The holobiont transcriptome of teneral tsetse fly species of varying vector competence

Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

Tsetse flies have a viviparous reproductive biology [6] characterized by the 'in utero' development of a single larva during each gonotrophic cycle. Here, the larva receives nutritional lipids and proteins through maternal secretions from modified female accessory reproductive glands known as milk glands. These milk gland secretions also seed progeny with the core bacteria of the tsetse digestive tract microbiota [7][8][9], specifically the obligate mutualist Wigglesworthia glossinidia [10] and the commensal Sodalis glossinidius [11] (hereafter Wigglesworthia and Sodalis, respectively). Although a more complex microbial diversity in the digestive tracts of adult flies has been reported, these environmentally acquired bacteria are lacking within tenerals (newly emerged adults which have not yet fed), are transient, and not integrated into tsetse biology due to their random occurrence [12][13][14].
Despite sharing a common route of maternal inheritance, the Wigglesworthia and Sodalis endosymbionts have distinct coevolutionary histories with their tsetse host. The [14] ancient mutualist, Wigglesworthia, likely established at the commencement (or soon thereafter) of tsetse species radiation with subsequent codiversification across host species spanning the course of 50-80 million years [15]. The extraordinary significance of the Wigglesworthia mutualism towards tsetse biology is symbolized by the bacteriome structure [16], which exclusively harbors Wigglesworthia intracellularly within specialized tsetse epithelial cells (known as bacteriocytes) in an immunotolerant niche [17] in return for nutrient supplementation of the limited blood diet. In contrast, Sodalis symbionts have a wide tropism being found in the tsetse digestive tract, muscle, hemolymph, salivary glands and fat body [9,18]. Unlike Wigglesworthia, Sodalis has established much more recently as reflected by its stochastic distribution in wild tsetse populations [19][20][21][22][23] which also indicates that this symbiosis is not a requisite for tsetse survival although its role towards tsetse vector competence remains contentious [24].
Age impacts tsetse vector competence as teneral flies have higher permissiveness towards trypanosome infections upon acquisition of their first blood meal (referred to as the "teneral phenomenon" reviewed in [25]). Although numerous microbiota contributions towards tsetse biology have been functionally characterized [26][27][28], the contributions of Wigglesworthia and Sodalis to the "teneral phenomenon" remain poorly understood. Here, we characterize and compare the Wigglesworthia, Sodalis and tsetse transcriptomes within teneral flies of low (Glossina brevipalpis) versus high (G. morsitans) vector competence. We present differences in the gene expression profiles of tsetse, Wigglesworthia and Sodalis that likely contribute towards unique immunological, physiological and metabolic landscapes that may impact vector competence. In particular, we focus on genes involved in nutrient provisioning with Wigglesworthia, genes that may disrupt tsetse physiology and facilitate trypanosome infections with Sodalis, and immunity in teneral tsetse of different species. These findings have significance not only towards understanding the basic biology of tsetse and its microbiota during the incipient stages of adulthood, but also applied merit towards identifying biological factors which may predispose certain tsetse species towards higher trypanosome infection rates which can be used in the design of novel control mechanisms.

General transcriptome features
Eighteen cDNA libraries were generated from the total homogenates of either sex-specific bacteriomes or midguts from G. brevipalpis and G. morsitans teneral (1 day old, unfed virgin) tsetse flies. For each species, we had a collection of 6 bacteriome libraries and 3 midgut libraries. Each library consisted of a pool of either 20 bacteriomes or midguts. The libraries were sequenced using Illumina HiSeq technology, resulting in an average of 17, 390,200 ± 2,558,126 (Std. dev) of paired-end reads (2 × 51 bp in length) from each sample. All reads were scored as "very good" quality (Phred scores > 30, Additional file 1: Figure S1). Reads were mapped to a reference dataset consisting of the collective genomes of tsetse species (G. brevipalpis and G. morsitans), Wigglesworthia isolates (W. glossinidia morsitans and W. glossinidia brevipalpis), and S. glossinidius (Additional file 1: Figure S2) using Salmon [29]. Importantly, the majority of reads mapped back only to their corresponding genomes serving as a control for specificity. A total of 112, 895,620 reads (corresponding to~63.2% of mapped reads) were identified as having tsetse origin, 65,550,668 reads (corresponding to~36.7% of mapped reads) mapped to Wigglesworthia and 214,292 reads (corresponding to~0.1% of mapped reads) were recognized as Sodalis. The lower abundance of Sodalis reads relative to Wigglesworthia likely arises due to a significantly lower population density at the teneral stage [30]. Nonspecific reads (i.e. reads that mapped back to the genomes which were not of origin) were excluded from further analyses. There were no significant differences in either the mean abundance of total reads (total G.  Figure S3B) between tsetse species libraries.

