimulated flies; 3 days), 8,032 (infected vs. refractory flies; 10 days) and 8,101 Gpg genes (infected vs. refractory flies; 20 days) on the Gmm reference genome (International Glossina Genome Initiative, 2014). Further, analyses to reveal differential expression (DE) were performed using the bioinformatics tools HTseq and EdgeR from Bioconductor (http://www.bioconductor.org/), which use the R statistical programming language and are widely accepted for modeling the inherent variation between biological replicates. Figure 2 presents the log₂ fold-change (stimulated vs. non-stimulated flies at day-3 post-infected blood meal) against the log₂ of the reads concentration (log-counts-per-million) for each gene after normalization. The generated cloud shows a log fold-change centered on 0 (ordinate axis), signifying that the libraries are properly normalized. Genes that are differentially expressed between the S and NS samples (*p* < 0.05) are represented in red. Similar results were obtained for the other experimental conditions.

## Identification of DEGs and Functional Annotation

The EdgeR method identified a total of 284, 139, and 59 Gmm genes corresponding respectively to the Gpg DEG samples S3

<table><caption>TABLE 1 | Assembly quality of Gpg libraries at the three different sampling times.</caption><thead><tr><th>Samples</th><th>Number of crude clusters (CC)</th><th>Number of clusters after filtering (CAF)</th><th>% CAF/CC</th></tr></thead><tbody><tr><td>NS 3-day sample<sup>a</sup></td><td>36,002,596</td><td>34,386,734</td><td>95.51</td></tr><tr><td>NS 3-day sample<sup>b</sup></td><td>41,153,580</td><td>39,330,015</td><td>95.57</td></tr><tr><td>S 3-day sample</td><td>32,726,727</td><td>31,257,269</td><td>95.51</td></tr><tr><td>S 3-day sample</td><td>33,386,646</td><td>31,848,385</td><td>95.39</td></tr><tr><td>NI 10-day sample</td><td>33,159,650</td><td>31,593,962</td><td>95.28</td></tr><tr><td>NI 10-day sample</td><td>30,632,671</td><td>29,185,036</td><td>95.27</td></tr><tr><td>I 10-day sample</td><td>42,223,049</td><td>40,108,756</td><td>94.99</td></tr><tr><td>I 10-day sample</td><td>43,418,918</td><td>41,279,341</td><td>95.07</td></tr><tr><td>NI 20-day sample</td><td>41,882,170</td><td>39,688,764</td><td>94.76</td></tr><tr><td>NI 20-day sample</td><td>38,192,692</td><td>36,205,087</td><td>94.80</td></tr><tr><td>I 20-day sample</td><td>40,587,354</td><td>38,401,915</td><td>94.62</td></tr><tr><td>I 20-day sample</td><td>46,189,793</td><td>43,693,837</td><td>94.60</td></tr><tr><td>Total</td><td>459,555,846</td><td>436,979,101</td><td>—</td></tr><tr><td>Mean</td><td>38,296,320</td><td>36,414,925</td><td>95.08</td></tr></tbody></table>

The superscripts *a* and *b* are two replicates of the “non-stimulated samples” at day 3. *Idem* for the other sampling conditions. *S*, stimulated; *NS*, non-stimulated; *NI*, non-infected; *I*, infected.
<figure><img src="image_5.png" /><figcaption>**FIGURE 2** | Smear plot of libraries. Samples are from trypanosome stimulated-/-non-stimulated tsetse fly midguts sampled 3 days post-infected blood meal, after normalization using the upper quartile method.</figcaption></figure>

<table><caption>TABLE 2 | Number of differentially expressed genes in Gpg.</caption><thead><tr><th rowspan="3">Experimental conditions</th><th rowspan="3">Number of identified genes</th><th colspan="4">Significantly differentially expressed genes</th></tr><tr><th rowspan="2">Overall</th><th rowspan="2">Overexpressed</th><th colspan="2">Fold-change</th></tr><tr><th>2 &lt; log<sub>2</sub> FC or log<sub>2</sub> FC &lt; -2</th><th>3 &lt; log<sub>2</sub> FC or log<sub>2</sub> FC &lt; -3</th></tr></thead><tbody><tr><td>S vs. NS (3 days)</td><td>8,286</td><td>284</td><td>229 (80.6%)</td><td>97 (34.1%)</td><td>44 (15.5%)</td></tr><tr><td>I vs. NI (10 days)</td><td>8,032</td><td>139</td><td>119 (85.6%)</td><td>60 (43.1%)</td><td>35 (25.2%)</td></tr><tr><td>I vs. NI (20 days)</td><td>8,101</td><td>59</td><td>37 (62.7%)</td><td>19 (32.2%)</td><td>6 (10.2%)</td></tr></tbody></table>

S, stimulated; NS, non-stimulated; NI, non-infected; I: infected.

vs. NS3 (Supplementary Table S1), I10 vs. NI10 (Supplementary Table S2), and I20 vs. NI20 (Supplementary Table S3), at a *p* < 0.05. Most of these genes were overexpressed regardless of the experimental condition. Specifically, there were 229 out of 284 genes (80.6%) in the day-3 samples (S3 vs. NS3), 119 out of 139 genes (85.6%) in I-10 vs. NI-10 samples, and 37 out of 59 genes (62.7%) in I20 vs. NI20. Furthermore, the number of DEGs were highly differentially overexpressed (log₂ FC > 2) or underexpressed (log₂ FC < -2). Specifically, there were 97 out of 284 DEGs (34%; S3 vs. NS3), 60 out of 139 DEGs (43%; I10 vs. NI10), and 19 out of 59 DEGs (32%; I20 vs. NI20). These data are summarized in **Table 2**. Genes exhibiting a highly differential overexpression or underexpression under the different experimental conditions (i.e., S vs. NS, I10 vs. NI10, and I20 vs. NI20) are grouped together in **Table 3**. Most DEGs encode a wide range of proteases, although 91 DEGs presented in Supplementary Tables S1-S3 could not be properly annotated

(i.e., best hit description = “hypothetical”), signifying that the panel of databases used for the annotation process should be enlarged or that the genes may be specific to the Gmm genome. In addition, several of the DEGs were very highly overexpressed. For example the log₂ FC of GMOY009756, which encodes a trypsin, had a fold-change of 7.14 in S3 vs. NS3 samples, and GMOY002278, which encodes the proteinase inhibitor I2, had a fold-change of 9.47 in I10 vs. NI10. In contrast, some DEGs were underexpressed: the log₂ FC of GMOY005345, which encodes an aspartic peptidase, had a fold-change of -6.51 in I20 vs. NI20 samples. **Table 3** is presented so as to facilitate comparison of differential expression levels for a given gene along the three sampling times. For instance, the levels (in log₂ FC) of GMOY005345, which encodes an aspartic peptidase, are 3.39 (S3 vs. NS3), 2.70 (I10 vs. NI10), and -6.51 (I20 vs. NI20).