Wigglesworthia-based analyses
As Wigglesworthia shapes multiple aspects of tsetse physiology, immune system maturation and metabolism [17,[26][27][28][31][32][33][34][35][36], we were particularly interested in identifying genes expressed by Wigglesworthia within teneral flies and determining whether these may differ between sexes and tsetse species. Our rationale for these analyses is that distinct expression profiles may likely be a product of host-symbiont interdependency, representing points of diversification in the symbiosis during the course of coevolution of tsetse and their respective Wigglesworthia which may then impact tsetse biology including vector competence.
Principal Component Analysis (PCA) was used to compare the global gene expression of Wigglesworthia between female and male libraries within a host species ( Fig. 2A & B). In both tsetse species, the female and male Wigglesworthia libraries separated along the second principal component, which explained~22% of variance in expression. An additional PCA on a core gene set (consisting of 591 genes) was performed to compare Wigglesworthia gene expression between the tsetse species. Upon the comparison of Wigglesworthia expression between G. brevipalpis and G. morsitans bacteriomes, species-specific libraries separated along the first principal component, which explained 71% of variance (Fig. 2C).
Tsetse sex drives differential expression of Wigglesworthia genes. DESeq [41] was used to identify differentially expressed genes (DEGs). A total of 26 DEGs were identified between Wigglesworthia libraries obtained from female versus male G. morsitans bacteriomes (Fig. 3A, Additional file 2: Table S1), with all of these significantly upregulated within female bacteriomes. Eleven of these genes (42%) are involved in metabolic roles including B vitamin synthesis such as bioA and bioD, both components of the biotin (B1) synthesis pathway, and pdxB in pyridoxal 5′-phosphate (B6) metabolism. The sigma 28 regulator of class III flagella genes (fliA) involved in controlling the assembly of the final components of flagellum and WIGMOR_RS00305, a homolog of fliJ, were also significantly increased in expression. The fliJ gene within Wigglesworthia isolated from G. morsitans was previously characterized as a pseudogene due to truncation [42], but a 41% amino acid identity between the two Wigglesworthia homologs with a particularly high retention in residue identity within the two critical binding domains (aa 39-51 for FlgN binding and aa 65-82 for FliT binding) [43] suggests at least some preservation of function which merits further investigation. The gene purF, also significantly upregulated, encodes amidophosphoribosyltransferase, the enzyme that catalyzes the initial step in de novo purine biosynthesis [44,45].
An elevated expression of Wigglesworthia hslU, a homolog of a chaperone-related protease [46,47], was also observed within G. morsitans female bacteriomes. Interestingly, the oligomerization of HslU subunits is associated with the regulation of cell growth [48] and may support the significant increase in Wigglesworthia density during early adulthood observed in females but lacking in male tsetse flies [30]. We further confirmed the upregulation of a subset of these Wigglesworthia genes (fliA, purF, bioA and bioD) within female bacteriomes through qRT-PCR (Fig. 3B). Although PCA analyses showed a sex separation between G. brevipalpis libraries, DESeq found no significant difference on a gene by gene basis when comparing Wigglesworthia expression.
Differential expression of Wigglesworthia genes between tsetse species. Within the 16 Clusters of Orthologous Groups (COG, [49]) that were shared between the two tsetse species, the proportion of genes within each COG did not significantly differ, neither for the highly expressed genes (TPM > 100, Kolmogorov-Smirnov test, p > 0.9999, Fig. 4A), nor for the DEGs (Kolmogorov-Smirnov test, p = 0.9718; Fig. 4B). An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, 'translation, ribosomal structure and biogenesis' followed by 'coenzyme transport and metabolism' were the most enriched COGs, highlighting the preservation of Wigglesworthia's vitamin provisioning role following host speciation. A total of 326 orthologs (55% of 591 protein coding genes, Fig. 4B) were identified as DEGs between the Wigglesworthia isolates of the two tsetse species (Additional file 3: Table S2). The Wigglesworthia symbionts within the G. morsitans bacteriomes demonstrate upregulation of 174 genes (53% of DEG genes, which corresponds to 29% of orthologous genes), while 152 genes (47% of DEG genes, which corresponds to 26% of orthologous genes) are upregulated within G. brevipalpis (Fig. 4B). Three COG categories which were unique to G. brevipalpis each only housed a single DEG that was significantly upregulated; "RNA processing and modification" (yjeR), "Intracellular trafficking, secretion and vesicular transport" (fliI), and "Defense mechanisms" (yadH). Both Wigglesworthia isolates (Fig. 4B) had a significant proportion of differentially expressed genes (i.e. 22.1% of DEG) falling within COGs associated with "Energy production and conversion" and towards the "Transport and metabolism of …" "lipids", "amino acids", and "carbohydrates". Additionally,~11% of DEGs fell in the COG of "Coenzyme transport and metabolism".
The Wigglesworthia genomes encode a complete flagellar apparatus [42,50] which likely facilitate its   . Values higher than the row mean are represented in yellow, and values lower than the row mean are represented in blue. Gene names are shown on the right. B Validation of selected Wigglesworthia genes found to be differentially expressed between G. morsitans isolates of females and males. Fold change as estimated by the 2 -ΔΔCt method via qRT-PCR supports that these genes are upregulated in females. The bacteriome is within the blue arrowheads and the midgut section is within the pink arrowheads (Created from Biorender.com) evolutionary persistence through vertical transmission using a milk gland route [7][8][9]. Interestingly, the expression patterns of flagellar genes cluster by host species. A total of 22 genes out of 37 flagellum genes examined (6 0%) significantly differed in their expression between the tsetse species, with one belonging to Class I, 18 to Class II and three to Class III (Fig. 5). The operon flhDC is a master regulator of flagellar genes [51] that activates the expression of Class II flagellar components. A significantly higher expression of the Wigglesworthia flhDC operon was observed in G. morsitans isolates. Counterintuitively, the corresponding expression levels of Class II flagellar genes downstream to flhDC were downregulated. Previous tsetse-Wigglesworthia studies demonstrated that the flagellar apparatus is downregulated within the bacteriome population, while it is actively synthesized by the milk gland population [42]. Interestingly, that previous study was based on the detection of the genes fliC and motA, which have a lower expression in our G. morsitans bacteriomes isolates, but a significantly higher expression in our G. brevipalpis isolates. A differential spatial and temporal regulation of flagella components of Wigglesworthia between tsetse host species merits further investigation.

Sodalis-based analyses
To further test the hypothesis that host species impact transcriptomic profiles in their microbiota and that these will be more pronounced the older the symbiosis, we also characterized and compared Sodalis gene expression in the two tsetse species. In all our midgut libraries, the reads mapping to the Sodalis genome constitute a very small proportion of total reads (~0.1%). The percentage of genes that show some level of expression varies widely across libraries, ranging from 58 to 64% in the G. morsitans midgut isolates, with an even greater span in the G. brevipalpis midgut isolates of 28-79% (over a total of 4541 protein coding genes) (Fig. 6A). If a gene was expressed, it likely had a transcription level of < 100 TPM (i.e. > 99% of 4541 genes, Fig. 6A). Only a small number of genes, constituting less than 1% of the total genes, is highly expressed (TPM > 100). More specifically, the proportion of genes that were highly expressed is significantly larger in the G. morsitans isolates (26.33 + 4.410) when compared to G. brevipalpis isolates (1.333 + 0.333, Welch's test, p = 0.0291, Additional file 4: Table S3). For those genes with > 100 TPM, the average TPM does not differ between the two tsetse host species (275.9 + 26.87 in G. morsitans isolates vs. 208.2 + 9.799 Fig. 4 COG classification of highly expressed genes and DEGs between Wigglesworthia isolates. A Clustering of highly expressed genes (TPM > 100) into orthologous groups. Each COG shows columns for highly expressed genes in each Wigglesworthia isolate. The numbers on top of the bars indicate the percentage of genes included in that particular COG relative to the total number of genes with TPM > 100 for each Wigglesworthia isolate. B Top, the horizontal bar partitions the fractions of non-differentially and differentially expressed genes among the 591 orthologs shared between the two Wigglesworthia isolates. Bottom, clustering of the differentially expressed genes into orthologous groups. Each COG shows columns for genes upregulated in each Wigglesworthia isolate. The numbers on top of the bars for each Wigglesworthia isolate indicate the percentage of genes included in that particular COG relative to the total of differentially expressed genes (n = 326). If a gene had more than one COG, it was placed into each respective COG (i.e., these genes have more than one representation). There are three categories in which the Wigglesworthia isolate from G. morsitans did not have genes that were significantly upregulated in G. brevipalpis isolates, nested t-test, p = 0.6042). Further, a PCA analysis shows that tsetse host species can account for 41% of gene expression variation (PC1) between the Sodalis isolates ( Fig. 6B), with no clustering by host sex. The analysis of read counts via IDEAmex [52] identified 235 genes to be differentially expressed between Sodalis isolates of the two tsetse species (Fig. 6C, Additional file 5: Table S4). These genes represent a very small proportion of the total protein coding genes (5.2%), particularly when contrasted with the high fraction of orthologs differentially expressed between the two Wigglesworthia isolates (55%).

Sodalis exochitinase expression profile may contribute to tsetse vector competence
The higher susceptibility of teneral tsetse to trypanosome infections is thought to partly arise due to the immaturity of the peritrophic matrix (PM). The PM of adult tsetse is continuously produced by the cardia (characteristic of a Type II PM) and forms a protective semipermeable barrier within the intestinal tract by surrounding the blood bolus within an endoperitrophic space. Due to the PM being rich in chitin [53] coupled with the higher susceptibility of tsetse flies towards trypanosome infection as tenerals [54], Sodalis genes encoding chitin-associated proteins may compromise PM integrity and thereby facilitate trypanosome infection [55][56][57]. Two genes involved in chitin-associated activities were among the most differentially expressed between the two Sodalis isolates ( Fig. 6D and E). The genes encoding the predicted chitin-binding protein (NCBI Protein ID: BAE74790) and exochitinase (chiA; NCBI Protein ID: BAE74749) are significantly upregulated within the Sodalis isolate of G. morsitans relative to that of G. brevipalpis during the teneral host stage ( Fig. 6D and E). The exochitinase gene is essential for Sodalis persistence within tsetse [58] as its chitinolytic activity produces N-acetyl-D-glucosamine (GlcNAc) which is the principal carbon source for this bacterium. Additionally, the GlcNAc monosaccharides inhibit antitrypanosomal lectins present in tsetse midgut [59] impeding their binding to trypanosome surface carbohydrates promoting parasite establishment [60]. The Sodalis exochitinase activity may also facilitate trypanosomes crossing into the ectoperitrophic space by disrupting the physical integrity of the PM.
Highly expressed Sodalis genes contain a large proportion of DEGs. Interestingly, the highly expressed genes (TPM > 100) in the Sodalis transcriptome contain also a disproportionately high number of DEGs (Fig. 7). As mentioned before, only about 5.2% of all Sodalis coding sequences are differentially expressed between the host species isolates; however, the subset of genes with TPM > 100 (Fig. 7) contains 55.3% of the total DEGs, which may indicate genes important for the Sodalis-tsetse symbiosis. For example, orthologs of type II toxin-antitoxin systems (SGP1_RS14115) along with a formation regulator BssS (SGP1_RS09085) facilitate biofilm formation [61,62]; hypothetically this may support close proximity of Sodalis to the PM lining of the gut lumen, where the bacterium may then deploy exochitinase (SGP1_RS13055), likely aided by lytic polysaccharide monooxygenase (SGP1_RS14050) and glycoside hydrolase family protein (SGP1_RS23580), in order to degrade chitin [63,64] for access to GlcNAc as nutritional source. Although the main known role of chaperones is towards correcting misfolded proteins during stress response [65][66][67][68], they are also hypothesized to be important in mediating insectbacteria interactions [69]. For example, groES constitutes one of the most abundant transcripts of the ant symbiont Blochmannia [70], Wigglesworthia in wild tsetse populations [71]) and Buchnera within aphids [72]. Sodalis transcriptomic profiles within teneral tsetse flies. A Scatterplot with TPM distribution within each tsetse midgut library; x axis is in log 10 scale, only genes with TPM > 0 are plotted (left). Dots around the median indicate TPM values of individual genes. Bar graphs partition the percentage of the total number of genes according to their expression levels (low expression < 100 TPM, high expression > 100 TPM or no expression with TPM = 0) with corresponding percentage of genes with > 100 TPM indicated by the call-outs on the bottom of each corresponding bar (right). Across midgut libraries of both tsetse species, the Sodalis isolates exhibit low gene expression. B PCA analysis indicates that 41.1% of the variability across Sodalis expression may be accounted for by tsetse host species. C Output of differential expression analyses to obtain the consensus set of DE genes between tsetse species isolates. The intersect at the center of the Venn diagram contains the genes found to be differentially expressed by the four informatic approaches indicated on each oval (n = 235). D-E Average TPM from three gut RNASeq libraries of selected Sodalis genes involved in the metabolism of chitin; asterisks indicate significant differential expression (*; p < 0.0001)