**Table 3** also provides the functional annotation data for each gene at each sampling time. To obtain an overview of the
<table><caption>TABLE 3 | Annotation on the *Glossina morsitans morsitans* genome of Gpg genes differentially expressed in response to Tbg infection.</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change<br>log<sub>2</sub> FC</th><th rowspan="2">-encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td colspan="6"><strong>PROTEASES AND PROTEASE INHIBITORS</strong></td></tr><tr><td>GMOY005345</td><td>3.39</td><td>Aspartic peptidase</td><td>GO:0006508 proteolysis</td><td>GO:0004190 aspartic-type<br>endopeptidase activity</td><td>No terms assigned</td></tr><tr><td>GMOY005345</td><td>2.70</td><td>Aspartic peptidase</td><td>GO:0006508 proteolysis</td><td>GO:0004190 aspartic-type<br>endopeptidase activity</td><td>No terms assigned</td></tr><tr><td>GMOY005345</td><td>-6.51</td><td>Aspartic peptidase</td><td>GO:0006508 proteolysis</td><td>GO:0004190 aspartic-type<br>endopeptidase activity</td><td>No terms assigned</td></tr><tr><td>GMOY007305</td><td>2.09</td><td>Destabilase</td><td>No terms assigned</td><td>GO:0003796 lysozyme activity</td><td>No terms assigned</td></tr><tr><td>GMOY007305</td><td>3.00</td><td>Destabilase</td><td>No terms assigned</td><td>GO:0003796 lysozyme activity</td><td>No terms assigned</td></tr><tr><td>GMOY000103</td><td>2.64</td><td>Fat body c-type lysozyme</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY000103</td><td>2.74</td><td>Fat body c-type lysozyme</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY002036</td><td>-2.75</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY003273</td><td>2.30</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY003994</td><td>4.19</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY006266</td><td>3.72</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008964</td><td>3.32</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008965</td><td>3.52</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008966</td><td>3.35</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008966</td><td>4.41</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY009436</td><td>2.11</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY009757</td><td>2.76</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY010768</td><td>2.73</td><td>Peptidase S1A, chymotrypsin-type</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY010768</td><td>2.06</td><td>Peptidase S1</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY002729</td><td>3.01</td><td>Serine protease 1</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY000672</td><td>6.95</td><td>Serine protease 6</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY000672</td><td>6.78</td><td>Serine protease 6</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY009756</td><td>7.14</td><td>Trypsin</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY009756</td><td>3.48</td><td>Trypsin</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008967</td><td>2.55</td><td>Trypsin</td><td>GO:0006508 proteolysis</td><td>GO:0004252 serine-type endopeptidase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY008967</td><td>3.37</td><td>Trypsin-like cysteine/serine peptid.<br>domain</td><td>No terms assigned</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY010488</td><td>6.83</td><td>Immune reactive putative protease<br>inhibitor</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010488</td><td>4.69</td><td>Immune reactive putative protease<br>inhibitor</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change<br>log<sub>2</sub> FC</th><th rowspan="2">Encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td>GMOY002277</td><td>2.31</td><td>Proteinase inhibitor I2, Kunitz<br>metazoa</td><td>No terms assigned</td><td>GO:0004867 serine-type endopeptidase<br>inhibit. Activ.</td><td>No terms assigned</td></tr><tr><td>GMOY002277</td><td>4.10</td><td>Proteinase inhibitor I2, Kunitz<br>metazoa</td><td>No terms assigned</td><td>GO:0004867 serine-type endopeptidase<br>inhibit. Activ.</td><td>No terms assigned</td></tr><tr><td>GMOY002278</td><td>6.54</td><td>Proteinase inhibitor I2, Kunitz<br>metazoa</td><td>No terms assigned</td><td>GO:0004867 serine-type endopeptidase<br>inhibit. Activ.</td><td>No terms assigned</td></tr><tr><td>GMOY002278</td><td>9.47</td><td>Proteinase inhibitor I2, Kunitz<br>metazoa</td><td>No terms assigned</td><td>GO:0004867 serine-type endopeptidase<br>inhibit. Activ.</td><td>No terms assigned</td></tr><tr><td>GMOY008344</td><td>2.94</td><td>Trypsin Inhibitor-like</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008344</td><td>4.38</td><td>Trypsin Inhibitor-like</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td colspan="6"><strong>ESTERASES-HYDROLASES</strong></td></tr><tr><td>GMOY000067</td><td>3.35</td><td>Alkaline phosphatase</td><td>GO:0008152 metabolic<br>process</td><td>GO:0016791 phosphatase activity</td><td>No terms assigned</td></tr><tr><td>GMOY000067</td><td>3.60</td><td>Alkaline phosphatase</td><td>GO:0008152 metabolic<br>process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY004731</td><td>2.06</td><td>Alkaline phosphatase-like,<br>alpha/beta/alpha</td><td>GO:0008152 metabolic<br>process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY006875</td><td>-2.02</td><td>Alkaline phosphatase-like,<br>alpha/beta/alpha</td><td>GO:0008152 metabolic<br>process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY004236</td><td>2.36</td><td>Acylphosphatase-like</td><td>No terms assigned</td><td>GO:0003998 acylphosphatase activity</td><td>No terms assigned</td></tr><tr><td>GMOY006958</td><td>2.60</td><td>Carboxylesterase</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY011249</td><td>2.83</td><td>Carboxylesterase</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012368</td><td>2.53</td><td>Exonuclease</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY007402</td><td>3.85</td><td>Extracellular Endonuclease, subunit<br>A</td><td>No terms assigned</td><td>GO:0016787 hydrolase activity</td><td>No terms assigned</td></tr><tr><td>GMOY012360</td><td>-2.93</td><td>Extracellular Endonuclease, subunit<br>A</td><td>No terms assigned</td><td>GO:0016787 hydrolase activity</td><td>No terms assigned</td></tr><tr><td>GMOY009375</td><td>7.56</td><td>Glycoside hydrolase</td><td>GO:0005975 carbohyd.<br>metabolic process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY012361</td><td>-2.55</td><td>Tsal2 protein precursor</td><td>No terms assigned</td><td>GO:0016787 hydrolase activity</td><td>No terms assigned</td></tr><tr><td>GMOY004309</td><td>2.44</td><td>Thiolase-like</td><td>GO:0008152 metabolic<br>process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td>GMOY007148</td><td>2.10</td><td>Thiolase-like</td><td>GO:0008152 metabolic<br>process</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr><tr><td colspan="6"><strong>BINDING</strong></td></tr><tr><td>GMOY010194</td><td>4.12</td><td>Araucan</td><td>No terms assigned</td><td>GO:0003677 DNA binding</td><td>No terms assigned</td></tr><tr><td>GMOY009525</td><td>7.53</td><td>Armadillo-type fold</td><td>No terms assigned</td><td>GO:0005488 binding</td><td>No terms assigned</td></tr><tr><td>GMOY009611</td><td>2.60</td><td>Barrier- to-autointegration factor,<br>BAF</td><td>No terms assigned</td><td>GO:0003677 DNA binding</td><td>No terms assigned</td></tr><tr><td>GMOY009394</td><td>-5.56</td><td>Basic-leucine zipper domain</td><td>GO:0006355 regulation<br>of transcription</td><td>GO:0003700 sequence-specific</td><td>No terms assigned</td></tr><tr><td>GMOY010195</td><td>5.99</td><td>Caupolican</td><td>GO:0006355 regul. of<br>transcrip,DNA-templated</td><td>GO:0003677 DNA binding</td><td>GO:0005634 nucleus</td></tr><tr><td>GMOY002708</td><td>7.89</td><td>Chitin binding</td><td>GO:0006030 chitin<br>metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY005278</td><td>2.93</td><td>Chitin binding domain</td><td>GO:0006030 chitin<br>metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY003840</td><td>4.12</td><td>Chitin binding domain</td><td>GO:0006030 chitin<br>metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY011054</td><td>6.08</td><td>Chitin binding domain</td><td>GO:0006030 chitin<br>metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY011810</td><td>6.55</td><td>Chitin binding domain</td><td>GO:0006030 chitin<br>metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change log<sub>2</sub> FC</th><th rowspan="2">-encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td>GMOY011809</td><td>8.08</td><td>Pro1 (Chitin related)</td><td>GO:0006030 chitin metabolic process</td><td>GO:0008061 chitin binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY004647</td><td>4.24</td><td>Cupredoxin</td><td>No terms assigned</td><td>GO:0005507 copper ion binding</td><td>No terms assigned</td></tr><tr><td>GMOY004364</td><td>2.90</td><td>Haemolymph juvenile hormone binding</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005487</td><td>3.48</td><td>Lim3</td><td>No terms assigned</td><td>GO:0008270 zinc ion binding</td><td>No terms assigned</td></tr><tr><td>GMOY007084</td><td>2.39</td><td>NAD(P)-binding domain</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY002356</td><td>2.31</td><td>Nucleotide-binding</td><td>No terms assigned</td><td>GO:0000166 nucleotide binding</td><td>No terms assigned</td></tr><tr><td>GMOY002825</td><td>4.47</td><td>Odorant binding protein 2</td><td>No terms assigned</td><td>GO:0005549 odorant binding</td><td>No terms assigned</td></tr><tr><td>GMOY002825</td><td>2.03</td><td>Odorant binding protein 2</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005548</td><td>2.99</td><td>Odorant binding protein 7</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY001476</td><td>2.20</td><td>Odorant binding protein 22</td><td>No terms assigned</td><td>GO:0005549 odorant binding</td><td>No terms assigned</td></tr><tr><td>GMOY008769</td><td>4.95</td><td>Small GTPase</td><td>GO:0007165 signal transduction</td><td>GO:0005525 GTP binding</td><td>GO:0016020 membrane</td></tr><tr><td>GMOY004228</td><td>5.44</td><td>Transferrin family</td><td>GO:0006879 cellular iron ion homeostasis</td><td>GO:0008199 ferric iron binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY004228</td><td>2.63</td><td>Transferrin family, iron binding site</td><td>GO:0006879 cellular iron ion homeostasis</td><td>GO:0008199 ferric iron binding</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY008315</td><td>2.05</td><td>Winged helix-turn-helix DNA-binding domain</td><td>GO:0006355 regul. of transcrip,DNA-templated</td><td>GO:0043565 sequence-specific DNA binding</td><td>No terms assigned</td></tr><tr><td colspan="6" style="background-color: #cccccc; font-weight: bold;">TRANSPORT/TRANSFERASE ACTIVITY</td></tr><tr><td>GMOY004684</td><td>-2.39</td><td>Cellul. retinaldehyde binding/a-tocopherol transport</td><td>GO:0006810 transport</td><td>GO:0005215 transporter activity</td><td>GO:0005622 intracel</td></tr><tr><td>GMOY008601</td><td>2.58</td><td>Fatty acid synthase 3</td><td>GO:0008152 metabolic process</td><td>GO:0016740 transferase activity</td><td>No terms assigned</td></tr><tr><td>GMOY008601</td><td>4.11</td><td>Fatty acid synthase 3</td><td>GO:0008152 metabolic process</td><td>GO:0016740 transferase activity</td><td>No terms assigned</td></tr><tr><td>GMOY008602</td><td>2.02</td><td>Fatty acid synthase 4</td><td>GO:0008152 metabolic process</td><td>GO:0016740 transferase activity</td><td>No terms assigned</td></tr><tr><td>GMOY008602</td><td>-2.55</td><td>Fatty acid synthase 4</td><td>GO:0008152 metabolic process</td><td>GO:0016740 transferase activity</td><td>No terms assigned</td></tr><tr><td>GMOY005442</td><td>2.35</td><td>Lipid transport protein</td><td>GO:0006869 lipid transport</td><td>GO:0005319 lipid transporter activity</td><td>No terms assigned</td></tr><tr><td>GMOY005442</td><td>2.40</td><td>Lipid transport protein</td><td>GO:0006869 lipid