Tsetse fly based analyses
Multiple studies have demonstrated that trypanosome and bacterial infections in tsetse flies involve the expression of antimicrobial peptides (AMPs), reactive oxygen species (ROS) and other key anti-pathogen genes [73][74][75][76][77]. Yet, little has been done to contrast immunological profiles at specific developmental timepoints between tsetse species and integrating with the activity of the core microbiota. To complement the characterization of the symbiont transcriptomes, and to obtain a holistic picture of the genomic interplay within tsetse that may enhance our understanding of interspecific differences in host traits, we also examined the transcriptomes of G. brevipalpis and G. morsitans.

Tsetse transcriptomic profiles show a distinct clustering by species and tissues
If a tsetse gene was expressed, it likely had a transcription level of < 100 TPM (Fig. 8A). Only a relatively small proportion (~8.2%) of the total gene count is highly   Table S5) and 3134 DEG between G. brevipalpis and G. morsitans in midgut libraries (Fig. 8D, Additional file 7: Tables S6).
Tsetse species differ in the expression of immunity related pathways as tenerals Given that immunity plays an essential role in mediating a spectrum of host-microbe interactions [78,79], we compared the immunological transcriptomes of G. brevipalpis and G. morsitans bacteriomes and midguts. Immunity-related genes were identified via orthology with D. melanogaster [80] and interspecies comparison performed [81]. These genes are critical components of various immunological responses including cellular, humoral, melanization and RNAi pathways (as identified in [80]). A heat map of immunity gene expression demonstrates clustering that distinguishes guts from bacteriomes and also by tsetse species (Fig. 9, Additional files 8 and 9: Tables S7 and S8 respectively). On average, genes involved in the various immunological mechanisms, are upregulated in midguts relative to the bacteriomes supporting the immunopermissive space of the bacteriome serving to protect essential Wigglesworthia symbionts (37.61 + 3.442 TPM in midguts vs. 15.88 + 1.154 TPM in bacteriomes; n = 86, nested t-test, p < 0.0001). This immune tolerance is further  23.8% of the variability may be accounted for by tsetse species (PC2). C Output of IDEAmex which depicts DE genes within the bacteriomes of tsetse species isolates; the intersect at the center of the Venn diagram contains the tsetse genes found to be differentially expressed by the four informatic approaches as identified adjacent to each oval; n = 3246 DE genes. D Output of IDEAmex which depicts DE genes within the midguts of tsetse species isolates; the intersect at the center of the Venn diagram contains the tsetse genes found to be differentially expressed by the four informatic approaches as identified adjacent to each oval; n = 3134 DE genes exemplified by the expression of pgrp-lb which is significantly higher within the bacteriomes of both tsetse species relative to midguts. Within the bacteriome the peptidoglycan recognition protein-LB (PGRP-LB) scavenges peptidoglycan released during Wiggleworthia cell division, thus preventing the activation of the hostile IMD pathway and offering symbiont protection [36,82].
The cellular immunity category contains the only DEG identified between intraspecific bacteriomes and midguts and also between tsetse species, LpR2 (GMOY006504, GBRI030794). LpR2 has a significantly higher expression in the midgut libraries of both tsetse species when compared to the corresponding bacteriome expression levels.
Interspecies comparisons also indicate that LpR2 is significantly upregulated within G. morsitans midguts and bacteriomes, when compared to the corresponding G. brevipalpis libraries. LpR2 encodes a lipophorin receptor involved in the regulation of the Toll pathway [83]. An additional cellular immunity gene expressed significantly higher in G. brevipalpis midgut libraries was pvr which coordinates immunity responses through the inhibition of humoral immunity while stimulating hemocyte distribution, an early event in cellular immunity [84].
Humoral immunity pathways, such as Toll and Imd, are generally activated upon infection by Gram-positive and Gram-negative bacteria, respectively, and lead to the Fig. 9 Expression profile of immunity genes in teneral G. morsitans and G. brevipalpis. Heatmap with row-normalized expression levels are shown where each row represents a gene and each cell represents the relative expression level for a sample of midguts or bacteriomes in terms of Zscores [observed transcripts per million (TPM) minus row mean TPM, divided by the standard deviation of TPMs for that row]. Values higher than the row mean are represented in green, and values lower than the row mean are represented in red. VectorBase gene ID for the two tsetse species is provided at the right of each row, including the gene symbol for the ortholog in D. melanogaster according to FlyBase. A black square next to the gene indicates a significant differential expression (adjusted p < 0.05) according to the comparison on the column headers located at the: DE = differentially expressed; G. BR. = G. brevipalpis; G. MO. = G. morsitans; BAC = bacteriome; GUT = midgut. The p-values for the corresponding comparisons are included in Supplementary Additional file 8: Table S7 and Additional file 9: Table S8. Blocks at the right group genes by immunity class, the unknown category indicates genes that were differentially expressed in D. melanogaster upon challenge with pathogenic bacteria, but are not genes associated with the other classes, immunity classes are provided only as a guidance, as cross-talk between pathways exists; blocks at the bottom indicate tsetse tissue and species of origin production of distinct sets of AMPs, such as drosomycin, defensin and metchnikowin (Toll pathway) and attacin, cecropin and diptericin (Imd pathway) [85][86][87][88][89]. The imd gene is highly expressed both in G. brevipalpis midguts and bacteriomes. The gene imd is a strong regulator of antimicrobial responses against invading Gram-negative bacteria by inducing the expression of transcripts that encode antimicrobial effector proteins upon the recognition of specific microbial-associated molecular patterns [90], which is also consistent with the higher expression of cecropin within G. brevipalpis midguts [91]. A higher expression of cecropin may confer greater protection to G. brevipalpis against trypanosome infections, as products of orthologs in other insect species have killing activity against the related Trypanosoma cruzi [92,93].
Interestingly, effete is highly expressed within midguts, but it is particularly higher in G. morsitans. The effete protein (Ubc5) mediates the polyubiquitination of IMD, leading to its degradation within the proteasome [94] which aligns with a lower imd expression. UevA joins Ubc5 in the polyubiquitination of IMD [94]; Uev1A is highly expressed across libraries, however its expression is not significantly different between any of the library comparisons. These results may suggest a decreased potency of the IMD pathway in G. morsitans in the teneral state, which merits further investigation.
Within the Toll pathway, G. brevipalpis bacteriomes have a higher abundance of spirit transcripts in comparison to G. morsitans. The serine protease Spirit functions as a processing enzyme for the cytokine-like molecule Spätzle [95], a required initial step towards the activation of the Toll receptor for countering Gram-positive and fungal pathogens. The Toll pathway inhibitors pellino and cactus are upregulated in G. brevipalpis bacteriomes when compared to midguts. The proteins Pellino [96] and Cactus [97]) inhibit the Toll pathway by impeding signal transduction from the cell surface [96]) and by decreasing transcription of antimicrobial peptide coding genes [98], respectively. This may suggest that G. brevipalpis flies offer a more permissive microenvironment for bacterial growth in their bacteriomes. Additionally, annexin B11 (AnxB11) was also upregulated in the bacteriomes of both species. Mammalian annexins are inhibitors of immune responses, where they suppress inflammatory responses during apoptosis [99,100]. This observation further supports a more tolerant microenvironment in both tsetse species G. brevipalpis and G. morsitans likely aimed to sustain high bacterial densities within bacteriomes when compared to midguts.
Overall, genes involved in the JAK/STAT pathway show a low expression (TPM < 100) at the teneral stage [12.62 + 1.401 TPM in JAK/STAT genes (n = 10) vs. 23.12 + 1.405 TPM in all immune-related genes (n = 86); nested t-test, p = 0.0403], however, comparative functional studies will be needed to validate differences in activation of the pathway between tsetse species. The cytokine Upd3 mediates cellular immune response and is an activator of the JAK/STAT pathway [101]. Expression levels for upd3 are consistently higher in G. brevipalpis bacteriome and midgut libraries relative to G. morsitans. Concurrently, the orthologs of the Signaltransducer and activator of transcription protein at 92E (stat92E) (GMOY008510 and GBRI003186) exhibit a higher expression in G. brevipalpis midguts. Upon pathogenic bacterial infection, Stat92E translocates to the nuclei of fat body cells where it drives antimicrobial peptide expression [102]. In contrast, a feedback inhibitor of the JAK/STAT pathway Apontic (apt) [103]) has relatively lower expression within G. brevipalpis libraries. It is puzzling that the ortholog set of the stat92E gene (GMOY003394 and GBRI003185) shows an opposite pattern of expression, where these have a higher level in G. morsitans bacteriomes and midguts when compared to the corresponding libraries within G. brevipalpis. This divergence in expression highlights the importance of functional characterization of genes across tsetse species.
In the melanization branch of immunity, both the Melanization Protease 1 [104] and Serine protease 7 (MP1-Sp7) may catalyze the initial activation of the pathway by cleaving the prophenoloxidase (PPO) zymogen to its active form phenoloxidase [105]. The serpin Spn27A is a serine protease inhibitor that negatively regulates melanization to limit the response to only the site of injury or infection preventing self-harm from excessive induction [106]. Interestingly, G. brevipalpis exhibits a simultaneously higher transcript abundance of both MP1-Sp7 (ortholog pair GMOY002535-GBRI013999) and Spn27A, which may allow this fly species to be poised to better respond should a pathogenic invasion occur and melanization required. Four tsetse ortholog pairs mapped to MP1-Sp7 in D. melanogaster, but the ortholog pairs GMOY002008-GBRI008730, GMOY006266-GBRI009793, and GMOY008308-GBRI030131 do not show a significant differential expression between tsetse species.