transport</td><td>GO:0005319 lipid transporter activity</td><td>No terms assigned</td></tr><tr><td>GMOY003490</td><td>4.50</td><td>Major Facilitator Superfamily transporter</td><td>GO:0055085 transmembrane transport</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY003491</td><td>3.97</td><td>Major Facilitator Superfamily transporter</td><td>GO:0055085 transmembrane transport</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY005103</td><td>2.77</td><td>Major Facilitator Superfamily transporter</td><td>GO:0055085 transmembrane transport</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY007627</td><td>2.09</td><td>Major Facilitator Superfamily transporter</td><td>GO:0055085 transmembrane transport</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY005102</td><td>6.28</td><td>N-acetylgalactosaminytransferase</td><td>GO:0008152 metabolic process</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY011877</td><td>2.37</td><td>Na+ channel, amiloride-sensitive</td><td>GO:0006814 sodium ion transport</td><td>GO:0005272 sodium channel activity</td><td>GO:0016020 membrane</td></tr><tr><td>GMOY009903</td><td>2.75</td><td>Neurotransmitter-gated ion-channel</td><td>GO:0006811 ion transport</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY005934</td><td>2.72</td><td>Pyridoxal phosphate-dependent transferase</td><td>No terms assigned</td><td>GO:0003824 catalytic activity</td><td>No terms assigned</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change log<sub>2</sub> FC</th><th rowspan="2">-encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td>GMOY009343</td><td>8.14</td><td>Sodium:neurotransmitter symporter</td><td>GO:0006836<br>neurotransmitter<br>transport</td><td>GO:0005328 neurotransmitter:Na<br>symporter act</td><td>GO:0016021 integral</td></tr><tr><td>GMOY009343</td><td>6.43</td><td>Sodium:neurotransmitter symporter</td><td>GO:0006836<br>neurotransmitter<br>transport</td><td>GO:0005328 neurotransmitter:Na<br>symporter act</td><td>GO:0016021 integral</td></tr><tr><td>GMOY009386</td><td>2.44</td><td>Sodium:neurotransmitter symporter</td><td>GO:0006836<br>neurotransmitter<br>transport</td><td>GO:0005328 neurotransmitter:Na<br>symporter act</td><td>GO:0016021 integral</td></tr><tr><td>GMOY002486</td><td>2.59</td><td>Two pore domain K channel, TASK<br>family</td><td>GO:0071805 K ion<br>transmemb, transport</td><td>GO:0005267 potassium channel activity</td><td>GO:0016020<br>membrane</td></tr><tr><td>GMOY012088</td><td>4.05</td><td>Tyrosine aminotransferase</td><td>GO:0009072 aromatic<br>amino acid<br>family metabolic process</td><td>GO:0004838 L-tyrosine:2-oxoglutarate<br>aminotransferase Activity</td><td>No terms assigned</td></tr><tr><td colspan="6"><strong>OXIDO-REDUCTION PROCESS</strong></td></tr><tr><td>GMOY001939</td><td>2.50</td><td>Cytochrome P450-4g1</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY002598</td><td>2.15</td><td>Cytochrome P450</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY006475</td><td>2.28</td><td>Cytochrome P450-4g1</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY006761</td><td>2.42</td><td>Cytochrome P450-4g1</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY006761</td><td>-2.08</td><td>Cytochrome P450-4g1</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY007181</td><td>3.49</td><td>Cytochrome P450</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY007652</td><td>3.43</td><td>Cytochrome P450</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY009767</td><td>3.96</td><td>Cytochrome P450</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY009909</td><td>3.35</td><td>Cytochrome P450</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016705 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY007529</td><td>4.49</td><td>Dehydrogenase/reductase</td><td>GO:0008152 metabolic<br>process</td><td>GO:0016491 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY004332</td><td>2.12</td><td>Fatty acyl-CoA reductase</td><td>No terms assigned</td><td>GO:0080019 fatty-acyl-CoA reductase<br>activity</td><td>No terms assigned</td></tr><tr><td>GMOY007497</td><td>6.25</td><td>NADH-cytochrome b-5 reductase 2</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0016491 oxidoreductase activity</td><td>No terms assigned</td></tr><tr><td>GMOY010446</td><td>2.40</td><td>2-oxoglutarate dioxygenase</td><td>GO:0055114<br>oxidation-reduction<br>process</td><td>GO:0050353 trimethyllysine dioxygenase<br>activity</td><td>No terms assigned</td></tr><tr><td colspan="6"><strong>HYPOTHETICAL</strong></td></tr><tr><td>GMOY000215</td><td>4.17</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY000215</td><td>5.24</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY000257</td><td>2.90</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change log<sub>2</sub> FC</th><th rowspan="2">Encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td>GMOY001239</td><td>2.53</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY002434</td><td>3.22</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY002933</td><td>2.07</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY002986</td><td>2.69</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003011</td><td>2.30</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003030</td><td>4.38</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003034</td><td>-2.10</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003158</td><td>2.70</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003197</td><td>2.04</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003830</td><td>2.75</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003830</td><td>3.68</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003974</td><td>2.28</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY003976</td><td>3.89</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY004022</td><td>-2.06</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY004337</td><td>5.80</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY004337</td><td>6.61</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005055</td><td>6.08</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005606</td><td>6.32</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005797</td><td>6.60</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005797</td><td>6.49</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005798</td><td>3.98</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005798</td><td>6.25</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY005799</td><td>2.24</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY006671</td><td>4.00</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY006276</td><td>2.33</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY007187</td><td>3.59</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY007637</td><td>4.06</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008016</td><td>4.65</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008016</td><td>6.67</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008627</td><td>3.64</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY009539</td><td>2.28</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY009540</td><td>2.19</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY009541</td><td>2.40</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY009951</td><td>3.11</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010224</td><td>6.87</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010224</td><td>3.57</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010232</td><td>-2.44</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>xGMOY012069</td><td>9.07</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012069</td><td>5.33</td><td>Hypothetical</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008956</td><td>2.90</td><td>hypothetical conserved protein</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td colspan="6"><strong>MISCELLANEOUS</strong></td></tr><tr><td>GMOY008458</td><td>5.74</td><td>Actin-related protein</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY008368</td><td>2.22</td><td>Adipokinetic hormone receptor isoform A</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY004147</td><td>4.32</td><td>Apolipophorin-III superfamily</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY011562</td><td>2.29</td><td>Cecropin</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY011562</td><td>2.36</td><td>Cecropin</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY011563</td><td>2.77</td><td>Cecropin</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY010882</td><td>3.02</td><td>Chemosensory protein 3</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY007457</td><td>2.47</td><td>Cytochrome b561/ferric reduct transmembrane</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th rowspan="2">Genes</th><th rowspan="2">Fold-change<br>log<sub>2</sub> FC</th><th rowspan="2">Encoded proteins (best hits)</th><th colspan="3">Gene Ontology (GO)</th></tr><tr><th>Biological process</th><th>Molecular function</th><th>Cellular component</th></tr></thead><tbody><tr><td>GMOY003354</td><td>2.48</td><td>Elongase 9</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY003354</td><td>6.25</td><td>Elongase 9</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY008821</td><td>3.10</td><td>Elongase 4</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY009277</td><td>3.72</td><td>Insect cuticle protein</td><td>No terms assigned</td><td>GO:0042302 structural constituent of<br>cuticle</td><td>No terms assigned</td></tr><tr><td>GMOY003876</td><td>2.08</td><td>Insect cuticle protein</td><td>No terms assigned</td><td>GO:0042302 structural constituent of<br>cuticle</td><td>No terms assigned</td></tr><tr><td>GMOY011216</td><td>2.40</td><td>Insect cuticle protein</td><td>No terms assigned</td><td>GO:0042302 structural constituent of<br>cuticle</td><td>No terms assigned</td></tr><tr><td>GMOY002258</td><td>2.03</td><td>Insulin-like</td><td>No terms assigned</td><td>GO:0005179 hormone activity</td><td>GO:0005576 extracel</td></tr><tr><td>GMOY003944</td><td>9.22</td><td>LIM and senesc, cell<br>antigen-like-protein 1</td><td>GO:0009987 cellular<br>process</td><td>GO:0005198 structural molecule activity</td><td>GO:0043226<br>organelle</td></tr><tr><td>GMOY011997</td><td>3.02</td><td>Mammalian NeuroPept, Y like<br>receptor</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY012052</td><td>3.40</td><td>Mammalian NeuroPept, Y like<br>receptor</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral</td></tr><tr><td>GMOY009745</td><td>2.02</td><td>Milk gland protein 1</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY001342</td><td>-2.40</td><td>Milk gland protein 2</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY001342</td><td>2.14</td><td>Milk gland protein 2</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012125</td><td>3.57</td><td>Milk gland protein 3</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY001343</td><td>2.50</td><td>Milk gland protein 6</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012016</td><td>-4.00</td><td>Milk gland protein 8</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012016</td><td>2.36</td><td>Milk gland protein 8</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY012369</td><td>2.24</td><td>Milk gland protein 10</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010160</td><td>2.78</td><td>Mpv17/PMP22</td><td>No terms assigned</td><td>No terms assigned</td><td>GO:0016021 integral<br>membrane</td></tr><tr><td>GMOY009494</td><td>-3.95</td><td>Rhodanese-like domain</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY010675</td><td>2.12</td><td>Single domain Von Willebrand<br>factor type C</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr><tr><td>GMOY007078</td><td>2.52</td><td>Single domain Von Willebrand<br>factor type C</td><td>No terms assigned</td><td>No terms assigned</td><td>No terms assigned</td></tr></tbody></table>