Discussion
Simultaneous host and symbiont examination enhances the understanding of their integrative biology RNAseq enables the parallel assessment of transcriptomes from co-occurring species (many of which are unable to be separated without compromising the vitality of one or more partners) such as hosts and their microbiota. This approach is especially suitable for the discovery of novel points of interaction in host-microbe symbioses and the synthesis of robust hypotheses pertaining to how traits may be generated through host/ microbiota activities. In this study, our main objective was to identify differentially expressed host and symbiont bacteria genes, between two tsetse fly species at the teneral stage, and to characterize how these transcriptional profiles may impact host biology including vector competence. Additionally, we hypothesized that symbionts (i.e. Wigglesworthia) with a lengthier host coevolution would show more distinctions in gene expression between tsetse species than recently acquired symbionts (i.e. Sodalis) which likely represent a greater extent of host adaptation.
The tsetse fly is recognized as a valuable animal model for enhancing our understanding of host-microbe symbiosis while also having high public health and agricultural significance as the obligate vector of African trypanosomes. As tenerals, tsetse have the highest susceptibility to trypanosome infections [25,54,[107][108][109][110] likely due to a compilation of low levels of anti-trypanosomal binding midgut lectins, depleted fat reserves following metamorphosis, and the immature structural integrity of the PM crucial for both physical containment of trypanosomes within the endoperitrophic space and also towards midgut epithelial immune regulation [53,60,111]. Furthermore, vector competence varies between tsetse species with members of the Morsitans subgenus, including G. morsitans, exhibiting higher susceptibility to trypanosome infections, while those of the Fusca subgenus, such as G. brevipalpis, are comparatively poor vectors [112][113][114]. A deeper understanding of the molecular interactions between tsetse and its endogenous microbiota and how these may facilitate the establishment of trypanosomes during the teneral stage offer pillars for the development of novel and specific vector control strategies.
We expected to find that genes and pathways promoting trypanosome infection as tenerals would be enriched in expression in G. morsitans relative to G. brevipalpis which would associate a characteristic transcriptome profile of tsetse and its symbionts with vector competence. These interspecific distinctions may be further studied to understand functional diversification and relevancy towards tsetse biology and ecology. For example, differential transcriptomic profiles may help distinguish degrees of reliance among symbionts and tsetse species, likely influenced by differences in symbiont history with tsetse, and help to identify targets for disruption of flysymbiont interactions in the context of novel vector control strategies targeting critical aspects of symbiosis.
The Wigglesworthia symbiont is not only essential for tsetse fly biology, but also its interaction with other members of the microbiota including Sodalis [115] and trypanosomes [27,31]. Intriguingly, between 72 and 83% of Wigglesworthia genes are expressed within teneral tsetse flies indicating high activity following adult metamorphosis.
The acquisition of nutrients by trypanosomes must be strategically orchestrated with host metabolism requiring a fine balance between obtaining sufficient nutrients to complete its lifecycle but not sacrificing tsetse fitness to the extent that transmission to a naïve vertebrate host is compromised. Trypanosomes are auxotrophs for metabolites that are also essential for the fly and are available in very low amounts within blood, such as B vitamins [116,117]. For example, G. brevipalpis harbors a Wigglesworthia isolate incapable of folate (B9) biosynthesis for which trypanosomes are also deficient. The exogenous supplementation of the G. brevipalpis diet with folate makes this tsetse species significantly more permissive to trypanosome establishment [31], highlighting trypanosome reliance on symbiont generated nutrients for successful vector infection. Here, metabolic properties of both tsetse and its bacterial symbionts may play key roles in trypanosomal nutrition and may signal parasite developmental cues that may ultimately impact the outcome of an infection. Genes in this category include those directly involved in vitamin and cofactor biosynthesis, such as those in the COG category of "Coenzyme transport and metabolism". Furthermore, a higher expression of Sodalis genes involved in chitin metabolism, such as the high expression of exochitinase and chitinbinding protein observed in the G. morsitans isolates, may have direct effects on trypanosome-tsetse interactions facilitating parasite establishment via competitive interference with lectins, while compromising the physical robustness of the PM which is primarily constituted of peritrophins and chitin. Lectin abundance within the midgut increases with tsetse age [110], therefore the breakdown of chitin by Sodalis would be less damaging towards anti-trypanosomal efforts as tsetse age [118]. Furthermore, chitin-binding proteins are thought to work in conjunction with exochitinases facilitating cell adhesion to cellular targets (reviewed in [119]), and orthologs have been implicated as virulence factors in various bacterial infections (reviewed in [120]). The transcriptomic profile of Wigglesworthia flagellar genes represents two different stages of flagella synthesis between the species isolates. Flagella facilitate fine-tune responses to environmental stimuli by bacteria [121] and their regulation and synthesis is metabolically expensive, stressing the importance of global and master regulators genes to control their expression. The master regulator flhDC is transcribed into a single mRNA and translated into two distinct proteins FlhD and FlhC which assemble into the hexameric complex FlhD 4 C 2 [122]. This complex attaches to promoter regions upstream of class II flagellar genes [51] facilitating ribosomal recruitment and transcription. Given the temporal regulation of this cascade (reviewed in [123,124]), the stability of FlhD 4 C 2 is under tight control [125] with even the degradation of the flhDC mRNA controlled by global regulators such as CsrA [126,127]. However, as csrA orthologs are absent in Wigglesworthia genomes, the regulation of master control genes such as flhDC and the potential role tsetse may play towards mediating flagellar control deserves further investigation. With this understanding, our results which show contrasting levels of flhDC transcription by Wigglesworthia