Gpg genes that were previously shown to be differentially expressed (DEGs) 3, 10, and 20 days after being challenged with Tbg were mapped on the Gmm genome and annotated on this reference genome. The table presents Gmm genes that are heterologs of the Gpg DEGs, in addition to the annotation results and the gene ontology. Only highly differential expressed genes (log<sub>2</sub>FC < -2 or log<sub>2</sub>FC > 2) have been considered. **Black** fonts: genes that are differentially expressed in stimulated vs. non-stimulated flies (at day 3 after fly challenge). **Blue** and **Red** fonts: genes differentially expressed at day 10 and day 20 after fly challenge, respectively.

functional groups and categories, we used the GO assignment to classify the functions of the unigenes. According to this process the genes expressed at high levels were classified into three GO groups (Figure 3) and further subdivided into categories: biological process (14 categories), molecular functions (22 categories), and cellular component (6 categories). The category "No terms assigned" was predominant across all GO groups at any investigated time.

## Comparing Gpg Gene Annotation on the Gmm Genome and on a Previously Used Panel of Genomes

The global and detailed results of this comparative approach are presented in Supplementary Table S4. Table 4, which is a refined list of Supplementary Table S4, focuses on the expression of Gmm genes that are similar to Gpg genes previously identified as

differentially expressed in response to Tbg infection. The results indicate that a high number of Gpg DEGs have orthologs in the Gmm genome. Furthermore, a large number of Gpg (22) and Gmm genes (23) encoding serine proteases were identified. Similarly, nine Gpg and nine Gmm genes were identified as encoding chitin binding proteins. Finally, whereas 14 Gmm genes encoding a "Major Facilitator Superfamily transporter" were identified, only one such gene was characterized in Gpg.

## Homologies between Identified Gmm Genes That Are Heterologous to Gpg DEGs with Genes from Other Organisms

In order to identify genes previously annotated as "uncharacterized" or "hypothetical," we used the BLASTx program to identify heterologous genes among various organisms listed in the NCBI databases. Homologies with a
<figure><img src="image_6.png" /></figure>

cut-off $E < 10^{-5}$ and-/-or displaying the highest hits score were selected; the minimum accepted homology level was 60%. **Table 5** presents the results of the recorded annotation, and **Figure 4** presents the species from which genomes the genes to be annotated displayed the best match.

Among the 284 Gmm genes heterologous to the day-3 Gpg DEGs samples, 54 genes showed significant matches with other organisms in the investigated databases. The top homology matches were *Drosophila* sp. (11.1%), *Ceratitis capitata* (13%), and *Musca domestica* (68.5%). The remaining 7.4% of genes matched with either *Homo sapiens* (1.8%), *Lucilia sericata* (3.5%), or *Volvox carteri* (2.1%). Similarly, among the 139 Gmm genes heterologous to the day-10 Gpg DEGs samples, 33 genes showed significant matches with other organisms. The top homology matches were *Drosophila* (18.2%), *Ceratitis capitata* (21.3%), and *Musca domestica* (51.5%). The remaining 9% of genes matched with either *Loxodonta africana* (2.9%) or *Volvox carteri* (6.1%). Finally, among the 59 DEGs from day-20 samples, 12 DEGs displayed significant matches with *Musca domestica* (58.7%), *Ceratitis capitata* (8.3%), or *Drosophila* sp (33%).

Several trends appear when comparing results from the annotation reported in **Table 5** with those reported in Supplementary Tables S1–S3 (or in **Table 3**, regarding the

genes in which the differential expression level was $-2 < \log_2$ FC or $\log_2$ FC > 2). First, many genes were not annotated; second, for genes that were annotated, the fold-change was identical; and finally, several genes that were annotated as “Hypothetical” when mapped on the Gmm genome could be identified when mapped on other databases. This was the case regarding the genes *GMOY003830* (i.e., => *Pherophorin-dz1* protein, when annotated on *Volvox carteri*), *GMOY004337* (i.e., => *Bardet-Biedl syndrome* 4 protein homolog, when annotated on *D. willistoni* or *Ceratitis capitata*), *GMOY005606* (i.e., => *leucine-rich repeat-containing protein 15-like*, when annotated on *Musca domestica*), *GMOY007560* (i.e., => *N-acetylgalactosaminyl transferase 2-like*, when annotated on *C. capitata*), *GMOY007584* (i.e., => *Synaptotagmin-1-like*, when annotated on *C. capitata*), and *GMOY008070* (i.e., => *Pherophorin-dz1 protein*, when annotated on *Volvox carteri*).