may be due to measuring gene expression at a single time point. Despite the great advantage of looking at all the genes present in a pathway simultaneously, RNASeq lacks the temporal resolution necessary to reflect the dynamic nature of regulatory processes. This temporal regulation may explain our results, as for example, the G. morsitans isolates expression pattern may represent a stage where the flhDC mRNA is being highly transcribed, but it has yet to be translated to go on and exert its activation on class II gene transcription. This would account for a comparatively high expression of flhDC operon while the class II genes are still at low levels. Conversely, the pattern observed in G. brevipalpis may reflect a stage further in the flagellar synthesis cascade where the flhDC mRNA was already transcribed and mostly degraded, while the corresponding protein complex is activating (or has already) the transcription of downstream class II flagellar genes. Further studies following the mRNA stability and protein levels of flhDC across tsetse development, specifically encompassing the late pupae to early adult transition, may clarify the diverging regulation dynamics behind these expression patterns with likely implications towards acquisition of the obligate Wigglesworthia critical for tsetse developmental biology. Inhibiting the vertical transmission of Wigglesworthia renders female progeny sterile and may serve as a novel angle for next generation pesticides with sole specificity to the tsetse fly.
The low number of read counts arising from Sodalis is not surprising given that the abundance of 16S rRNA sequences belonging to Sodalis is relatively low in G. morsitans [12,128]. The small proportion of differentially expressed genes in Sodalis, in comparison to Wigglesworthia, may reflect differences in symbiont acquisition times where the ancient Wigglesworthia is particularly finetuned to its host species due to their extensive coevolutionary history. In contrast, Sodalis is in an incipient stage of co-diversification [129,130]. Two other notable features of the Sodalis transcriptome include the significantly greater proportion of highly expressed genes (> 100 TPM) within the G. morsitans midgut isolates and the wide range of Sodalis genes expressed within G. brevipalpis individuals.
Tsetse fly fitness requires a balanced interaction with its microbiome and the environment Invertebrates depend on innate immunity to mediate responses to microorganisms. These interactions may antagonize an infection, neutralize a pathogen, or even establish permissive (i.e. tolerance) conditions, thus promoting beneficial symbioses [78]. Beneficial symbionts have been found to orchestrate a coordinated development of the host immune system that simultaneously allows persistence while still enabling protection from pathogens. For example, Burkholderia symbionts differentially suppress host immunity to allow persistence [131] while playing a critical role in the proper functioning of the immune system in the bean bug Riptortus pedestris [132]. Similarly, Wigglesworthia is essential for the development of the immune system in tsetse [33,34], given that larvae reared in the absence of this symbiont exhibit compromised humoral and cellular immune responses to pathogens as adults [34,35].
The transcriptomic profiles of immunity related genes we observe in tsetse midguts and bacteriomes are consistent with an attenuated response to symbionts, rather than with "on" or "off" states, as for example regarding humoral immunity. The Toll pathway generally responds to challenge by Gram-positive bacteria and fungi, while the IMD pathway is dedicated towards the surveillance and protection against Gram-negative bacteria and fungi [89]. Our bacteriome libraries indicate that the Toll pathway is inactive in tenerals, not surprising, given that the sequencing libraries were generated from laboratory reared teneral flies lacking exposure to exogenous bacterial challenges. However, the Imd pathway is active within tenerals, as may be expected given that the tsetse microbiota is dominated by the Gram-negative bacteria, Sodalis and Wigglesworthia. However, these natural infections represent a physiological challenge for the tsetse, particularly due to the essential nature of its association with Wigglesworthia. Tsetse appear to have evolved mechanisms to circumvent this challenge by differential activation of the pathway within bacteriomes versus midguts and through the targeted expression of genes such as pgrp-lb, which when translated scavenges peptidoglycan released during Wiggleworthia cell division, thus preventing the activation of the hostile IMD pathway [36] which would prove detrimental towards Wigglesworthia.
Interestingly, several observations support an enhanced readiness in G. brevipalpis to deter trypanosome infections as tenerals. Previous transcriptome analyses in tsetse have found that activators of both Imd and Toll pathways are present in the fat body [133] and are necessary to counteract trypanosome infections and decrease their density [134]. The imd gene is highly expressed in G. brevipalpis midguts along with a concomitant high expression of the antimicrobial peptide cecropin. The cecropin peptide has potent killing activity against the American trypanosome Trypanosoma cruzi, [92,93], so similar deleterious effects against African trypanosomes may thus be hypothesized. Additionally, genes for enzymes that initiate the melanization cascade (i.e. MP1-Sp7) and their corresponding inhibitors (Serpin 27A) both have a higher expression in G. brevipalpis, which may enable a faster deployment should a trypanosome infection occur, as phenoloxidases have been implicated in response to trypanosome challenges in other insects such as triatomines [135] and bumble bees [136]). Consistent with this rationale, a pre-activation of immunity via artificial bacterial challenge, enables tsetse to respond more efficiently to a trypanosome challenge [74]. It is plausible that tsetse species provide microenvironments with different degrees of biological hostility toward trypanosomes, which would translate into distinctions in vector competence.
The differential expression observed in the set of genes arising from tsetse and its symbionts becomes even more relevant, as these occur at the teneral state. It seems possible that following adult metamorphosis, G. brevipalpis is comparatively better suited than G. morsitans to counter trypanosome infections. These gene expression patterns warrant a deeper investigation. For example, selective knockdown of genes or their activity, through RNA interference or chemically mediated inhibition, followed by trypanosome challenge would provide avenues targeted at assessing the contribution of identified genes towards vector competence.