DISCUSSION

The chronic and acute forms of sleeping sickness endemic to sub-Saharan Africa are caused by two *Trypanosoma* sub-species, Tbg and Tbr, which are, respectively, transmitted to their
<table><caption>TABLE 4 | Identification of Gmm gene orthologs of Gpg genes on the basis of their expression products.</caption><thead><tr><th>Glossina palpalis gambiensis genes</th><th>Best hit description-/name of the encoded proteins</th><th>Glossina morsitans morsitans genes</th></tr></thead><tbody><tr><td>GLOS ARP3.1.1</td><td>Actin-related protein [Drosophila melanogaster]</td><td></td></tr><tr><td></td><td>Actin-related protein</td><td>GMOY008458</td></tr><tr><td>GLOS DVIR GJ17549.1.1</td><td>Acyltransferase—GJ17549 [Drosophila virilis]</td><td></td></tr><tr><td></td><td>Acyl-CoA N-acyltransferase</td><td>GMOY003123</td></tr><tr><td>GLOS LOC101462532.1.1</td><td>Adenylosuccinate lyase-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Adenylosuccinase</td><td>GMOY002461</td></tr><tr><td>GLOS LOC101450467.1.1</td><td>Alkaline phosphatase-like—membrane-bound [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Alkaline phosphatase</td><td>GMOY000067</td></tr><tr><td>GLOS LOC101455841.1.3</td><td>Alpha-2-macroglobuline—CD109 antigen-like isoform X5 [C. capitata]</td><td></td></tr><tr><td></td><td>Alpha-2-macroglobulin</td><td>GMOY010996</td></tr><tr><td>GLOS LOC101461571.2.2</td><td>Aspartic protease-like (lysosomal) [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Aspartic peptidase</td><td>GMOY005345; GMOY010103</td></tr><tr><td>GLOS DVIR GJ18228.1.1; GLOS FDL.1.2</td><td>Beta-hexosaminidase—GJ18228 [Drosophila virilis/D. melanogaster]</td><td></td></tr><tr><td></td><td>Beta-hexosaminidase domain 2-like</td><td>GMOY001794</td></tr><tr><td>GLOS KCC2A.2.2</td><td>Ca2+/calmodulin-dependent protein kinase type II</td><td></td></tr><tr><td></td><td>Ca2+/calmodulin-dependent protein kinase</td><td>GMOY006719</td></tr><tr><td>GLOS CEC.2.2; GLOS CECC.1.1; GLOS CG10252.2.2</td><td>Cecropin [G. m. morsitans/D. yakuba/D. melanogaster]</td><td></td></tr><tr><td></td><td>Cecropin (anti-microbial peptide)</td><td>GMOY011562; GMOY011563</td></tr><tr><td>GLOS DANA GF24496.1.1; GLOS DANA GF24494.3.12</td><td>Chitin binding—GF24496 [Drosophila ananassae]</td><td></td></tr><tr><td>GLOS DGRI GH11353.5.6; GLOS DGRI GH14440.1.1</td><td>Chitin binding—GH11353 [Drosophila grimshawi]</td><td></td></tr><tr><td>GLOS DMOJ GI10981.2.2; GLOS DMOJ GI13574.3.3</td><td>Chitin binding—GI10981 [Drosophila mojavensis]</td><td></td></tr><tr><td>GLOS DWIL GK11657.1.1; GLOS DWIL GK13541.1.5</td><td>Chitin binding—GK11657 [Drosophila willistoni]</td><td></td></tr><tr><td>GLOS DPER GL15114.1.3</td><td>Chitin binding—GL15114 [Drosophila persimilis]</td><td></td></tr><tr><td></td><td>Chitin binding</td><td>GMOY002708; GMOY003840; GMOY005251; GMOY005278; GMOY009806; GMOY009807; GMOY011054; GMOY011809; GMOY011810</td></tr><tr><td>GLOS LOC101462140.1.1</td><td>Chitinase 3-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Chitinase-like protein Idgf5</td><td>GMOY009161</td></tr><tr><td>GLOS CP305.1.2; GLOS C4AC3.1.1; GLOS CP6G1.1.1; GLOS CP9F2.9.9; GLOS CP6W1.1.1</td><td>Cytochrome P450 305a1 [D. melanogaster]</td><td></td></tr><tr><td></td><td>Cytochrome P450 9f2 [D. melanogaster]</td><td></td></tr><tr><td></td><td>cytochrome P450-4g1</td><td>GMOY001150; GMOY001939; GMOY006475; GMOY006761</td></tr><tr><td></td><td>Cytochrome P450</td><td>GMOY002598; GMOY002627; GMOY005461; GMOY007064; GMOY007181; GMOY007270; GMOY007652; GMOY009767; GMOY009909</td></tr><tr><td>GLOS RN181.1.1</td><td>E3 ubiquitin-protein ligase RNF181 homolog</td><td></td></tr><tr><td></td><td>E3 ubiquitin-protein ligase SINA like</td><td>GMOY002938; GMOY008903</td></tr><tr><td>GLOS ELP2.1.1</td><td>Elongator complex protein 2; D. m. GN = Elp2 PE = 1 SV = 1</td><td></td></tr><tr><td></td><td>Elongase 4; 9</td><td>GMOY008821; GMOY003354</td></tr><tr><td>GLOS LOC101461359.1.1</td><td>Endoplasmic reticulum metallopeptidase 1-like isoform X1 [C. capitata]</td><td></td></tr><tr><td></td><td>Endoplasmic reticulum metallopeptidase 1</td><td>GMOY009845; GMOY010241</td></tr><tr><td>GLOS LOC101454791.1.1</td><td>Enkurin-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Enkurin</td><td>GMOY009600</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 4 | Continued</caption><thead><tr><th>Glossina palpalis gambiensis genes</th><th>Best hit description-/name of the encoded proteins</th><th>Glossina morsitans morsitans genes</th></tr></thead><tbody><tr><td>GLOS LOC101459623.1.5</td><td>Exonuclease 3'-5' domain-containing protein 2-like [C. capitata]</td><td></td></tr><tr><td></td><td>Exonuclease</td><td>GMOY012368</td></tr><tr><td>GLOS LOC101459395.1.1</td><td>Gamma-glutamyl hydrolase-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Gamma-glutamyl hydrolase</td><td>GMOY000946</td></tr><tr><td>GLOS GSTT1.2.5; GLOS GST.1.1</td><td>Glutathione S-transferase 1-1 [L. cuprina/Musca domestica]</td><td></td></tr><tr><td></td><td>Glutathione S-transferase</td><td>GMOY002000; GMOY009373</td></tr><tr><td>GLOS LOC101449625.1.1</td><td>Glyoxalase domain-containing protein 4-like isoform X1 [C. capitata]</td><td></td></tr><tr><td></td><td>Glyoxylase</td><td>GMOY008525</td></tr><tr><td>GLOS DVIR GJ19325.1.1</td><td>G-protein receptor activity—GJ19325 [Drosophila virilis]</td><td></td></tr><tr><td></td><td>G-protein coupled receptor</td><td>GMOY009447</td></tr><tr><td>GLOS DWIL GK15016.1.2</td><td>Haemolymph juvenile hormone binding- GK15016- [D. willistoni]</td><td></td></tr><tr><td></td><td>Haemolymph juvenile hormone binding</td><td>GMOY004364</td></tr><tr><td>GLOS H12.1.1</td><td>Histone H1.2 [Drosophila virilis]</td><td></td></tr><tr><td></td><td>Histone H1</td><td>GMOY002746</td></tr><tr><td>GLOS IMDH.2.2</td><td>Inosine-5'-monophosphate dehydrogenase [D. melanogaster]</td><td></td></tr><tr><td></td><td>Inosine-5'-monophosphate dehydrogenase</td><td>GMOY006458</td></tr><tr><td>GLOS LECA.10.13</td><td>Lectin subunit alpha [Sarcophaga peregrina]</td><td></td></tr><tr><td></td><td>Lectin-like C-type</td><td>GMOY001011; GMOY009274</td></tr><tr><td>GLOS LRRX1.1.4</td><td>Leucine-rich repeat-containing protein— [D. discoideum]</td><td></td></tr><tr><td></td><td>Leucine rich repeat containing protein</td><td>GMOY010344</td></tr><tr><td>GLOS LSD1.1.1</td><td>Lipid storage droplets surface-binding protein 1 lipid storage droplet-1</td><td></td></tr><tr><td></td><td>Lipid transporter—GK13707 [Drosophila willistoni]</td><td>GMOY007510</td></tr><tr><td>GLOS DWIL GK13707.1.1</td><td>Lipid transporter protein</td><td></td></tr><tr><td></td><td>Lipid transport protein</td><td>GMOY002410; GMOY005442; GMOY005442;</td></tr><tr><td>GLOS LOC101449088.1.1</td><td>lysM—peptidoglycan-binding domain-containing protein 1-like [C. capitata]</td><td></td></tr><tr><td></td><td>LysM domain</td><td>GMOY008891</td></tr><tr><td>GLOS DPER GL12526.1.1</td><td>Major Facilitator Superfamily-type transporter / — [D. persimilis]</td><td></td></tr><tr><td></td><td>Major Facilitator Superfamily transporter</td><td>GMOY001742; GMOY003428; GMOY003490; GMOY003491;</td></tr><tr><td></td><td></td><td>GMOY003839; GMOY004738; GMOY005103; GMOY005106;</td></tr><tr><td></td><td></td><td>GMOY005109; GMOY005501; GMOY007545; GMOY007627;</td></tr><tr><td></td><td></td><td>GMOY012075; GMOY012352</td></tr><tr><td>GLOS LOC101462556.1.1</td><td>MD-2-related lipid-recognition protein-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>MD-2-related lipid-recognition domain</td><td>GMOY006406</td></tr><tr><td>GLOS MYSN.1.1</td><td>Myosin heavy chain, non-muscle [D. melanogaster]</td><td></td></tr><tr><td></td><td>Myosin heavy chain</td><td>GMOY007533; GMOY008852</td></tr><tr><td>GLOS DMOJ Gl24301.1.1</td><td>Neuropeptide Y receptor—Gl24301 [Drosophila mojavensis]</td><td></td></tr><tr><td></td><td>NeuroPeptide Y like receptor / mammalian (Putative)</td><td>GMOY011997; GMOY012052</td></tr><tr><td>GLOS DGRI GH13991.1.1; GLOS DVIR GJ10540.1.1</td><td>Odorant binding—GH13991 [Drosophila grimshawi]/Drosophila virilis</td><td></td></tr><tr><td>GLOS OB99B.1.1</td><td>Odorant-binding protein 99b</td><td></td></tr></tbody></table>

(Continued)
<table><caption>TABLE 4 | Continued</caption><thead><tr><th>Glossina palpalis gambiensis genes</th><th>Best hit description-/name of the encoded proteins</th><th>Glossina morsitans morsitans genes</th></tr></thead><tbody><tr><td></td><td>Odorant binding protein 1; 2; 7</td><td>GMOY000890; GMOY002825; GMOY005548;</td></tr><tr><td></td><td>Odorant binding protein 21; 22</td><td>GMOY006418; GMOY001476</td></tr><tr><td>GLOS LOC101453268.1.1</td><td>Period circadian protein-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Period circadian protein</td><td>GMOY012110</td></tr><tr><td>GLOS LOC101458811.1.1</td><td>Pyridoxal kinase-like [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>Pyridoxal phosphate-dependent transferase</td><td>GMOY005488; GMOY005934</td></tr><tr><td>GLOS_DMOJ_GI20119.1.1; GLOS_DMOJ_GI16517.2.2;</td><td>Serine proteases (see details in Supplementary Table S4)</td><td>GMOY000672; GMOY002036; GMOY002729; GMOY003271</td></tr><tr><td>GLOS_DSEC_GM17695.1.1; GLOS LOC101456159.4.4;</td><td></td><td>GMOY003273; GMOY003280; GMOY003693; GMOY003994</td></tr><tr><td>GLOS_DMOJ_GI18413.1.2; GLOS_DANA_GF15448.2.3;</td><td></td><td>GMOY006266; GMOY006369; GMOY006991; GMOY008468</td></tr><tr><td>GLOS_AAEL_AAEL007969.1.1; GLOS LOC101461009.2.2;</td><td></td><td>GMOY008469; GMOY008958; GMOY008962; GMOY008964</td></tr><tr><td>GLOS_EAST.2.2; GLOS LOC101457953.1.5;</td><td></td><td>GMOY008965; GMOY008966; GMOY009418; GMOY009436</td></tr><tr><td>GLOS LOC101462986.1.1; GLOS_DWIL_GK19454.1.1</td><td>Serine protease inhibitor (Serpin) GI19420 [D. mojavensis/D. virilis]</td><td>GMOY009757; GMOY010502; GMOY010768</td></tr><tr><td>GLOS LOC101459895.9.9; GLOS_DWIL_GK24139.1.1;</td><td>Serine-type endopeptidase inhibitor— [D. ananassae/D. erecta]</td><td></td></tr><tr><td>GLOS LOC101455430.10.10; GLOS LOC101455604.4.10;</td><td>Serine-type endopeptidase inhibitor /Metalloendopeptidase [D. willistoni]</td><td></td></tr><tr><td>GLOS_DMOJ_GI21244.4.5; GLOS_DMOJ_GI24442.1.1;</td><td>Serine proteinase inhibitors (Kazal domain)</td><td>GMOY010058</td></tr><tr><td>GLOS_DVIR_GJ21497.1.3; GLOS_DVIR_GJ22718.8.10;</td><td>Single domain von Willebrand factor type C [D. willistoni]</td><td></td></tr><tr><td>GLOS_DVIR_GJ21498.1.1; GLOS_DVIR_GJ21499.1.1;</td><td>Single domain von Willebrand factor type C</td><td>GMOY003774; GMOY005237; GMOY007078; GMOY010675</td></tr><tr><td>GLOS_DMOJ_GI19420.1.1; GLOS_DVIR_GJ17584.1.1</td><td>Sulfate transmembrane transporter—GK22031 [Drosophila willistoni]</td><td></td></tr><tr><td>GLOS_DWIL_GK10999.1.1; GLOS LOC101459846.1.2</td><td>Sulfate transporter</td><td>GMOY000550</td></tr><tr><td>GLOS_DWIL_GK15974.5.7; GLOS_DMOJ_GI22128.1.1</td><td>Timeless-like isoform X1 protein (circadian rhythm regulation) [C. capitata]</td><td></td></tr><tr><td>GLOS_DWIL_GK22031.1.1</td><td>Timeless protein</td><td>GMOY006112</td></tr><tr><td>GLOS_TRF.1.1</td><td>Transferrin [Sarcophaga peregrina]</td><td></td></tr><tr><td>GLOS LOC101463325.1.1</td><td>Transferrin family, iron binding site</td><td>GMOY004228</td></tr><tr><td>GLOS_DWIL_GK18237.1.1</td><td>Trypsin-like [Ceratitis capitata]</td><td></td></tr><tr><td>GLOS LOC101451574.1.1</td><td>Trypsin-like cysteine/serine peptidase domain</td><td>GMOY002535; GMOY008308</td></tr><tr><td>GLOS LOC101458997.1.1</td><td>UDP-glucuronosyl/UDP-glucosyltransferase—GK18237 [D. willistoni]</td><td></td></tr><tr><td></td><td>UDP-glucuronosyl/UDP-glucosyltransferase</td><td>GMOY007046</td></tr><tr><td></td><td>WD repeat-containing protein 81-like isoform X1 [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>WD40/YVTN repeat-like-containing domain</td><td>GMOY010092</td></tr><tr><td></td><td>Yellow-like protein (protein of the gelly) [Ceratitis capitata]</td><td></td></tr><tr><td></td><td>yolk protein 3</td><td>GMOY006227</td></tr></tbody></table>