Conclusions
The tsetse fly, similar to other animals, has to balance protection against pathogens with the biological integration of its essential microbiome. Our results indicate that this equilibrium may be, at least partially, achieved via a comparative downregulation of immunity in the compartments that harbor essential symbionts (i.e. bacteriomes in tsetse) relative to a direct route for pathogen entry, such as the digestive tract. Here, we show that bacterial symbionts exhibit transcriptomic profiles that reflect the duration of their respective host co-evolutionary histories, with a high percentage of DEGs in the ancient Wigglesworthia and a significantly lower proportion of DEGs in the more recent Sodalis when comparing tsetse species isolates. Furthermore, observed differences in the metatranscriptomes of the two tsetse species considered, such as the putatively higher deployment of antimicrobial peptide cecropin by G. brevipalpis, or higher transcription of enzymes with predicted chitinolytic activity in the Sodalis isolate from G. morsitans, offer insight into mechanisms that may predispose tsetse species to trypanosome establishment.

Insect rearing
Glossina morsitans morsitans pupae were provided by the Institute of Zoology, Slovak Academy of Sciences (Bratislava, Slovakia), and Glossina brevipalpis pupae were supplied by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (Vienna, Austria). Pupae were maintained in the Department of Biology insectary at West Virginia University at 24 ± 1°C with 55% relative humidity on a 12-h light/12h dark schedule until adult eclosion. Teneral flies (newly emerged adults prior to blood meal acquisition) were collected < 24 h post emergence from pupae and sorted by sex. Flies included in this study were all trypanosome free.

Dissections and RNA extraction
Bacteriomes and intestinal tracts (flanked by the bacteriome and the Malpighian tubules) were microscopically dissected and placed in RNAlater (Invitrogen, Carlsbad, CA) at − 20°C. The bacteriomes or intestinal tracts of 20 teneral tsetse of each sex and species were pooled for one biological sample, resulting in a total of 18 biological samples used in our analyses. Bacteriomes and guts were homogenized and total RNA was extracted using a Mas-terPure RNA purification kit (Epicentre, Madison, WI) according to the manufacturer's protocol for tissue samples. DNA was removed from the RNA samples using a Turbo DNA-free kit (Ambion, Austin, TX) following the rigorous DNase treatment option. The RNA concentration was measured using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA) with an Agilent 2000 Bioanalyzer RNA Nano chip used to validate RNA sample quality and integrity. mRNA library preparation, sequencing, and genome alignment Library preparation was performed at the WVU Genomics Core Facility by using 1 μg of total RNA and Ribo-Zero Gold Epidemiology kit (Illumina, San Diego, CA) following the manufacturers recommended protocol. Following cDNA synthesis, libraries were quantified via Qubit fluorometer with high sensitivity DNA reagent and run on an Agilent high sensitivity DNA chip to determine average library size. The libraries were pooled in equimolar amounts and sequenced using the Illumina HiSeq 1500 platform (2 by 51 bp) at Marshall University. Following sequencing, raw reads were postprocessed to remove Illumina adapter and barcode sequences.

Wigglesworthia
(GCA_ 000008885. 1) and Sodalis glossinidius (GCA_ 000010085.1) genomes using Salmon [29]. Gene transcription level was converted to Transcripts per Million (TPM) fragments mapped and used for statistical comparison of expression levels between libraries [138]. Statistically significant differences were accepted at p < 0.05 with adjusted p-values for multiple testing.

Principal Component Analysis (PCA)
PCAs comparing gene TPM values were performed with the prcomp package (version 3.6.3) in the R software suite. PCA excluded genes that lacked expression across all libraries. PCA plots were visualized using the R package ggbiplot.

Differential expression analyses
IDEAmex [52], which implements the R packages DESeq2 [139], edgeR [140], limma [141] and NOISeq [142], with an adjusted p-value cut-off of 0.05 was used to compare gene expression profiles between Wigglesworthia isolates, between Sodalis isolates and between tsetse species. To compare Wigglesworthia gene expression between sexes within a species, the mapped read counts were initially used as input for DESeq. Additionally, EggNOG mapper [143] was used to assign Clusters of Orthologous Groups (COG, [49]) to categorically summarize annotation data into specific biological categories that were enriched within specific libraries.

Validation of differential expression through qRT-PCR
A subset of genes identified to be differentially expressed between libraries was verified via quantitative reverse transcription PCR (qRT-PCR). For the validation of the differential expression of Wigglesworthia genes, biological samples that consisted of six individual bacteriomes from either tsetse sex were collected in RNAlater (ThermoFisher Scientific, Waltham, MA) following manufacturer's protocol. Total RNA was isolated using Mas-terPure™ RNA (Epicentre, Madison, WI) and treated with TURBO™ DNase (ThermoFisher Scientific, Waltham, MA) following the rigorous treatment protocol to remove contaminant DNA. Linearized plasmid standards used for the quantification of gene copy number were made for respective genes using the pGEM®-T Vector Systems (Promega, Madison, WI) according to manufacturer instructions. Table S1 includes a list of primers used for cloning and qPCR amplification. First-strand cDNA synthesis was performed with SuperScript™ II Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA). Second-strand cDNA synthesis was performed with a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using the SsoFast™ PCR cocktail (Bio-Rad) and following the conditions used in Additional file 10: Table S9. Three technical replicates were performed for each biological sample and averages obtained. The relative gene expression was determined for selected genes using the 2 -ΔΔCt method [144].

Immunity
Putative orthologs of D. melanogaster immunity genes [80] in tsetse were identified by using FlyBase (https:// flybase.org). Orthologs between G. morsitans and G. brevipalpis were identified through VectorBase (https:// vectorbase.org). The list of immune-related orthologs was validated and placed into pathways according to [81].

Graphs and statistical analyses
Heatmaps were generated with the 'heatmap.2 function' in the gplots R package with clustering by species/tissue type based on the distinct expression patterns between isolates. GraphPad Prism was used for statistical analyses with p-values < 0.05 considered statistically significant. Infante for mapping of the raw reads. We thank Adam Pollio for contributions to the Wigglesworthia flagella gene analyses, and Ying Zhang for help in rearing the tsetse flies. All authors approved the final version of the manuscript.