The table compares the results of the previous Gpg DEG annotation on a panel of various genomes with the corresponding annotation on the Gmm genome. A comparison is presented between the Gpg and the Gmm genes on the basis of the identified proteins they encode.
<table><caption>TABLE 5 | Gmm gene heterologs of Gpg DEGs matching genes from other organism databases.</caption><thead><tr><th>Genes</th><th>log<sub>2</sub> FC</th><th>Homology with other organisms (&gt;60%)</th></tr></thead><tbody><tr><td>GMOY000550</td><td>-0.90</td><td>Musca domestica sodium-independent sulfate anion transporter-like (LOC101893700), mRNA</td></tr><tr><td>GMOY001137</td><td>1.09</td><td>Musca domestica aminomethyltransferase, mitochondrial-like (LOC101895864), mRNA</td></tr><tr><td>GMOY001742</td><td>-0.9</td><td>Ceratitis capitata synaptic vesicle glycoprotein 2B-like (LOC101452461), transcript variant X3, mRNA</td></tr><tr><td>GMOY002004</td><td>-1.78</td><td>Musca domestica putative fatty acyl-CoA reductase CG5065-like (LOC101898308), mRNA</td></tr><tr><td>GMOY002004</td><td>1.8</td><td>Musca domestica putative fatty acyl-CoA reductase CG5065-like (LOC101898308), mRNA</td></tr><tr><td>GMOY002024</td><td>0.96</td><td>Musca domestica phosphotriesterase-related protein-like (LOC101890186), mRNA</td></tr><tr><td>GMOY002356</td><td>2.31</td><td>C. capitata CUGBP Elav-like family member 2-like (LOC101455154), transcript variant X1 to X3, mRNA</td></tr><tr><td>GMOY002461</td><td>0.82</td><td>Musca domestica adenylosuccinate lyase-like (LOC101900029), mRNA</td></tr><tr><td>GMOY002486</td><td>2.6</td><td>Musca domestica potassium channel subfamily K member 9-like (LOC101895107), mRNA</td></tr><tr><td>GMOY002535</td><td>1.5</td><td>Ceratitis capitata serine protease easter-like (LOC101451852), mRNA</td></tr><tr><td>GMOY002729</td><td>1.91</td><td>Lucilia sericata clone LScDNA1 putative salivary trypsin mRNA, complete cds</td></tr><tr><td>GMOY002729</td><td>3.0</td><td>Musca domestica serine proteinase stubble-like (LOC101890358), mRNA</td></tr><tr><td>GMOY002938</td><td>-1.10</td><td>Musca domestica uncharacterized LOC101893009 (LOC101893009), mRNA</td></tr><tr><td>GMOY003158</td><td>2.70</td><td>Musca domestica uncharacterized LOC101895341 (LOC101895341), mRNA</td></tr><tr><td>GMOY003161</td><td>1.44</td><td>Musca domestica thyrotropin receptor-like (LOC101887582), mRNA</td></tr><tr><td>GMOY003354</td><td>2.48</td><td>Ceratitis capitata elongation of very long chain fatty acids protein AAEL008004-like (LOC101449680), mRNA</td></tr><tr><td>GMOY003354</td><td>6.3</td><td>Musca domestica elongation of very long chain fatty acids protein AAEL008004-like (LOC101893043), mRNA</td></tr><tr><td>GMOY003443</td><td>1.27</td><td>Musca domestica uncharacterized LOC101889318 (LOC101889318), partial mRNA</td></tr><tr><td>GMOY003590</td><td>1.77</td><td>Musca domestica collagen alpha-1(IV) chain-like (LOC101897761), transcript variant X3, mRNA</td></tr><tr><td>GMOY003590</td><td>1.3</td><td>Musca domestica collagen alpha-1(IV) chain-like (LOC101897761), transcript variant X3, mRNA and variant X1, X2</td></tr><tr><td>GMOY003830</td><td>2.75</td><td>Homo sapiens BAC clone CH17-465115 from chromosome unknown, complete sequence (=hypothetical)</td></tr><tr><td>GMOY003830</td><td>3.7</td><td>Volvox carteri f, nagariensis mRNA for pherophorin-dz1 protein</td></tr><tr><td>GMOY003839</td><td>0.95</td><td>Musca domestica putative inorganic phosphate cotransporter-like (LOC101889974), mRNA</td></tr><tr><td>GMOY003949</td><td>1.01</td><td>M. domestica glutamine-fructose-6-phosphate aminotransferase [isomerizing] 2-like (LOC101889985), transcript</td></tr><tr><td>GMOY004309</td><td>2.44</td><td>Musca domestica fatty acid synthase-like (LOC101893120), mRNA</td></tr><tr><td>GMOY004332</td><td>2.1</td><td>Drosophila willistoni GK20732 (Dwil\GK20732), mRNA /Fatty acyl-CoA reductase</td></tr><tr><td>GMOY004337</td><td>5.8</td><td>D. willistoni GK20950 (Dwil\GK20950), mRNA Bardet-Biedl syndrome 4 protein homolog (=hypothetical on Gmm genome)</td></tr><tr><td>GMOY004337</td><td>6.6</td><td>Ceratitis capitata Bardet-Biedl syndrome 4 protein homolog (LOC101449311), mRNA (=hypothetical on Gmm genome)</td></tr><tr><td>GMOY004589</td><td>1.09</td><td>Musca domestica muscle M-line assembly protein unc-89-like (LOC101890868) mRNA</td></tr><tr><td>GMOY004712</td><td>1.65</td><td>Musca domestica acyl-protein thioesterase 1-like (LOC101890399), mRNA</td></tr><tr><td>GMOY004738</td><td>-0.78</td><td>Musca domestica facilitated trehalose transporter Tret1-like (LOC101891733), transcript variant X1, Mrna</td></tr><tr><td>GMOY004873</td><td>1.6</td><td>Musca domestica transmembrane and TPR repeat-containing protein CG4341-like (LOC101893859), mRNA</td></tr><tr><td>GMOY005102</td><td>6.28</td><td>Musca domestica N-acetylgalactosaminyltransferase 4-like (LOC101894376), mRNA</td></tr><tr><td>GMOY005106</td><td>-1.22</td><td>Drosophila willistoni GK13266 (Dwil\GK13266), mRNA / Major facilitator superfamily transporter</td></tr><tr><td>GMOY005278</td><td>2.93</td><td>Musca domestica mucin-5AC-like (LOC101899868), mRNA</td></tr><tr><td>GMOY005278</td><td>1.9</td><td>Musca domestica mucin-5AC-like (LOC101899868), mRNA</td></tr><tr><td>GMOY005345</td><td>-6.5</td><td>Musca domestica lysosomal aspartic protease-like (LOC101894831), mRNA</td></tr><tr><td>GMOY005487</td><td>3.48</td><td>Musca domestica LIM/homeobox protein Lhx4-like (LOC101900654), mRNA</td></tr><tr><td>GMOY005488</td><td>1.81</td><td>Musca domestica alpha-methyldopa hypersensitive protein-like (LOC101888467), mRNA</td></tr><tr><td>GMOY005527</td><td>0.87</td><td>Musca domestica c-1-tetrahydrofolate synthase, cytoplasmic-like (LOC101891351), transcript variant X2, mRNA</td></tr><tr><td>GMOY005606</td><td>6.3</td><td>Musca domestica leucine-rich repeat-containing protein 15-like (LOC101899894), mRNA (=hypothetical on Gmm genome)</td></tr><tr><td>GMOY005934</td><td>2.72</td><td>Ceratitis capitata cysteine sulfinic acid decarboxylase-like (LOC101455610), mRNA</td></tr><tr><td>GMOY006111</td><td>1.1</td><td>Drosophila willistoni GK14673 (Dwil\GK14673), mRNA (Gonadal trypsine)</td></tr><tr><td>GMOY006205</td><td>1.2</td><td>Ceratitis capitata DNA replication licensing factor Mcm5-like (LOC101458261), mRNA</td></tr><tr><td>GMOY006406</td><td>1.69</td><td>Musca domestica ecdysteroid-regulated 16 kDa protein-like (LOC101898283), mRNA</td></tr><tr><td>GMOY006406</td><td>1.7</td><td>Musca domestica ecdysteroid-regulated 16 kDa protein-like (LOC101898283), mRNA</td></tr><tr><td>GMOY006458</td><td>-0.78</td><td>Musca domestica inosine-5'-monophosphate dehydrogenase-like (LOC101895820), mRNA</td></tr><tr><td>GMOY006671</td><td>4.0</td><td>Musca domestica uncharacterized LOC101890025 (LOC101890025), mRNA</td></tr><tr><td>GMOY006761</td><td>1.99</td><td>Musca domestica cytochrome P450 CYP4G13v2 mRNA, complete cds</td></tr><tr><td>GMOY006761</td><td>2.4</td><td>Musca domestica cytochrome P450 CYP4G13v2 mRNA, complete cds</td></tr><tr><td>GMOY006761</td><td>-2.1</td><td>Musca domestica cytochrome P450 CYP4G13v2 mRNA, complete cds</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 5 | Continued</caption><thead><tr><th>Genes</th><th>log<sub>2</sub> FC</th><th>Homology with other organisms (&gt;60%)</th></tr></thead><tbody><tr><td>GMOY006875</td><td>-2.0</td><td>Musca domestica membrane-bound alkaline phosphatase-like (LOC101896753), mRNA</td></tr><tr><td>GMOY006875</td><td>-1.8</td><td>Musca domestica membrane-bound alkaline phosphatase-like (LOC101896753), mRNA</td></tr><tr><td>GMOY006979</td><td>1.08</td><td>Musca domestica phosrestin-2-like (LOC101892743), mRNA</td></tr><tr><td>GMOY007046</td><td>2.0</td><td>Ceratitis capitata UDP-glucuronosyltransferase 2B13-like (LOC101462823), transcript variant X2, mRNA</td></tr><tr><td>GMOY007131</td><td>0.97</td><td>Musca domestica inositol-3-phosphate synthase-like (LOC101889622), mRNA</td></tr><tr><td>GMOY007148</td><td>2.10</td><td>Ceratitis capitata fatty acid synthase-like (LOC101463409), mRNA</td></tr><tr><td>GMOY007497</td><td>6.3</td><td>Musca domestica NADH-cytochrome b5 reductase 3-like (LOC101897795), transcript variant X2, mRNA</td></tr><tr><td>GMOY007523</td><td>1.44</td><td>Musca domestica collagen alpha-1(IV) chain-like (LOC101895032), transcript variant X3, mRNA</td></tr><tr><td>GMOY007523</td><td>1.1</td><td>Musca domestica collagen alpha-1(IV) chain-like (LOC101895032), transcript variant X3, mRNA</td></tr><tr><td>GMOY007560</td><td>-1.0</td><td>C. capitata polypeptide N-acetylgalactosaminyltransferase 2-like (LOC101448408), mRNA (=hypothetical on Gmm genome)</td></tr><tr><td>GMOY007584</td><td>1.1</td><td>Ceratitis capitata synaptotagmin-1-like (LOC101450559), mRNA (=hypothetical on Gmm genome)</td></tr><tr><td>GMOY008017</td><td>1.88</td><td>Volvox carteri f. nagariensis mRNA for pherophorin-dz1 protein (=hypothetical when mapped on Gmm genome)</td></tr><tr><td>GMOY008017</td><td>1.2</td><td>Volvox carteri f. nagariensis mRNA for pherophorin-dz1 protein (=hypothetical when mapped on Gmm genome)</td></tr><tr><td>GMOY008266</td><td>-1.3</td><td>Drosophila willistoni GK24772 organic anion transporter (Dwil\GK24772), mRNA</td></tr><tr><td>GMOY008308</td><td>1.9</td><td>Drosophila melanogaster easter (ea), transcript variant A, mRNA</td></tr><tr><td>GMOY008458</td><td>5.74</td><td>Musca domestica actin, indirect flight muscle-like (LOC101895248), mRNA</td></tr><tr><td>GMOY008525</td><td>0.97</td><td>Drosophila willistoni GK21885 (Dwil\GK21885), mRNA</td></tr><tr><td>GMOY008601</td><td>2.58</td><td>Musca domestica fatty acid synthase-like (LOC101893120), mRNA</td></tr><tr><td>GMOY008601</td><td>4.1</td><td>Drosophila willistoni GK12914 (Dwil\GK12914), mRNA</td></tr><tr><td>GMOY008602</td><td>2.02</td><td>Drosophila pseudoobscura pseudoobscura GA26263 (Dpse\GA26263), mRNA</td></tr><tr><td>GMOY008602</td><td>-2.5</td><td>Drosophila willistoni GK12914 (Dwil\GK12914), mRNA</td></tr><tr><td>GMOY008852</td><td>0.93</td><td>Musca domestica myosin heavy chain, non-muscle-like (LOC101892851), transcript variant X1 to X3, mRNA</td></tr><tr><td>GMOY008966</td><td>4.4</td><td>Loxodonta africana kallikrein-11-like (LOC100667195), mRNA</td></tr><tr><td>GMOY008973</td><td>0.80</td><td>Lucilia cuprina alpha esterase (LcaE7) mRNA, implicated in organophosphate resistance, complete cds</td></tr><tr><td>GMOY009018</td><td>1.59</td><td>Ceratitis capitata uncharacterized LOC101448539 (LOC101448539), transcript variant</td></tr><tr><td>GMOY009018</td><td>1.8</td><td>Musca domestica uncharacterized LOC101899326 (LOC101899326), mRNA</td></tr><tr><td>GMOY009079</td><td>1.23</td><td>Musca domestica fatty acid synthase-like (LOC101893120), mRNA</td></tr><tr><td>GMOY009375</td><td>7.56</td><td>Musca domestica uncharacterized LOC101900740 (LOC101900740), mRNA</td></tr><tr><td>GMOY009394</td><td>-5.56</td><td>Musca domestica CCAAT/enhancer-binding protein-like (LOC101898926), mRNA</td></tr><tr><td>GMOY009447</td><td>-1.22</td><td>Ceratitis capitata calcitonin gene-related peptide type 1 receptor-like (LOC101462563), mRNA</td></tr><tr><td>GMOY009600</td><td>1.20</td><td>Musca domestica enkurin-like (LOC101897351), mRNA</td></tr><tr><td>GMOY009845</td><td>0.6</td><td>Musca domestica endoplasmic reticulum metallopeptidase 1-like (LOC101898765), transcript variant X3, mRNA</td></tr><tr><td>GMOY009903</td><td>2.75</td><td>M. domestica strain rspin nicotinic acetylcholine receptor beta 3 subunit (nAChRbeta3) gene, nAChRbeta3-C allele, complete cds</td></tr><tr><td>GMOY009983</td><td>1.7</td><td>Drosophila grimshawi GH17190 (Dgri\GH17190), mRNA</td></tr><tr><td>GMOY010224</td><td>6.9</td><td>Musca domestica uncharacterized LOC101889990 (LOC101889990), partial mRNA</td></tr><tr><td>GMOY010224</td><td>3.6</td><td>Musca domestica uncharacterized LOC101889990 (LOC101889990), partial mRNA (=> Hypothetical)</td></tr><tr><td>GMOY010241</td><td>0.8</td><td>Musca domestica endoplasmic reticulum metallopeptidase 1-like (LOC101898765), transcript variant X3, mRNA</td></tr><tr><td>GMOY010481</td><td>-1.53</td><td>Musca domestica protein Wnt-5-like (LOC101892275), mRNA</td></tr><tr><td>GMOY010972</td><td>0.73</td><td>Musca domestica phenoloxidase subunit A3-like (LOC101897997), transcript variant X1 and X2, mRNA</td></tr><tr><td>GMOY011232</td><td>1.32</td><td>Drosophila melanogaster PAPS synthetase (Papss), transcript variant A, mRNA variant A to H</td></tr><tr><td>GMOY011418</td><td>0.87</td><td>Drosophila willistoni GK13980 (Dwil\GK13980), mRNA / glycogen synthase</td></tr><tr><td>GMOY011618</td><td>-0.9</td><td>Ceratitis capitata putative fatty acyl-CoA reductase CG5065-like (LOC101456246), transcript variant X2, mRNA</td></tr><tr><td>GMOY012052</td><td>3.4</td><td>Drosophila pseudoobscura pseudoobscura GA30114 Neuropeptide Y (Dpse\GA30114), mRNA</td></tr><tr><td>GMOY012069</td><td>9.1</td><td>Musca domestica uncharacterized LOC101891108 (LOC101891108), mRNA</td></tr><tr><td>GMOY012069</td><td>5.3</td><td>Musca domestica uncharacterized LOC101891108 (LOC101891108), mRNA</td></tr><tr><td>GMOY012075</td><td>1.56</td><td>Drosophila melanogaster CG31663 (CG31663), transcript variant B, mRNA (Major facilitator superfamily transporter)</td></tr><tr><td>GMOY012075</td><td>-1.7</td><td>Drosophila willistoni GK15555 (Dwil\GK15555), mRNA (Major facilitator superfamily transporter)</td></tr><tr><td>GMOY012352</td><td>0.94</td><td>Ceratitis capitata monocarboxylate transporter 10-like (LOC101448353), transcript variant X1, mRNA</td></tr></tbody></table>

Black fonts, day-3 samples; Blue fonts, day-10 samples; Red fonts, day-20 samples.
<figure><img src="image_7.png" /><figcaption>**FIGURE 4** | Species on which Gmm genes were mapped in order to characterize genes previously annotated as "hypothetical." (A) Mapping of the 284 Gmm genes heterologous to Gpg DEGs in trypanosome stimulated vs. non-stimulated flies (day-3 sampling). (B) Mapping of the 139 Gmm genes heterologous to Gpg DEGs in trypanosome infected vs. non-infected flies (day-10 sampling). (C) Mapping of the 59 Gmm genes heterologous to Gpg DEGs in trypanosome infected vs. non-infected flies (day-20 sampling).</figcaption></figure>

vertebrate hosts by the *Glossina* species Gpg and Gmm (Aksoy et al., 2014; Beschin et al., 2014). Nevertheless, the biological cycles, vertebrate transmission processes, and pathogenicity development of the two parasites are similar. Recently, in the context of an anti-vector strategy project to fight the disease, we performed a global transcriptomic analysis of Gpg gene expression associated with fly infection by Tbg. More precisely, we attempted to characterize genes that were differentially expressed according to the status of the fly at several sampling times (i.e., non-infected, infected, or self-cured). This included genes that could be involved in the fly's vector competence, and consequently genes that could possibly be manipulated in order to reduce or even suppress this competence.

The similarities between the Tbg and Tbr life cycles prompted us to determine whether the Gmm genome carried genes that could be heterologous to the Gpg DEGs, which could then allow the development of common molecular approaches. Accordingly, the Gpg sequences resulting from the previous RNA-seq *de novo* assembly (Hamidou Soumana et al., 2015) were mapped on the Gmm genome, the DEGs were characterized, and the corresponding genes were annotated.

When the Gpg sequences were mapped and annotated on a panel of various databases (C. capitata, D. melanogaster, D. willistoni, D. virilis, D. mojavensis, Acyrthosiphon pisum, Hydra magnipapillata, Anopheles sp., Bombyx sp., Aedes sp., and G. morsitans; Hamidou Soumana et al., 2015) we identified 553

(S vs. NS), 52 (I10 vs. NI10), and 143 (I20 vs. NI20) DEGs. In contrast, we identified 284 (S vs. NS), 139 (I10 vs. NI10) and 59 (I20 vs. NI20) DEGs when sequences were mapped and annotated on the *G. m. morsitans* database (using its whole genome annotated on the *Drosophila melanogaster*, *Aedes aegypti*, *Anopheles gambiae*, *Culex quinquefasciatus*, and *Phlebotomus papatasi* databases; International Glossina Genome Initiative, 2014). The differences in the number of identified DEGs, as well as the high number of “uncharacterized” genes, could be due to differences in the database panels used to annotate Gpg or Gmm. We cannot exclude the possibility that some of the Gpg DEGs do not have heterologous genes in Gmm, or that some of them could be specific to either Gpg or Gmm and consequently cannot be annotated yet. Nevertheless, regarding I10 vs. NI10 sampling (and in contrast to the two other experimental conditions), the number of recorded DEGs was more than 2-fold higher when the Gpg transcripts were mapped on the Gmm genome, prompting questions of how this is possible. However, at this stage of our research we cannot offer a satisfactory explanation.

We examined the potential influence of database panel composition by annotating the Gmm DEGs on a separate set of databases that included *D. melanogaster* as an internal control. The results (Table 5) clearly demonstrate the validity of the annotation process, since all Gmm genes (GMOY, etc.) were annotated (best hit description and fold-change) on the novel set of databases as they had been annotated on the former
set (Table 3), and that several genes could be identified thanks to their annotation primarily on the *Volvox carteri* or *Musca domestica* databases which had never been used before, and despite the fact these organisms (algae and mouse) are genetically distant from the tsetse fly.

The most important observation regarding our objective is that almost all of the Gpg genes previously considered to be potentially involved in tsetse fly vector competence (cf. Hamidou Soumana et al., 2015) had a "countrepart" (i.e., heterologous genes) in the Gmm genome, despite the fact that none of the Gpg DEGs matched with any Gmm genes. This was the case for the large array of genes encoding peptidases, especially serine peptidases (represented by more than 20 genes), identified in the genomes of both fly species. This was similarly observed for ~10 genes present in both genomes that encode chitin binding proteins, since chitin metabolism is involved in the ability of tsetse flies to host trypanosomes (Maudlin and Welburn, 1994; Welburn and Maudlin, 1999), in addition to cecropin (an antimicrobial peptide), among others (Weiss et al., 2014).

Here, we were particularly interested in detecting the presence or not of genes with a reported role in the immunity of tsetse flies or other organisms (Weiss et al., 2014). Genes encoding Pro3 protein (GMOY009756, GMOY000672) and transferrin (GMOY004228) were identified. Pro3 has a potential function as a serine protease (tyrosinase) and is specifically produced by the proventriculus, an organ that plays an important role in the tsetse immune response. This protein could be involved in the immune response via activation of the cascade of prophenol oxidase and melanization (Jiang et al., 1998). Moreover, the gene GMOY0010488 was identified as encoding an "immuno reactive putative protease inhibitor" that is overexpressed in trypanosome stimulated or infected Gpg flies. The transferrin gene was overexpressed in both stimulated and infected Gpg flies; this result is in agreement with Geiser and Winzerling (2012), who reported on the role of transferrin in the immune response of insects, as well as its role in iron transport. By reducing the oxidative stress in tsetse fly guts, transferrin may promote the survival of trypanosomes. Guz et al. (2012) observed transferrin overexpression after challenge with bacteria, even at a higher level than what is typically observed in the case of infection by trypanosomes.

The gene GMOY011809 encodes Pro 1 peritrophin, which is a constituent of the peritrophic membrane (PM). The PM is established after the fly takes its first blood meal, and it is

permanently renewed by the proventriculus (Moloo et al., 1970; Tellam et al., 1999). The PM primarily functions to envelop the blood meal and protect the intestinal epithelium against abrasion by ingested matter, although it can also represent an obstacle to the passage of ingested parasites into the ectoperitrophic space (Lehane, 1997; Hegedus et al., 2009). The gene GMOY005278 encodes mucin, which participates with peritrophin in the composition of the PM.

We have also identified genes encoding antimicrobial peptides: in Supplementary Table S1, GMOY01052 through GMOY010524 encode attacin, whereas GMOY0011562 and GMOY0011563 encode cecropin. Furthermore, both attacin and cecropin are overexpressed in Gpg trypanosome stimulated or infected flies.

Our work is the first comparison of its kind between the two *Glossina* species. This is primarily due to the fact that the different scientific teams working on HAT commonly focus on investigating either Gmm (and the acute form of trypanosomiasis) or Gpg (and the chronic form of trypanosomiasis), but not both together. Indeed, one of our most relevant findings is the observation that Gmm has the same genes at its disposal that Gpg may use to control its vector competence. Importantly, this comparison will assist future studies in revealing common molecular targets to increase the refractoriness of either fly species to infection by trypanosomes.

## AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: IH, AG. Performed the experiments: IH, BT, SR, HP. Analyzed the data: IH, SR, HP, AG. Wrote the paper: AG.

## ACKNOWLEDGMENTS

The authors thank the "Région Languedoc-Roussillon—Appel d'Offre Chercheur d'Avenir 2011" and the "Institut de Recherche pour le Développement" for their support.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2017.00540/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer CGFdL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Hamidou Soumana, Tchicaya, Rialle, Parrinello and Geiger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.